Datasets:

compositional-format-following

/

summarization_eval

like 0

over the past decade , protein separation techniques and peptide analysis by mass spectrometry have greatly improved , and qualitative and quantitative proteome comparisons have become powerful tools for defining the composition of complex proteomes and the functions of protein complexes.(1 ) current ion trap mass spectrometers , in particular , are very sensitive and have fast analysis times that enable the identification of hundreds of proteins in a single lc - ms / ms analysis . however , obtaining comprehensive protein profiles from very complex samples , such as biological fluids or mammalian cell or tissue lysates , remains challenging due to the large number of proteins present in the sample over wide concentration ranges . to improve proteome coverage , samples typically are fractionated at either the protein level or peptide level , or both . regardless of the initial fractionation methods used , the final step in most strategies involves analysis of tryptic peptides by lc - ms / ms . however , undersampling will occur , that is , only a subset of the peptides will be identified , if the complexity of these tryptic digests exceeds the analytical capacity of the mass spectrometer ( e.g. , when more peptides elute from the hplc column per unit time than can be analyzed , or low - abundance peptides are below the instrument detection limit ) . a common observation when complex proteomes are analyzed is that the lists of proteins identified are often quite variable , which has raised concerns about the general value of proteomics for most applications , if it actually does have poor reproducibility . typically , the identifications of abundant proteins are reproducible , while most of the variability is observed in lower - abundance proteins . one strategy of achieving more comprehensive proteome coverage actually exploits this variability by simply performing repetitive lc - ms / ms analyses on each sample . other strategies for increasing the number of proteins identified include prefractionating the proteome based on protein physical properties , improving separation of the peptide mixture at the hplc separation step , modifying the esi interface to enhance ionization , or adding an ion mobility spectrometry ( faims ) interface before the ion trap.(19 ) proteome coverage also can be improved at the data analysis stage by using high - mass - accuracy data(20 ) or improved data - process algorithms . other strategies include a two - step method that reanalyzes the same sample with an inclusion list ( eliminating redundant precursor selection in the second analysis ) and a replay run that analyzes a sample twice from a single injection with targeted analysis of undersampled features in the replay run.(26 ) although each of these methods can achieve increased sensitivity , it is difficult to assess the relative importance of strategies that have not been directly compared in a single study due to variations in samples , study design , mass spectrometry platforms , data - analysis strategies used , and other laboratory - to - laboratory variations . consequently , a number of direct comparisons of alternative analysis strategies have been conducted to identify the best analysis approaches for specific types of samples such as serum and cell or tissue lysates . factors that should be considered when comparing alternative analysis strategies are the amount of total protein required for the analysis and the total amount of mass spectrometry instrument time required by each method . it is now well - established that repetitive lc - ms / ms analyses , longer hplc gradients , or additional fractionation via either more fractions or more modes of separation of complex proteomes will usually confer some benefit in terms of depth of analysis . the key factor is to determine the most time- and resource - effective strategies for achieving a high depth of analysis , as these factors directly affect sample throughput and proteome analysis cost . in this study , we compared the effects of two commonly used strategies : gelc - ms / ms with repetitive lc - ms / ms analysis and additional protein prefractionation and assessed their effects on depth of analysis and reproducibility using cancer cell lysates . neither strategy requires expensive special hardware or reagents , and both have been integrated into diverse analyses of complex proteomes by multiple laboratories . because of its simplicity , repetitive analysis often has been used to increase protein identifications after different fractionation schemes , most of which involve two dimensions of separation at either the peptide or protein levels , such as mud - pit or gelc . utilizing a cell extract of the human metastatic melanoma cell line 1205lu,(31 ) the gelc - ms / ms analysis with and without repetitive injections was compared and these data sets were compared to a 3-d method that included the prefractionation of the cell lysate using microscale solution isoelectrofocusing ( microsol ief ) prior to the sds - page separation . to balance the total number of lc - ms / ms runs and total instrument time between the repetitive 2-d method and the 3-d method , the 20 fractions from the sds - page fractionation each were analyzed four times and the cumulative result was compared with the 80-fraction proteome analysis using the 3-d approach . other factors such as hplc gradient , mass spectrometry method , and data processing were kept uniform to demonstrate clearly the relative effects of repetitive analyses versus an additional prefractionation step at the protein level . these comparisons showed excellent reproducibility between the methods , and substantially improved proteome coverage using the 3-d method compared with the repetitive analysis of fractions from the 2-d method . rpmi-1640 cell culture medium , sypro ruby stain , and nupage precast gels were from invitrogen corporation , carlsbad , ca . ultra pure urea , thiourea , dtt , and chaps were from ge healthcare , ltd . , giles , u.k . the bradford protein assay kit was from thermo fisher scientific , waltham , ma , and sequencing - grade porcine trypsin was from promega corporation , madison , wi . the human melanoma 1205lu cell line was maintained in rpmi-1640 medium supplemented with 10% fetal calf serum . cells were harvested on ice by scraping in cold phosphate - buffered saline ( pbs ) with inhibitors ( 0.3 mm pmsf , 2 mm sodium orthovanadate , 50 mm sodium fluoride , 1 g / ml leupeptin , 1 g / ml pepstatin ) when 7090% confluence was reached . the tissue culture plates then were washed three times in cold pbs with inhibitors ; the washes were collected , combined with the initial scraped cells , collected by centrifugation , and the cell pellet was quick frozen in liquid nitrogen followed by storage at 80 c . cell lysates were prepared using 800 l of lysis buffer ( 25 mm tris , ph 8.0 , 8 m urea , 2 m thiourea , 4% chaps , 1 mm dtt ) per cell pellet collected from a 15-cm tissue culture dish . the lysate was sonicated with a probe sonicator on ice with a duty cycle setting of 50% and an output level < 5% . insoluble material was removed by centrifugation at 100 000 g for 60 min and was estimated to be < 1% of the total protein based on staining intensity of colloidal coomassie sds gels . the supernatant was reduced using dtt and alkylated with n , n - dimethylacrylamide ( dma ) , as previously described.(11 ) total protein in the supernatant was measured using the bradford method . cell lysates ( 1.5 mg ) were separated into four ph ranges by microsol ief using a zoom ief fractionator ( invitrogen corporation , carlsbad , ca ) . for these separations , the fractionator contained five immobiline gel disks , each sustaining a ph of 3.0 , 5.4 , 6.2 , 7.0 , and 12 , respectively . these disks delineated four separation chambers and the cell lysate in a sample buffer consisting of 8 m urea , 2 m thiourea , 4% chaps , 1% dtt , 1% ph 37 zoom focusing buffer , and 1% ph 712 zoom focusing buffer was loaded into the middle two separation chambers . the outer chambers were loaded with sample buffer only and standard electrode solutions were used . samples were focused at a constant 750 v and a maximum of 1 ma for 3 h. the solution in each sample chamber was removed and measured . each chamber was rinsed with a small volume of sample buffer so that , when combined with the original solution removed from that chamber , the final volume was 700 l . proteins trapped in the immobiline gel disks were recovered using two sequential 175 l extractions for 30 min each using 10 mm tris , ph 8 , 1% sds for 30 min . the cell lysate and microsol ief fractions were initially analytically separated on 10% nupage minigels ( with mes running buffer ) using standard separation conditions . preparative sds - page was run using unfractionated cell lysate and the four microsol ief fractions in an analogous manner to the analytical run , except the separation distance was limited to 40 mm to minimize required numbers of fractions and gel volume per fraction . typically , six lanes two lanes from the gelc ( 2-d ) method containing 60 g each of cell lysate and one lane each from the 3-d method were sliced into 4 mm 1 mm 1 mm thick gel pieces . two adjacent pieces in each lane were combined in a digestion well and were digested overnight with trypsin . corresponding digestions from the duplicate 2-d method lanes were combined , generating a total of 20 digestions for the 2-d method . trypsin digestions were injected into a 75 m i.d . 15 cm picofrit ( new objective , inc . , woburn , ma ) column packed with 5 m magic c18 resin and peptides were separated by nano - hplc ( eksigent technologies , dublin , ca ) interfaced with a ltq linear ion trap mass spectrometer ( thermo fisher scientific , waltham , ma ) . for each analysis , 8 l of trypsin digest was loaded onto the column using solvent a ( 0.1% formic acid in milli - q [ millipore corporation , billerica , ma ] water ) . peptides were subsequently eluted using the following gradient conditions with 0.1% formic acid in acetonitrile as solvent b : 128% b over 42 min , 2850% b over 25.5 min , 5080% b over 5 min , and 80% b for 5 min . to minimize carryover twenty 2-d samples were analyzed first , followed by another replicated injection of the same 2-d samples . then , 80 3-d samples were analyzed , followed by the third and fourth replicated injections of the 2-d samples . the ltq mass spectrometer was operated with dynamic exclusion enabled for 30 s , full scans from 4002000 m / z , and data - dependent ms / ms analysis on the six most intense ions . 28 , rev . 13 , university of washington , seattle , wa ) in bioworks ( ver . 3.1 , thermo fisher scientific , waltham , ma ) , and the human uniref 100 ( ver . may , 2007 ) protein database , which was downloaded from the protein information resource at georgetown university . to generate the decoy database , the protein amino acid sequence for each database entry was reversed and the entire reversed database was appended in front of the original forward sequences . the data were searched against the combined forward / reverse database using partial trypsin specificity with a 2.5 da precursor mass tolerance and 1 da fragment ion mass tolerance . consensus protein lists were generated by dtaselect ( ver 1.9 , licensed from scripps research institute , la jolla , ca ) after applying the following filters : full tryptic boundaries , xcorr 1.8 ( z = 1 ) , 2.1 ( z = 2 ) , 3.25 ( z = 3 ) , cn 0.05 . custom software was then used to ensure that each unique peptide sequence was used only once in assembling the protein list . to identify common and unique proteins found by 2-d and 3-d methods , protein and peptide data were put into a relational database ( mysql ) and matched using custom software . the strategy used to compare ( 1 ) the conventional gelc - ms / ms method ( protein separation by sds gel + reverse - phase peptide separation ) , ( 2 ) gelc - ms / ms with repetitive injections , and ( 3 ) a 3-d method that used microsol ief fractionation prior to the sds - page step ( protein ief + sds gel + reverse - phase peptide separation ) is summarized in figure 1 . to directly compare the relative impact on proteome coverage of repetitive injections versus an additional protein separation step , equal amounts of the same cell lysate were fractionated and an equal number of lc - ms / ms analyses were used . a cell lysate of 1205lu cell line was processed in such a manner that each common step was comparable . the 3-d method consisted of microsol ief , sds - page , and lc - ms / ms . subsequently , the final minimum consensus protein lists from the three data sets were compared to evaluate the ability of enhancing sensitivity of repetitive analysis and additional fractionation strategies . microsol ief initially was developed to prefractionate complex proteomes prior to narrow ph range 2-d gels(33 ) and was subsequently used prior to 1-d sds - page as part of a 4-d strategy for fractionating serum or plasma.(11 ) in this study , we evaluated its utility for prefractionating cell lysates prior to gelc - ms / ms , since this separation mode is orthogonal to sds - page and does not suffer from many of the limitations of 2-d gels . because the dynamic range of protein concentrations in cell lysates is substantially less than in plasma or serum , the microsol ief separation was designed to yield four final fractions rather than the larger number of fractions typically used for plasma . the immobiline membrane disks between chambers were extracted because , as shown previously , proteins with pi values close to the ph of the disk remain in the disk but can be recovered in high yield . extracts from disks with extreme ph values ( 3.0 and 12 ) showed only trace amount of proteins , as few proteins have pi s near these values . extractions from disks located between separation chambers contained a mixture of proteins unique to that disk and proteins present in adjacent chambers as previously observed . when similar samples were separated by microsol ief and analyzed on 2-d gels , comparisons of solution fractions and membrane fractions showed that most proteins recovered from membrane disks were either unique to the membrane or had pi s slightly lower than the ph of the membrane ( data not shown ) . since the goal in this experiment was to fractionate the entire proteome into a small number of fractions at the microsol ief step , disk extracts were combined with the fraction to its right to maximize recovery ( figure 2 ) . the extraction from the ph 12 disk was discarded as it contained a negligible amount of protein . thus , after microsol ief fractionation , the cancer cell proteome was divided into four fractions of roughly similar complexity and with a simpler protein content . separation of the melanoma 1205lu cell extract by microsol ief and pooling to produce four fractions . equal portions of each fraction and separation membrane disk extract were analyzed by sds - page to evaluate separation and relative amounts of total protein in each fraction . the solution recovered from individual chambers was pooled with the adjacent membrane disk extract on the low ph side of the pool , as shown at the bottom of the gel , to produce four fractions . the extract from the ph 12 membrane disk had negligible protein and was not used further . for the conventional gelc - ms / ms method , 60 g of the original cell lysate , which was close to a maximum load while avoiding band distortion , was loaded into each of two lanes of a nupage gel ( figure 3a ) . proteins were separated until the tracking dye migrated 40 mm . after staining the gel with colloidal coomassie , the 40-mm lane was divided into 20 equal fractions and digested with trypsin . digests from corresponding positions in the two replicate lanes were combined to yield sufficient volume for four 8 l injections . sds - page separation of melanoma 1205lu cell lysate and microsol fractions for proteome analysis . samples were electrophoresed until the tracking dye migrated 4 cm ; gels were stained with colloidal coomassie and individual lanes were cut into 20 equal - sized slices , as shown . ( a ) for the 2-d method , the unfractionated lysate of the 1205lu cells was separated in two lanes ( 60 g / lane ) . ( b ) microsol ief fractions derived from 120 g of cell lysate were separated for the 3-d method . for the 3-d method , the amount of each fraction loaded onto the preparative gel was the protein recovered from 120 g of total cell lysate ( figure 3b ) . hence , the total amount of protein in the four gel lanes in figure 3b should be close to the amount of protein in the two lanes in figure 3a , provided sample losses during the microsol ief procedure were low , as has been previously demonstrated . as noted above , the gel lanes from the microsol fractions were cut and digested in the same manner as the gel slices from figure 3a , except in this case there were no duplicate gel lanes to be combined . hplc and mass spectrometer performance were carefully monitored to ensure consistent performance through these experiments . to further minimize effects of minor instrument performance variations , samples were injected in an order described in materials and methods . consistent performance of the autoinjector was monitored by weighing each sample vial before and after injection . there was no significant difference in the injection amount between the two methods ( t - test p > 0.05 ) . all data were consistently analyzed and filtered as described in materials and methods , which resulted in estimated false - positive rates ( fpr ) calculated by dividing reverse - hit peptide counts by forward - hit peptide counts of 1.6% and 1.4% for 2-d and 3-d data , respectively . figure 4 shows the nonredundant peptide and protein counts for the 3-d data set as well as differing numbers of replicates for the 2-d sample set . similar numbers of peptide and protein identifications were obtained for all individual 2-d data sets ( data not shown ) . as expected , the total number of nonredundant peptides increased moderately as additional replicate data sets were combined , and the incremental increase diminished as each new replicate was added . it was evident from the curve shown in figure 4a that the data set resulting from combining four replicate runs was approaching a plateau . parallel trends were observed for the peptide data sets defined by proteins identified by 3 , 2 , or 1 peptide(s ) . a total of 25 641 nonredundant peptides were identified after combining all four replicates of the 2-d analysis , which was 26% less than the 32 216 peptides identified by the 3-d method . ( a ) nonredundant peptide counts from a single 2-d analysis , combined data from increasing numbers of replicate analyses , and the 3-d method . ( b ) corresponding nonredundant protein counts for the same data sets as shown in panel a. the total number of lc - ms / ms runs that each data set contains is shown at the bottom of the figure . protein counts showed a similar trend as the peptide counts ( figure 4b ) , that is , adding a second replicate increased the number of nonredundant proteins more markedly than adding a third and fourth replicate . for example , the number of proteins identified by two or more peptides increased by 594 , 305 , and 162 when two , three , and four replicates were combined . the most appropriate comparison of the 3-d and 2-d methods is between the 3-d data set and four replicates of the 2-d samples because they both involve a total of 80 lc - ms / ms runs using equal amounts of initial cell lysate . when protein identifications based on two or more peptides were counted , the 3-d method identified 3486 proteins compared with 2850 proteins for the 2-d method . these 636 additional proteins ( 22.3% increase ) indicate a clear advantage of the 3-d method compared with the 2-d / replicate run method , even when equal amounts of mass spectrometer time are utilized . the proteomes produced by the 2-d / four repetitive - run and the 3-d methods were compared using the lists of proteins identified by two or more peptides . this comparison was facilitated by the fact that both data sets were searched using the same database and that dtaselect lists all protein names identified in the search . if a protein identification carried one protein name that could be found in the other data set , we deduced that both data sets had identified the same protein . this comparison at the protein name level showed extensive overlap between the two data sets even at this first level comparison ( figure 5 ) . specifically , the two data sets shared 2555 proteins , which corresponded to 90% of the smaller 2-d / repetitive - run proteome . the 295 proteins that appeared to be unique to the less in - depth 2-d / replicate run data set were further investigated to determine the basis for this apparent lack of reproducibility between in - depth analyses of the same biological sample . further analysis showed that 184 of these proteins were present in the 3-d data set as single - hit proteins . a majority ( 135 ) of these 184 proteins were two - peptide proteins in the 2-d data , indicating that the 3-d method identified these proteins by one less peptide . when the unfiltered data for the 3-d proteome was examined using randomly selected proteins in this group , we found that a substantial number of these proteins had been identified by the same second peptide as in the 2-d data set , but one or more of the filtering parameters was slightly lower than the cutoff values selected for global data filtering . as a result , these moderate changes in scoring parameters reduced the two- or three - hit protein identification in the 2-d data set to a 1-hit protein in the 3-d data set . we also further evaluated the 111 proteins found exclusively in the 2-d method . more than three - quarters ( 88 ) of them were two - peptide proteins . among these identifications , the 11 proteins identified by more than three unique peptide sequences were a particular concern , as the number of peptide identifications suggested that these proteins were not near the detection threshold and should have been identified in the more in - depth 3-d proteome . we found that the majority of the peptide sequences associated with these 11 proteins in the 2-d data set also existed in the 3-d data set , but they were assigned to highly homologous proteins ( supplemental table 1 ) . although a large number of common sequences for these proteins were observed in both data sets , a small number of unique sequences led the dtaselect program to assign these peptides to different proteins for each data set . of the 385 peptides assigned to the 111 proteins unique to the 2-d / replicate run data set , 226 peptides were present in the filtered 3-d data the two methods identified 2555 common proteins with two or more peptides per protein ( 90% of the total proteins identified in the smaller 2-d / replicate - run data set ) . of the 295 apparently unique proteins in the 2-d method , 184 proteins were identified in the 3-d data set by one peptide . the pie charts in the lower panels show the number of peptide hits for the 2-d method for the proteins that were not directly identified in the 3-d data set ( 111 proteins or 3.9% of the proteins in the 2-d data set ) and those identified by a single peptide in the 3-d data set ( 183 proteins or 6.5% of the proteins in the 2-d data set ) . these proteins , which were identified by two or three peptides in the 2-d / replicate data set , were identified by a single peptide in the 3-d data set . in each case , a second peptide was detected in the 3-d data set but failed to pass the data filter cutoff due to slightly lower values for xcorr or cn ( bolded values in table ) . the filtering cutoff values used were xcorr 1.8 ( + 1 ) ; 2.1 ( + 2 ) ; 3.25 ( + 3 ) , cn 0.05 . the bold values indicate they are below cutoff value . - indicates not found . to a first approximation , those proteins identified in the 2-d / repetitive - runs data set by only two or three peptides can be regarded as likely low - abundance proteins in the sample analyzed , since sequence coverage is usually a rough indicator of protein abundance level . the relative depth of coverage for these putative low - abundance proteins was compared in the 2-d / repetitive - run and the 3-d methods . as shown in figure 6 , for those proteins identified in both data sets , the majority showed greater sequence coverage in the 3-d data set . that is , of the 491 common proteins identified by two peptides in the 2-d data , the 3-d method found more peptides for 317 proteins ( 64.6% ) and the remaining proteins in this group were identified with an equal number of peptides . furthermore , in 114 cases , the 3-d method found at least three additional unique peptides , indicating a substantially greater depth of analysis . of the 394 common proteins identified by the 2-d method with three peptides , 325 proteins ( 82.5% ) were identified by an equal or larger number of peptides in the 3-d method . these data clearly indicate that , in most cases , the 3-d method had the ability to detect more peptides for low - abundance proteins than did the 2-d method . comparison of the number of peptides identified in the 2-d / repetitive and 3-d methods . ( a ) among proteins common to both data sets , 491 proteins were identified by two peptides in the 2-d / repetitive data set . the 3-d method found an equal number of peptides for 174 proteins and more peptides for 317 proteins . ( b ) among 394 proteins identified with three peptides by the 2-d method , the 3-d method found one less peptide for 69 proteins ( i.e. , + 1 for 2-d ) , an equal number of peptides for 99 proteins , and more peptides for 226 proteins . in this study , we systematically evaluated the relative merits of repetitive lc - ms / ms runs compared with introduction of an additional protein level separation step for increasing proteome coverage of cancer cell lysates . factors that affect apparent reproducibility between proteome analyses performed on the same sample also were evaluated . the basic analysis platform used for repetitive analyses was the commonly utilized gelc - ms / ms method , which can be considered to be a 2-d proteomics method as it involves two dimensions of separation , that is , protein separation using sds - page and reverse - phase hplc separation of tryptic peptides . this method was compared to a 3-d method consisting of solution ief at the protein level followed by the gelc - ms / ms method . it is important when comparing alternative analysis platforms to consider the total number of lc - ms / ms runs per proteome , because improved proteome coverage typically can be achieved by lengthening the hplc gradient or by repeating lc - ms / ms analysis of complex samples . similarly , many separation modes prior to lc - ms / ms can be at least incrementally improved by simply increasing the number of fractions collected , provided that the resolution of the separation method exceeds the initial fraction size used . but in some cases , increases in protein coverage may be too small to be considered advantageous when total analysis time per proteome is considered . hence , evaluation of the merits of greater depth of analysis , particularly small improvements , must be constantly weighted relative to overall throughput . furthermore , total mass spectrometer instrument time frequently is the limiting resource , and when fractionation prior to the lc - ms / ms step is used , it is the rate - limiting step in proteome analysis throughput . hence , the most meaningful comparisons are those where the total mass spectrometer analysis time per proteome is held constant . in this study , we used a consistent gradient time and 80 lc - ms / ms runs for both the 3-d method and the 2-d / repetitive - run method ( four repeat injections ) . similarly , all other experimental variables were held as constant as possible , including use of replicate aliquots of a single cell lysate preparation , gel separation lengths , gel volumes per trypsin digestion reaction , instrument tuning , and data - analysis methods . our goal was to determine , quantitatively , which method represents the more efficient utilization of mass spectrometer time when analyzing complex proteomes . robust proteome analysis methods should be reproducible in addition to identifying the majority of proteins present in a biological sample . one major cause of variations in proteins identified in replicate analyses of the same proteome is undersampling in the mass spectrometer , as discussed above . therefore , high proteome coverage should be linked to good reproducibility of proteome analysis results because extensive proteome coverage will occur only if undersampling is minimized . a second factor that will contribute to poor reproducibility between proteome protein lists is use of data filtering conditions that result in high false - peptide and protein - identification rates , since false positives usually are random . hence , data filtering stringency is another tradeoff that must be considered when selecting a proteome analysis strategy . while low stringency filters contribute to noise and low reproducibility , excessively stringent filters will greatly diminish the number of protein identifications and hence the value of the experiment . in the current study , data filters were used that yielded peptide false - positive rates between 1 and 2% as estimated using a decoy reverse database , thereby minimizing apparent poor reproducibility between data sets . this level of stringency results in very few false positives for proteins identified by two or more peptides and , while there are some false positives within the one - hit protein list , a majority of these identifications are correct . the repetitive analyses of 2-d data showed increased peptide and protein counts ( figure 4 ) indicative of undersampling in the basic gelc - ms / ms method used here . the overall gain from four repetitive analyses of proteins identified by two or more peptides was 1061 ( 59% ) compared to the initial single analysis . a similar increase ( 61% ) was observed in repetitive mudpit using nine analyses.(10 ) as expected , the greatest positive impact on proteome coverage was use of a second replicate run , which increased the number of proteins identified by two or more peptides by 33% while doubling instrument time . in contrast , adding a third and fourth replicate only increased protein coverage by 13% and 6% , respectively . these data indicate that performing a second analysis of each fraction when using gelc - ms / ms would be a positive tradeoff between instrument time and protein coverage . however , further doubling of instrument time by performing four repetitive runs is unlikely to represent optimal use of instrument time for most types of experiments . of course , an alternative to performing a repetitive analysis of gel fractions would be to obtain more slices per gel lane . in analogous experiments where gel lanes were divided into 40 or 60 fractions , we observed increases in the number of proteins identified that were similar to those obtained in this study for duplicate and triplicate analyses of the 20 fractions per gel lane ( data not shown ) . although producing more fractions per gel lane increases the number of in - gel digestions , the overall increase in total analysis time for a proteome is minor . hence , we generally prefer to use more fractions per lane rather than to replicate analyses when greater depth of analysis is desired using gelc - ms / ms experiments . longer gels and larger numbers of fractions per lane were not used in the current study because we wanted to keep total mass spectrometer time per proteome ( approximately 160 h per proteome ) within practical limits while simultaneously matching gel lengths , gel volumes , and other parameters . that is , extrapolating from other 2-d and 3-d experiments that we have performed , we expect that the total number of proteins for each data set ( 2-d , 2-d / repetitive runs , and 3-d ) would have increased moderately if we would have used 40 or 60 slices per gel lane for all samples . but use of 40 or 60 fractions per gel lane would have increased total instrument time to about 320 and 480 h per proteome , which represents an impractically low throughput for most studies . interestingly , as we increase the number of fractions per gel lane to 40 or 60 fractions , the incremental increases in new proteins identified diminish , analogous to the diminishing benefits of adding each additional replicate in the repetitive - run approach ( figure 4a ) . although similar trends are observed for these two approaches , the mechanisms for increasing protein coverage are quite different . that is , using a larger number of gel fractions increases protein separation and simplifies the mixture of proteins present in each fraction , while repetitive runs exploit subtle variations in peptide separations in replicate hplc runs and subtle variations in data - dependent selection of low - level ions for ms / ms fragmentation and analysis . the 3-d method clearly provided superior protein and peptide coverage compared with the 2-d / repetitive method , which indicates that adding an additional protein separation step represents a more efficient use of mass spectrometer instrument time . this method identified 3486 proteins with two or more peptides , which is 22% more than the 2-d / repetitive method that used equal instrument time . at the peptide level , the 3-d method identified 30 385 high - confidence , nonredundant peptides , which is nearly 2.5 times more than that found in a single survey using the 2-d method ( 12 160 ) and 28% more than the cumulative count in four repetitive analyses ( 23 648 ) . furthermore , more unique peptides were found for most low - abundance proteins in the 3-d method data compared with the cumulative 2-d method data ( figure 6 ) . it is not surprising that adding solution ief as an additional orthogonal protein separation step to a gelc - ms / ms method is an efficient strategy for increasing proteome coverage and sequence coverage of lower - abundance proteins . microsol ief separates the proteins that would normally be in a single gel slice into four gel slices ( see figure 3 ) . , the simpler samples should decrease ion suppression effects and reduce dynamic range within each digest . finally , in some cases , improved scores for ms2 spectra in the 3-d method probably resulted from a lower probability of interfering ions being isolated with the target ion for fragmentation . while the repetitive analysis strategy also improved proteome coverage , it had neither a built - in mechanism to reduce repeated sampling of abundant ions between replicates , nor could it explore the ions below the ms2 triggering threshold . an alterative technique that has sometimes been used to improve replicate runs is to scan different mass ranges in each replicate . however , pilot experiments suggested that this approach was less productive than the simple repetitive analysis method used here . one frequent criticism of proteomics methods is that the proteins identified on repeat analyses often are not very reproducible . a recent study suggested good reproducibility was achievable across 27 laboratories on a simple 20-protein mixture after uniform data processing was used.(35 ) but this simple sample of abundant proteins at the same concentration does not reflect real biological complexity . hence , in the current study , we compared the reproducibility between different analysis methods using a very complex sample of biological interest , that is , a human cancer cell lysate . among four replicate analyses of the 2-d samples , this indicated at least 76% of the proteins observed in one analysis were reproducibly detected despite significant undersampling . more importantly , at least 90% of the proteins observed in the 2-d / four - replicate data set based on two or more peptides directly matched to a corresponding protein 3-d data set , and most of the apparent mismatches were caused by trivial data analysis issues . a more rigorous comparison of the two comprehensive data sets showed that greater than 96% of the proteins identified in the 2-d / repetitive - run proteome were actually observed within the complete 3-d data set . one reason for the initially apparent , lower reproducibility when protein names were compared was slight variations in peptides scores together with use of rigid data filter cutoff values ( see above and figure 5 ) . that is , the 10% of proteins that were apparently unique to the 2-d / repetitive - run data set included 184 proteins ( 6.4% of the 2-d / repetitive protein list ) that were identified by a single peptide in the 3-d data set . reasons why these proteins were only identified by a single peptide in the 3-d data set include run - to - run variations in automated selection of low - abundance signals for ms / ms and run - to - run variations in sequest scores coupled with use of rigid data filters . a second contributing factor to the initially apparent , lower reproducibility at the protein list level is database redundancy and limitations of current software for consistently producing consensus protein lists from identified peptides . of the 111 proteins apparently unique to the 2-d / repetitive - run data set and not identified by a single - hit protein in the 3-d data set , most were highly homologous to proteins identified in the 3-d data set ( see results and supplemental table 1 ) . among the 385 peptides belonging to the 111 unique proteins in the 2-d data , although these 111 unique proteins comprised 4% of all proteins identified , the 159 unique peptides were only 0.7% of all 2-d filtered peptides . this illustrates that very small variations in identified peptides can have a proportionally higher apparent since we used each unique peptide a single time during assembly of consensus protein lists , the common sequences were assigned to the protein with the most unique sequences . consequently , for a group of proteins that have high sequence identity , one or two unique peptides could determine which protein in the protein family emerged in the final consensus protein list . this illustrates that better software tools are needed for identifying and displaying putative unique proteins within protein families . similarly , improved databases with uniform names or other labels that clearly indicate membership within a protein family would be beneficial . in conclusion , additional prefractionation with microsol ief substantially increased proteome coverage and sequence coverage compared with a gelc - ms / ms - repetitive run method that utilized an equal amount of mass spectrometer time . furthermore , the reproducibility of protein lists between the two methods was quite high because undersampling during data acquisition had been minimized . most of the apparent differences in protein identifications were due to limitations of current sequence databases and protein naming conventions , as well as software limitations for filtering database search results and building consensus protein lists .

in - depth , reproducible coverage of complex proteomes is challenging because the complexity of tryptic digests subjected to lc - ms / ms analysis frequently exceeds mass spectrometer analytical capacity , which results in undersampling of data . in this study , we used cancer cell lysates to systematically compare the commonly used gelc - ms / ms ( 1-d protein + 1-d peptide separation ) method using four repetitive injections ( 2-d / repetitive ) with a 3-d method that included solution isoelectric focusing and involved an equal number of lc - ms / ms runs . the 3-d method detected substantially more unique peptides and proteins , including higher numbers of unique peptides from low - abundance proteins , demonstrating that additional fractionation at the protein level is more effective than repetitive analyses at overcoming lc - ms / ms undersampling . importantly , more than 90% of the 2-d / repetitive protein identifications were found in the 3-d method data in a direct protein level comparison , and the reproducibility between data sets increased to greater than 96% when factors such as database redundancy and use of rigid scoring thresholds were considered . hence , high reproducibility of complex proteomes , such as human cancer cell lysates , readily can be achieved when using multidimensional separation methods with good depth of analysis .

pubmed

although perioperative mortality from tonsillectomy and submucosal uvulopalatopharyngoplasty ( smuppp ) with tonsillectomy has decreased significantly , postoperative bleeding is still an important complication . the incidence of bleeding after tonsillectomy is approximately 0.5%-10% , with death occurring in 1 in 20,000 patients . primary bleeding refers to that occurring within the first 24 h postoperatively , and bleeding occurring 5 - 10 days after surgery has been classified as secondary or delayed bleeding . factors such as age , gender , and surgical technique have been evaluated as risk factors for posttonsillectomy bleeding . to date , no study has specifically compared the rate of bleeding after tonsillectomy and smuppp . we reviewed the medical records of all patients at our institution who underwent unipolar cautery tonsillectomy alone or in conjunction with smuppp during the last 10 years to compare the incidence of secondary , delayed postoperative hemorrhage between the two procedures . in this retrospective study , following approval from the institutional review board , we reviewed the medical records of 404 patients who underwent tonsillectomy and smuppp procedures at our institution between january 2001 and december 2010 . the patients were divided into two groups according to the surgical intervention they had gone through . group 1 , who underwent tonsillectomy , consisted of 198 patients ; group 2 , who underwent smuppp , consisted of 206 patients . patients younger than 18 years of age or those who underwent a tonsillectomy as part of the treatment of carcinoma of the head or neck were excluded from the study . all postoperative secondary , delayed ( 5 - 10 days after surgery ) hemorrhage which required intervention , such as electrocautery under local anesthesia or a second surgery under general anesthesia for both groups was recorded . it is based on standard surgical principles of epithelial preservation and tension - free closure of the epithelium . most of the mucosa of the soft palate and the anterior and posterior pillars are preserved . elimination of redundant palatal and pharyngeal tissue is accomplished by subepithelial and muscular stretching and closure . the smuppp technique comprises two steps : step 1 , tonsillectomy and excision of the soft palate and redundant tissue ; and step 2 , reconstruction . a detailed description of the technique is as follows . the patient is orally intubated , and the head is hyperextended with a crowe - davis tonsillectomy mouth gag in place . this helps to assess the lateral extent of the tonsil and the proper plane of dissection . the needle tip is then used in 35 w cutting mode to expose the tonsil capsule via a curvilinear incision . to further identify the capsule , yankauer suction the dissection proceeds using a bovie in 35 w coagulation mode to separate the tonsil capsule from the superior constrictor muscle and to cauterize the blood vessels entering the tonsil . constant traction of the tonsil is maintained , allowing the tissue to be separated as it is cauterized by the bovie . the removal of the tonsil proceeds within its anatomical boundaries , with the musculature of the tonsil fossa left intact . dissection proceeds inferiorly toward the base of the tongue until the tonsil is free of all attachments . at the end of the tonsillectomy , after the removal of both tonsils , the palate is addressed . the uvula is grasped with allis forceps and retracted anteriorly for an optimal approach to the posterior surface of the soft palate . a curvilinear horizontal incision is made on the mucosa at the base of the uvula posteriorly , preserving almost the entire posterior soft palate mucosa . using the needle tip in cautery coagulation mode , the mucosa is separated from the muscle , releasing the posterior soft palate mucosa . using the needle tip in cutting mode , a trapezoid incision is outlined at the anterior mucosa of the soft palate at a level previously identified below the dimpled area , as visualized preoperatively on the awake patient . the incision is extended bilaterally and horizontally across the soft palate to the downward slope of the anterior pillar . the uvula and the submucosal tissue of the lower edge of the soft palate are excised . the redundant pharyngeal folds are eliminated by approximation of the muscular tissue of the tonsillar fossa and the soft palate using interrupted 3 - 0 vicryl sutures through these muscles . the mucosal flap edges are loosely approximated , without undermining , using 3 - 0 vicryl sutures . particular attention is paid to the inferior parts of the tonsillar fossa , where the majority of secondary bleeding occurs . bipolar cautery is used to cauterize these parts , after which the inferior parts of the tonsillar fossas are sutured using inferolateral pharyngoplasty . inferolateral pharyngoplasty involves at least four interrupted sutures with 3 - 0 vicryl to tack the inferior pharyngeal redundancy laterally . this is accomplished by suturing the muscle layer of the inferior tonsillar fossa laterally to the base of the tongue as the first layer . the second layer of mucosal epithelial interrupted sutures then overlies this layer . during soft palate excision , particular attention is paid to the base of the uvula on both sides where the uvular artery enters the soft palate . bipolar cauterization followed by tacking with 3 - 0 vicryl sutures may have decreased the incidence of postoperative bleeding compared wth routine tonsillectomy . the tonsillectomy technique used in group 1 patients is also the same electrocautery technique and instrumentation described by drezner , and the tonsillar fossae are left intact for secondary healing [ videos 1 - 2 ] . the test was used for statistical analysis . except in cases with expected sample sizes of fewer than five , postoperative secondary bleeding from the tonsillectomy regions were recorded and compared . bleeding from the palate was excluded . the smuppp technique used in this study it is based on standard surgical principles of epithelial preservation and tension - free closure of the epithelium . most of the mucosa of the soft palate and the anterior and posterior pillars are preserved . elimination of redundant palatal and pharyngeal tissue is accomplished by subepithelial and muscular stretching and closure . the smuppp technique comprises two steps : step 1 , tonsillectomy and excision of the soft palate and redundant tissue ; and step 2 , reconstruction . a detailed description of the technique is as follows . the patient is orally intubated , and the head is hyperextended with a crowe - davis tonsillectomy mouth gag in place . this helps to assess the lateral extent of the tonsil and the proper plane of dissection . the needle tip is then used in 35 w cutting mode to expose the tonsil capsule via a curvilinear incision . to further identify the capsule , yankauer suction the dissection proceeds using a bovie in 35 w coagulation mode to separate the tonsil capsule from the superior constrictor muscle and to cauterize the blood vessels entering the tonsil . constant traction of the tonsil is maintained , allowing the tissue to be separated as it is cauterized by the bovie . the removal of the tonsil proceeds within its anatomical boundaries , with the musculature of the tonsil fossa left intact . dissection proceeds inferiorly toward the base of the tongue until the tonsil is free of all attachments . at the end of the tonsillectomy , and the muscle fibers are still covered by fascia . after the removal of both tonsils , the uvula is grasped with allis forceps and retracted anteriorly for an optimal approach to the posterior surface of the soft palate . a curvilinear horizontal incision is made on the mucosa at the base of the uvula posteriorly , preserving almost the entire posterior soft palate mucosa . using the needle tip in cautery coagulation mode , the mucosa is separated from the muscle , releasing the posterior soft palate mucosa . using the needle tip in cutting mode , a trapezoid incision is outlined at the anterior mucosa of the soft palate at a level previously identified below the dimpled area , as visualized preoperatively on the awake patient . the incision is extended bilaterally and horizontally across the soft palate to the downward slope of the anterior pillar . the uvula and the submucosal tissue of the lower edge of the soft palate are excised . the redundant pharyngeal folds are eliminated by approximation of the muscular tissue of the tonsillar fossa and the soft palate using interrupted 3 - 0 vicryl sutures through these muscles . the mucosal flap edges are loosely approximated , without undermining , using 3 - 0 vicryl sutures . particular attention is paid to the inferior parts of the tonsillar fossa , where the majority of secondary bleeding occurs . bipolar cautery is used to cauterize these parts , after which the inferior parts of the tonsillar fossas are sutured using inferolateral pharyngoplasty . inferolateral pharyngoplasty involves at least four interrupted sutures with 3 - 0 vicryl to tack the inferior pharyngeal redundancy laterally . this is accomplished by suturing the muscle layer of the inferior tonsillar fossa laterally to the base of the tongue as the first layer . the second layer of mucosal epithelial interrupted sutures then overlies this layer . during soft palate excision , particular attention is paid to the base of the uvula on both sides where the uvular artery enters the soft palate . bipolar cauterization followed by tacking with 3 - 0 vicryl sutures may have decreased the incidence of postoperative bleeding compared wth routine tonsillectomy . the tonsillectomy technique used in group 1 patients is also the same electrocautery technique and instrumentation described by drezner , and the tonsillar fossae are left intact for secondary healing [ videos 1 - 2 ] . the test was used for statistical analysis . except in cases with expected sample sizes of fewer than five , the mean age of group 1 patients was 38.1 ( 2.58 ) years , and that of group 2 was 37.7 ( 2.2 ) years . no statistically significant difference in age or gender distribution was found between groups 1 and 2 ( p > 0.05 ) . there were 198 patients in the tonsillectomy with unipolar electrocautery group ( group 1 ) and 206 in the smuppp group ( group 2 ) . the incidence of secondary , delayed hemorrhage was 5.05% ( 10 patients ) in group 1 and 1.45% ( 3 patients ) in group 2 ( p = 0.05 ) showing statistically significant difference . the incidence of delayed hemorrhage requiring surgical treatment was 4.54% ( nine patients ) in group 1 and 0.97% ( two patients ) in group 2 [ graph 1 ] . the rate of post - tonsillectomy hemorrhage reported in the literature is variable due to the lack of consensus on what is considered significant postoperative hemorrhage . in many studies , only if surgical intervention under general anesthesia is required to control the bleeding will a case be considered as having significant hemorrhage , and the studies that take this view find lower hemorrhage rates . but in our study , all patients who required medical attention for control of bleeding were considered as having postoperative hemorrhage , regardless of whether they required hemorrhage control under local anesthesia or full surgery under general anesthesia . posttonsillectomy hemorrhage incidence in adults is reported in the literature to be 1.5%-18.0% , but the more commonly given range is between 3.0% and 5.0% . in our study , the postoperative bleeding rate of the tonsillectomy group was similar to the rates mentioned in the literature . rates of hemorrhage after smuppp have not been studied as extensively as those after tonsillectomy . the range encountered in the literature varies from 0.6% to 14.0% , and in our study , the bleeding rate after smuppp was 1.45% which falls within that range . in the present study , when electrocautery tonsillectomy and smuppp groups were compared , a significant difference in the rate of hemorrhage was apparent . although the tonsillectomy portion of the procedures was performed in an identical manner for both of our study groups , the postoperative bleeding rates differed . some studies have found an increased rate of posttonsillectomy hemorrhage in males compared with females , but the gender distribution did not differ between study groups , which agrees with several other studies . the increased rate of hemorrhage in the tonsillectomy group could not be attributed to any factor other than the operative techniques used . however , the bleeding rates for tonsillectomy and smuppp patients remained significantly higher among tonsillectomy patients whose muscular tissue of the tonsillar fossa was not approximated using interrupted 3 - 0 vicryl sutures through the tissue . suturing both the muscular and mucosal layers of the tonsillar fossa in smuppp seemed to decrease the incidence of postoperative bleeding . furthermore , bipolar cauterization followed by tacking with 3 - 0 vicryl sutures may have . decreased the incidence of postoperative bleeding . suturing of the tonsillar pillars is the only influencing factor detected that may have lowered the bleeding incidence . double - layer closure and approximating muscular and mucosal layers after tonsillectomy might help postoperative oropharyngeal healing , resulting in decreased bleeding incidence . medical attention for control of bleeding were considered as having postoperative hemorrhage , regardless of whether they required hemorrhage control under local anesthesia or full surgery under general anesthesia . posttonsillectomy hemorrhage incidence in adults is reported in the literature to be 1.5%-18.0% , but the more commonly given range is between 3.0% and 5.0% . in our study , the postoperative bleeding rate of the tonsillectomy group was similar to the rates mentioned in the literature . rates of hemorrhage after smuppp have not been studied as extensively as those after tonsillectomy . the range encountered in the literature varies from 0.6% to 14.0% , and in our study , the bleeding rate after smuppp was 1.45% which falls within that range . in the present study , when electrocautery tonsillectomy and smuppp groups were compared , a significant difference in the rate of hemorrhage was apparent . although the tonsillectomy portion of the procedures was performed in an identical manner for both of our study groups , the postoperative bleeding rates differed . some studies have found an increased rate of posttonsillectomy hemorrhage in males compared with females , but the gender distribution did not differ between study groups , which agrees with several other studies . the increased rate of hemorrhage in the tonsillectomy group could not be attributed to any factor other than the operative techniques used . however , the bleeding rates for tonsillectomy and smuppp patients remained significantly higher among tonsillectomy patients whose muscular tissue of the tonsillar fossa was not approximated using interrupted 3 - 0 vicryl sutures through the tissue . suturing both the muscular and mucosal layers of the tonsillar fossa in smuppp seemed to decrease the incidence of postoperative bleeding . furthermore , bipolar cauterization followed by tacking with 3 - 0 vicryl sutures may have . suturing of the tonsillar pillars is the only influencing factor detected that may have lowered the bleeding incidence . double - layer closure and approximating muscular and mucosal layers after tonsillectomy might help postoperative oropharyngeal healing , resulting in decreased bleeding incidence . submucosal tonsillectomy , which is a part of smuppp , has a lower incidence of postoperative delayed hemorrhage compared with tonsillectomy using unipolar cautery in adult patients . we suggest routine use of the suturing technique for smuppp in all tonsillectomy patients to reduce the rate of postoperative hemorrhage .

background : the aim of this study was to compare the incidence of postoperative secondary hemorrhage for tonsillectomy and submucosal uvulopalatopharyngoplasty ( smuppp).materials and methods : in this retrospective case series , the medical records of 404 patients who underwent tonsillectomy with unipolar electrocautery and smuppp at our institution between january 2001 and december 2010 were reviewed . the patients were divided into two groups : group 1 ( 198 patients ) underwent tonsillectomy ; group 2 ( 206 patients ) underwent smuppp . main outcome measures were incidence of bleeding or complications after tonsillectomy and smuppp and the need for revision surgery.results:the mean age of group 1 patients was 38.1 ( 2.58 ) years and that of group 2 was 37.7 ( 2.25 ) years . males were 51.3% of group 1 and 46.7% of group 2 . no statistically significant difference in age or gender distribution was found between groups 1 and 2 . the incidence of secondary , delayed hemorrhage was 5.05% ( 10 patients ) in group 1 and 1.45% ( three patients ) in group 2 ( p = 0.05 ) . the incidence of delayed hemorrhage requiring surgical treatment was 4.54% ( nine patients ) in group 1 and 0.97% ( two patients ) in group 2.conclusion:in adults , smuppp , which includes tonsillectomy , has a lower incidence of postoperative delayed hemorrhage than does tonsillectomy with unipolar cautery .

pubmed

the - and -lactam scaffolds are found in various biologically relevant compounds . additionally , these core structures are crucial intermediates in the synthesis of various natural products as well as pharmaceuticals , prompting the development of new methods for the synthesis of these valuable structures . our group has explored the synthesis of -lactams by the reactions of aryl- , thioaryl- , and cyano - substituted anhydrides . furthermore , we have demonstrated that one - pot four - component reactions ( 4crs ) are possible in the case of thioaryl substitution . recent computational studies of the cyanosuccinic anhydride reactions demonstrate that they proceed by a mannich - like mechanism via the enol tautomer of the anhydride . related reactions reported by castagnoli , cushman , and haimova likely proceed by this mechanism as well . consistent with the proposed mechanism , those anhydrides are poorly enolizable and require forcing conditions . two decades later , cushman and haimova reported analogous reactions with homophthalic anhydride , which is highly enolizable . the high reactivity of this anhydride at lower temperatures is consistent with the mannich - like mechanism . these results prompted us to explore mannich - type reactions and related 4crs of variously substituted anhydrides . although aryl - substituted succinic anhydrides showed some variation in reactivity based on the substituents attached to the aromatic ring , direct attachment of an electron - withdrawing group resulted in higher reactivity . furthermore , in the case of cyanosuccinic anhydrides , the diastereoselectivity was reversed relative to the aryl and thioaryl cases and could be controlled by resident stereogenic centers . we envisioned that the electron - withdrawing capability of a sulfone would impart the anhydride with similar reactivity as the nitrile - substituted anhydride , albeit with drastically different steric requirements ( scheme 1 ) . herein we report the development of anhydride mannich reactions ( amrs ) using sulfone - substituted anhydrides for the synthesis of - and -lactams as well as a six - step formal synthesis of ( )-isoretronecanol . multigram - scale syntheses of anhydrides 3a d were achieved from readily available starting materials . anhydride 3a was synthesized by conjugate addition of benzenesulfinic acid sodium salt ( 7 ) to maleic anhydride to yield a diacid , which was cyclized in one step ( scheme 2a ) . the syntheses of anhydrides 3b d were achieved by michael addition of sulfone acetate 8 to various tert - butyl acrylates followed by ester cleavage and cyclization ( scheme 2b ) . anhydride 3a readily reacted with imines to form -lactams . as with previous amrs , the carboxylic acid 5 ( table 1 ) is the first - formed product . unlike all previous related intermediates , these carboxylic acids were prone to facile decarboxylation . mild heating drove the decarboxylation to completion , allowing isolation of the sulfone - substituted lactams in high yields with only the anti diastereomer detectable by h nmr spectroscopy . in all cases , non - enolizable substrates the lack of reactivity of ketones and enolizable aldehydes in the amr probably stems from the rapid equilibration to the enamine tautomer , which is unreactive in the mannich addition step . the stereochemical configuration of the products was assigned by x - ray crystallography . lactam 10e has an anti configuration between the aromatic ring and the sulfonyl group . the other lactams were assigned by comparison of the coupling constants of the adjacent protons ( e.g. , 10f ; figure 1a ) . furthermore , acid intermediate 5f , which leads to 10f by decarboxylation , was also crystalline and was found to have an anti configuration between the sulfone and aromatic substituents ( figure 1b ) . our previous mechanistic work on the cyano - substituted anhydrides suggests that the anti - configured carboxylic acids are the kinetic products of the amrs . subsequent decarboxylation leads to the anti - sulfones , which would also be expected to be thermodynamically preferred . x - ray structures of 5f , 10f , and 10 g and j values for 10e and 10j . the stereochemical outcome of the reactions of imines with 3a is adequately explained by a transition state that is analogous to what is operative for cyanosuccinic anhydrides the iminium ion is attacked by the enolate of the anhydride , and the cyclic transition state is stabilized by hydrogen bonding to the sulfone . after the mannich reaction , subsequent rapid transannular acylation forms the lactam product.1 the carboxylic acid intermediates could be trapped with trimethylsilyldiazomethane ( tmschn2 ) . after many esterification conditions were tried , treatment of crude mixtures of 5 with a commercially available solution of tmschn2 resulted in reasonable yields of methyl esters 11a c ( table 2 ) . these intermediates could also be smoothly desulfonylated with magnesium in methanol to produce the corresponding esters 12 in good yield . although this reaction was successful for 11a and 11b , no ester product was observed in the attempted desulfonylation of 11c . unlike the analogous -lactams , these intermediates were impossible to isolate , thus preventing assignment of the configuration of the predominant diastereomer . upon decarboxylation , sulfone lactams 13 were formed as single isomers in high yields ( table 3 ) . determined by h this change in diastereoselectivity upon decarboxylation suggests the formation of a planar anion followed by selective protonation from one face of the anion . this result is in accordance with experimental data and models of sulfone anions proposed by corey . similar to our results with the succinic anhydride , the reaction tolerated a variety of non - enolizable aldehydes and a variety of alkylamines and anilines . while some of our initial reactions gave low yields ( 4050% ) , the use of commercially available imines or those purified by distillation significantly increased the yields of the lactam products . methyl - substituted anhydrides 3c and 3d provided trisubstituted -lactams in high yields with good diastereomeric ratios ( scheme 3 ) . these results suggest that substitution on the anhydride provides excellent facial selectivity , resulting in the formation of only two diastereomers of the intermediate carboxylic acids . studies are ongoing in our laboratory to develop an enantioselective michael addition with sulfone acetate 8 to provide the anhydrides as single enantiomers , which would presumably provide enantiomerically pure lactam products . this new amr was applied to a formal synthesis of ( )-isoretronecanol ( scheme 4 ) . the reaction of imine 16 with anhydride 3a followed by ring - closing metathesis provides the pyrrolizidine core of the natural product from cinnamaldehyde , allylamine , and 3a in two steps . the elimination of 18 yields 21 , which has been converted to ( )-isoretronecanol in two steps in low yield . further reduction yields lactam ester 22 , which has been converted to ( )-isoretronecanol in one step . while this second route is redox - inefficient , it provides material in a much higher yield and avoids the high - pressure conditions necessary to reduce pyrrole 21 . this synthetic route showcases the versatility of the anhydride michael reaction of the sulfone - substituted anhydride , exemplifying its usefulness in the assembly of heterocyclic targets . this synthesis demonstrates that the amr of sulfone - substituted anhydrides will be useful for the assembly of many heterocyclic targets , natural and otherwise . in summary , we have successfully synthesized various sulfone - substituted - and -lactams in good yields with high diastereoselectivities ( > 95:5 ) by an anhydride mannich reaction . we have previously studied the reactivity of several different substituted succinic anhydrides , but these are the first new results employing glutaric anhydyrides since the original work of castagnoli . unlike previous reactions of this type to date , the carboxylic acid products were prone to decarboxylation . although the -lactam acids derived from the substituted succinic anhydride could be trapped as esters , the analogous -lactam products could not be intercepted . another important difference was observed in the diastereoselectivity of the initial mannich - type reaction . although the reactions of succinic anhydyrides were very selective , the acid products derived from the glutaric anhydrides were generally formed as diastereomeric mixtures . in both cases , we have shown that this methodology is highly robust and have showcased it in the formal synthesis of a biologically active natural product . unless otherwise specified , all commercially available reagents were used as received . all reactions using dried solvents were carried out under an atmosphere of argon in flame - dried glassware with magnetic stirring . dry solvent was dispensed from a solvent purification system that passes solvent through two columns of dry neutral alumina . h nmr spectra and proton - decoupled c nmr spectra were obtained on a 400 or 600 mhz nmr spectrometer . chemical shifts ( ) are reported in parts per million ( ppm ) relative to residual solvent ( chcl3 , s , 7.26 ) . multiplicities are given as s ( singlet ) , d ( doublet ) , t ( triplet ) , dd ( doublet of doublets ) , m ( multiplet ) , br m ( broad multiplet ) , or br s ( broad singlet ) ; c nmr chemical shifts are reported relative to cdcl3 ( t , 77.23 ) unless otherwise noted . high - resolution mass spectra were recorded in positive esi mode in methanol or acetonitrile . silica gel chromatographic purifications were performed by flash chromatography with silica gel packed in glass columns . the eluting solvent for each purification was determined by thin - layer chromatography ( tlc ) on glass plates coated with silica gel and visualized by uv light or by staining with ceric ammonium molybdate ( cam ) followed by gentle heating . the following abbreviations are used throughout : ethyl acetate ( etoac ) , hexanes ( hex ) , dichloromethane ( dcm ) , triethylamine ( tea ) , diisopropylethylamine ( dipea ) . benzenesulfinic acid sodium salt ( 10.0 g , 60.9 mmol ) was dissolved in 100 ml of h2o at 0 c . then 10% hcl was added , and the mixture was extracted two times with etoac . the combined organic layers were dried over na2so4 , and the solvent was removed in vacuo to yield a white solid , which was dissolved in 50 ml of toluene . ac2o ( 70 ml , 20 mmol ) was added , and the reaction mixture was heated at reflux for 3 h. the solvent was evaporated in vacuo , and the resulting brown solid was washed several times with cold dcm and collected in a fritted funnel , yielding a white to light - brown solid ( 6.8 g , 46% yield over the two steps ) . mp 132.4133.1 c ( dec . ) ; h nmr ( 400 mhz , cdcl3 ) 7.96 ( dd , j = 8.4 , 1.3 hz , 2h ) , 7.867.76 ( m , 1h ) , 7.727.63 ( m , 2h ) , 4.52 ( dd , j = 10.3 , 4.4 hz , 1h ) , 3.68 ( dd , j = 19.9 ( gem ) , 4.4 hz , 1h ) , 3.39 ( dd , j = 19.7 ( gem ) , 10.6 hz , 1h ) ; c nmr ( 100 mhz , cdcl3 ) 166.0 , 162.9 , 135.8 , 135.4 , 129.9 , 129.7 , 64.4 , 30.1 ; ir ( neat ) 1782 , 1739 ( c = o ) , 1169 ( s = o ) cm ; hrms ( esi - tof ) m / z [ m + h ] calcd for c10h9o5s 241.0171 , found 241.0165 . the amine and the aldehyde were dissolved in thf ( 0.10 m ) , and 3 equiv of triethyl orthoformate was added . the anhydride was added , and then the reaction mixture was allowed to stir for 3 h. water was added to the mixture ( final concentration 0.05 mmol ) , followed by the addition of 3 equiv of k2co3 and heating at reflux for 4 h , unless otherwise stated . the reaction mixture was allowed to cool to rt and then extracted two times with etoac . the solvent was evaporated in vacuo , and the crude mixture was purified by flash chromatography using an etoac/3050% gradient , unless otherwise stated . general procedure a was used with sulfone anhydride 3a ( 0.24 g , 0.99 mmol ) , benzylamine ( 109 l , 0.99 mmol ) , and benzaldehyde ( 102 l , 1.0 mmol ) to yield 10a ( 0.32 g , 83% ) as a foam ( 50:50 etoac / hex , rf = 0.59 ) . h nmr ( 600 mhz , cdcl3 ) 7.687.64 ( m , 2h ) , 7.62 ( tt , j = 7.5 , 1.3 hz , 1h ) , 7.487.41 ( m , 2h ) , 7.327.23 ( m , 6h ) , 7.097.06 ( m , 2h ) , 6.836.79 ( m , 2h ) , 5.09 ( d , j = 14.9 hz , 1h ) , 4.70 ( d , j = 3.3 hz , 1h ) , 3.66 ( ddd , j = 9.9 , 4.4 , 3.3 hz , 1h ) , 3.44 ( d , j = 14.9 hz , 1h ) , 3.03 ( dd , j = 18.2 ( gem ) , 4.3 hz , 1h ) , 2.95 ( ddd , j = 18.2 ( gem ) , 9.9 , 1.2 hz , 1h ) ; c nmr ( 101 mhz , cdcl3 ) 170.5 , 137.7 , 136.4 , 134.7 , 134.4 , 129.5 , 129.3 , 128.9 , 128.7 , 128.7 , 128.4 , 127.8 , 126.1 , 63.5 , 60.6 , 44.5 , 30.7 ; ir ( thin film ) 1692 ( c = o ) , 1322 ( s = o ) cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c23h21no3sna 414.1140 , found 414.1120 . general procedure a was used with sulfone anhydride 3a ( 0.30 g , 1.26 mmol ) and ( e)-n - methyl-1-phenylmethanimine ( 156 l , 1.26 mmol ) . because the commercially available imine was used in this experiment , the anhydride was added immediately after the imine was dissolved instead of waiting for 3 h or overnight . the reaction yielded 10b ( 0.332 g , 83% ) as a foam ( 50:50 etoac / hex , rf = 0.35 ) . h nmr ( 600 mhz , cdcl3 ) 7.89 ( d , j = 7.5 hz , 2h ) , 7.69 ( t , j = 7.5 hz , 1h ) , 7.58 ( t , j = 7.8 hz , 2h ) , 7.367.31 ( m , 3h ) , 7.067.02 ( m , 2h ) , 4.97 ( d , j = 3.5 hz , 1h ) , 3.66 ( dt , j = 9.8 , 4.1 hz , 1h ) , 2.90 ( dd , j = 18.1 ( gem ) , 4.7 hz , 1h ) , 2.82 ( dd , j = 18.1 ( gem ) , 9.8 hz , 1h ) , 2.64 ( s , 3h ) ; c nmr ( 150 mhz , cdcl3 ) 170.6 , 138.2 , 136.8 , 134.6 , 129.7 , 129.5 , 129.0 , 128.9 , 126.1 , 64.3 , 63.6 , 31.0 , 28.4 ; ir ( thin film ) 1690 ( c = o ) , 1308 ( s = o ) cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c17h17no3sna 338.0827 , found 338.0818 . general procedure a was used with sulfone anhydride 3a ( 0.240 g , 1.00 mmol ) , prop-2-yn-1-amine ( 64.0 l , 1.00 mmol ) , and benzaldehyde ( 102 l , 1.00 mmol ) to yield 10c ( 0.182 g , 54% ) as a foam . the residue was purified by flash column chromatography ( 50:50 etoac / hex , rf = 0.53 ) . h nmr ( 600 mhz , cdcl3 ) 7.89 ( m , 2h ) , 7.68 ( t , j = 7.4 hz , 1h ) , 7.607.53 ( t , 2h ) , 7.357.29 ( m , 3h ) , 7.077.02 ( m , 2h ) , 5.23 ( d , j = 4.0 hz , 1h ) , 4.51 ( dd , j = 17.6 , 2.6 hz , 1h ) , 3.75 ( ddd , j = 9.4 , 5.1 , 4.0 hz , 1h ) , 3.253.20 ( m , 1h ) , 2.96 ( dd , j = 18.2 ( gem ) , 5.1 hz , 1h ) , 2.89 ( ddd , j = 18.2 ( gem ) , 9.8 , 1.1 hz , 1h ) , 2.25 ( t , j = 2.5 hz , 1h ) ; c nmr ( 150 mhz , cdcl3 ) 169.9 , 137.3 , 136.6 , 134.6 , 129.6 , 129.4 , 129.0 , 128.9 , 126.4 , 76.2 , 73.3 , 63.9 , 60.9 , 31.1 , 30.5 ; ir ( thin film ) 1699 ( c = o ) , 1414 , 1322 ( s = o ) cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c19h17no3sna 362.0827 , found 362.0831 . general procedure a was used with sulfone anhydride 3a ( 0.24 g , 0.99 mmol ) , propylamine ( 82 l , 0.99 mmol ) , and furan-2-carbaldehyde ( 83 l , 1.00 mmol ) to yield 10d ( 0.167 g , 50% ) as a foam . the residue was purified by flash column chromatography ( 50:50 etoac / hex , rf = 0.47 ) . h nmr ( 600 mhz , cdcl3 ) 7.907.86 ( m , 2h ) , 7.707.65 ( m , 1h ) , 7.57 ( dd , j = 8.3 , 7.2 hz , 2h ) , 7.32 ( d , j = 1.8 hz , 1h ) , 6.27 ( dd , j = 3.3 , 1.9 hz , 1h ) , 6.19 ( d , j = 3.3 hz , 1h ) , 5.07 ( d , j = 4.1 hz , 1h ) , 3.96 ( ddd , j = 8.9 , 6.1 , 4.1 hz , 1h ) , 3.40 ( ddd , j = 13.9 , 8.8 , 7.1 hz , 1h ) , 2.952.85 ( m , 2h ) , 2.65 ( ddd , j = 13.9 , 8.6 , 5.2 hz , 1h ) , 1.471.37 ( m , 1h ) , 1.331.24 ( m , 1h ) , 0.82 ( t , j = 7.4 hz , 3h ) ; c nmr ( 75 mhz , cdcl3 ) 169.9 , 149.4 , 143.5 , 136.7 , 134.5 , 129.6 , 128.6 , 110.6 , 109.8 , 60.8 , 55.2 , 42.7 , 31.2 , 20.1 , 11.1 ; ir ( thin film ) 1690 ( c = o ) , 1416 , 1308 ( s = o ) cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c17h19no4sna 356.0932 , found 356.0929 . general procedure a was used with sulfone anhydride 3a ( 0.3 g , 1.2 mmol ) , propylamine ( 100 l , 1.2 mmol ) , and benzaldehyde ( 126 l , 1.2 mmol ) to yield 10e ( 0.36 g , 87% ) as a solid ( 50:50 etoac / hex , rf = 0.36 ) . mp 149.8150.4 c ; h nmr ( 400 mhz , cdcl3 ) 7.89 ( dt , j = 8.5 , 1.4 hz , 2h ) , 7.767.54 ( m , 3h ) , 7.437.24 ( m , 3h ) , 7.136.98 ( m , 2h ) , 5.09 ( d , j = 3.5 hz , 1h ) , 3.703.56 ( m , 2h ) , 2.972.76 ( m , 2h ) , 2.54 ( dddd , j = 13.7 , 8.4 , 5.1 , 1.1 hz , 1h ) , 1.551.31 ( m , 2h ) , 0.85 ( td , j = 7.3 , 1.2 hz , 3h ) ; c nmr ( 100 mhz , cdcl3 ) 170.6 , 138.4 , 136.7 , 134.5 , 129.6 , 129.3 , 128.8 , 128.7 , 126.0 , 63.8 , 61.1 , 42.6 , 30.9 , 19.9 , 11.1 ; ir ( thin film ) 1688 ( c = o ) cm ; hrms ( esi - tof ) m / z [ m h ] calcd for c19h20no3s 342.1164 , found 342.1155 . general procedure a ( except that the decarboxylation was performed at room temperature overnight ) was used with sulfone anhydride 3a ( 0.3 g , 1.2 mmol ) , propylamine ( 102 l , 1.2 mmol ) , and 4-cyanobenzaldehyde ( 0.164 g , 1.3 mmol ) to yield 10f ( 0.36 g , 80% ) as a foam ( 50:50 etoac / hex , rf = 0.35 ) . h nmr ( 600 mhz , cdcl3 ) 7.89 ( dt , j = 8.5 , 1.8 hz , 2h ) , 7.777.54 ( m , 5h ) , 7.26 ( m , 2h ) , 5.20 ( d , j = 3.6 hz , 1h ) , 3.67 ( dt , j = 13.7 , 8.0 hz , 1h ) , 3.57 ( dt , j = 10.0 , 4.3 hz , 1h ) , 2.88 ( dd , j = 18.2 ( gem ) , 4.5 hz , 1h ) , 2.76 ( dd , j = 18.1 ( gem ) , 10.0 hz , 1h ) , 2.52 ( ddd , j = 13.8 , 8.6 , 5.0 hz , 1h ) , 1.44 ( m , 2h ) , 0.87 ( t , j = 7.3 hz , 3h ) ; c nmr ( 600 mhz , cdcl3 ) 170.7 , 144.1 , 136.6 , 134.9 , 133.3 , 129.9 , 128.9 , 127.2 , 118.1 , 113.1 , 63.8 , 60.6 , 43.1 , 31.3 , 20.1 , 11.2 ; ir ( thin film ) 1692 ( c = o ) , 1445 , 1412 ( s = o ) cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c20h20n2o3sna 391.1092 , found 391.1098 . general procedure a was used with sulfone anhydride 3a ( 88 mg , 0.36 mmol ) , propylamine ( 30 l , 0.36 mmol ) , and aldehyde ( 50 mg , 0.37 mmol ) to yield 10 g ( 87 mg , 65% ) as a solid ( 50:50 etoac / hex , rf = 0.17 ) . mp 125.3126.1 c ; h nmr ( 600 mhz , cdcl3 ) 7.927.86 ( m , 2h ) , 7.697.62 ( m , 1h ) , 7.55 ( t , j = 7.9 hz , 2h ) , 7.28 ( m , 1h ) , 6.936.82 ( m , 3h ) , 5.16 ( s , 1h ) , 3.80 ( d , j = 9.8 hz , 1h ) , 3.69 ( s , 3h ) , 3.49 ( dt , j = 13.5 , 8.1 hz , 1h ) , 2.96 ( dd , j = 17.9 ( gem ) , 4.0 hz , 1h ) , 2.85 ( dd , j = 17.9 ( gem ) , 9.9 hz , 1h ) , 2.48 ( ddd , j = 13.7 , 8.5 , 5.2 hz , 1h ) , 1.461.28 ( m , 2h ) , 0.82 ( t , j = 7.3 hz , 3h ) ; c nmr ( 151 mhz , cdcl3 ) 171.2 , 157.5 , 137.7 , 134.2 , 130.5 , 129.5 ( 2c ) , 129.0 ( 2c ) , 125.7 , 121.1 , 111.5 , 62.8 , 55.6 , 42.8 , 31.7 , 20.3 , 11.3 ; ir ( thin film ) 1681 ( c = o ) cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c20h23no4sna 396.1245 , found 396.1252 . general procedure a was used with sulfone anhydride 3a ( 0.18 g , 0.73 mmol ) , benzylamine ( 76 l , 0.69 mmol ) , and aldehyde ( 0.1 ml , 0.79 mmol ) to yield 10h ( 0.25 g , 82% ) as a foam ( 50:50 etoac / hex , rf = 0.36 ) . h nmr ( 600 mhz , cdcl3 ) 7.817.74 ( m , 2h ) , 7.657.14 ( m , 13h ) , 6.12 ( d , j = 15.8 hz , 1h ) , 5.74 ( dd , j = 15.6 , 8.5 hz , 1h ) , 4.93 ( d , j = 15.0 hz , 1h ) , 4.38 ( dd , j = 8.8 , 4.3 hz , 1h ) , 3.90 ( d , j = 15.0 hz , 1h ) , 3.65 ( dt , j = 9.9 , 5.1 hz , 1h ) , 3.01 ( dd , j = 18.0 ( gem ) , 5.5 hz , 1h ) , 2.86 ( dd , j = 18.1 ( gem ) , 10.0 hz , 1h ) ; c nmr ( 151 mhz , cdcl3 ) 170.1 , 136.8 , 135.3 , 135.2 , 135.0 , 134.4 , 129.5 , 128.8 , 128.7 , 128.7 , 128.4 , 127.7 , 126.8 , 126.7 , 125.3 , 61.7 , 59.9 , 44.7 , 30.8 ; ir ( thin film ) 1690 ( c = o ) , 1146 ( s = o ) cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c25h23no3sna 440.1296 , found 440.1282 . general procedure a was used with sulfone anhydride 3a ( 0.3 g , 1.26 mmol ) , isopropylamine ( 100 l , 1.22 mmol ) , and benzaldehyde ( 128 l , 1.26 mmol ) to yield 10i ( 0.297 g , 70% ) as a foam ( 50:50 etoac / hex , rf = 0.42 ) . h nmr ( 600 mhz , cdcl3 ) 7.987.86 ( m , 2h ) , 7.72 ( tt , j = 7.2 , 1.5 hz , 1h ) , 7.687.56 ( m , 2h ) , 7.407.26 ( m , 3h ) , 7.176.97 ( m , 2h ) , 5.10 ( d , j = 2.3 hz , 1h ) , 4.203.98 ( m , 1h ) , 3.57 ( ddd , j = 9.6 , 2.6 , 2.1 hz , 1h ) , 2.93 ( ddd , j = 18.2 ( gem ) , 9.6 , 1.9 hz , 1h ) , 2.882.75 ( m , 1h ) , 1.21 ( dd , j = 6.8 , 2.0 hz , 3h ) , 0.85 ( dd , j = 6.9 , 1.9 hz , 3h ) ; c nmr ( 151 mhz , cdcl3 ) 170.7 , 140.7 , 136.6 , 134.5 , 129.6 , 129.2 , 128.8 , 128.7 , 125.9 , 64.5 , 60.1 , 45.5 , 31.2 , 20.8 , 19.5 ; ir ( thin film ) 1693 ( c = o ) , 1145 ( s = o ) cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c19h21no3sna 366.1140 , found 366.1147 . general procedure a was used with sulfone anhydride 3a ( 0.30 g , 1.26 mmol ) , allylamine ( 93 l , 1.24 mmol ) , and benzaldehyde ( 128 l , 1.26 mmol ) to yield 10j ( 0.33 g , 78% ) as a foam ( 50:50 etoac / hex , rf = 0.42 ) . h nmr ( 600 mhz , cdcl3 ) 7.88 ( dt , j = 8.5 , 2.0 hz , 2h ) , 7.777.67 ( m , 1h ) , 7.59 ( m , 2h ) , 7.437.27 ( m , 3h ) , 6.99 ( m , 2h ) , 5.55 ( dddd , j = 17.6 , 10.0 , 7.8 , 4.3 hz , 1h ) , 5.235.15 ( m , 1h ) , 5.145.02 ( m , 2h ) , 4.40 ( ddt , j = 15.4 , 3.9 , 1.9 hz , 1h ) , 3.753.65 ( m , 1h ) , 2.95 ( m , 3h ) ; c nmr ( 151 mhz , cdcl3 ) 170.42 , 138.2 , 136.8 , 134.5 , 131.0 , 129.7 , 129.5 , 129.4 , 128.9 , 126.2 , 119.0 , 63.9 , 60.7 , 43.4 , 31.0 ; ir ( thin film ) 1691 ( c = o ) , 1145 ( s = o ) cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c19h19no3sna 364.0983 , found 364.0974 . general procedure a was used with sulfone anhydride 3a ( 0.30 g , 1.2 mmol ) , propylamine ( 100 l , 1.2 mmol ) , and 4-methoxybenzaldehyde ( 151 l , 1.2 mmol ) to yield 10k ( 0.38 g , 84% ) as a foam ( 50:50 etoac / hex , rf = 0.27 ) . h nmr ( 600 mhz , cdcl3 ) 7.927.86 ( d , j = 7.8 hz , 2h ) , 7.69 ( m , 1h ) , 7.58 ( t , j = 7.8 hz , 2h ) , 6.96 ( dd , j = 9.2 , 2.5 hz , 2h ) , 6.84 ( dd , j = 9.0 , 2.5 hz , 2h ) , 5.04 ( d , j = 3.5 hz , 1h ) , 3.80 ( sharp s , 3h ) , 3.663.53 ( m , 2h ) , 2.90 ( dd , j = 18.2 ( gem ) , 4.7 hz , 1h ) , 2.852.76 ( dd , j = 18.2 ( gem ) , 10.2 hz , 1h ) , 2.52 ( m , 1h ) , 1.511.32 ( m , 2h ) , 0.880.79 ( t , j = 7.8 hz , 3h ) ; c nmr ( 150 mhz , cdcl3 ) 170.4 , 159.9 , 136.9 , 134.4 , 130.3 , 129.6 , 128.8 , 127.4 , 114.7 , 64.1 , 60.7 , 55.3 , 42.5 , 31.1 , 20.0 , 11.1 ; ir ( thin film ) 1687 ( c = o ) , 1354 , 1144 ( s = o ) cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c20h23no4sna 396.1245 , found 396.1252 . n - benzylidenebenzylamine ( 0.1 ml , 0.53 mmol ) was added to a round - bottom flask containing na2so4 ( 0.37 g , 2.6 mmol ) and thf ( 6 ml ) , and then sulfone anhydride 3a ( 0.13 g , 0.54 mmol ) was added . the reaction mixture was allowed to stir for 1.5 h , after which the na2so4 was removed by filtration and the solvent was removed in vacuo . the residue was redissolved in etoac , transferred to a separatory funnel , and washed with hcl ( 1.2 m or 10% ) and brine . the combined organic layers were dried over na2so4 , and the solvent removed in vacuo . the resulting acid was redissolved in a mixture of toluene and methanol ( 12 ml:7 ml ) and cooled to 0 c . to the cold solution , tmschn2 ( 0.53 ml , 1.1 mmol , 2 m solution in hexanes ) after 2 h the solvent was removed in vacuo , and the resulting material was purified by column chromatography ( 3060% etoac : hex ) to yield 11a ( 0.157 g , 66% over the two steps ) as an amorphous solid ( 50:50 etoac / hex , rf = 0.47 ) . h nmr ( 600 mhz , cdcl3 ) 7.647.18 ( m , 15h ) , 5.21 ( d , j = 14.5 hz , 1h ) , 4.89 ( s , 1h ) , 3.70 ( d , j = 18.7 ( gem ) hz , 1h ) , 3.383.30 ( m , 2h ) , 3.13 ( s , 3h ) ; c nmr ( 150 mhz , cdcl3 ) 170.0 , 164.9 , 134.9 , 134.8 , 134.8 , 134.4 , 131.1 , 129.6 , 129.5 , 129.5 , 129.0 , 128.9 , 128.9 , 128.2 , 76.2 , 63.9 , 52.7 , 44.9 , 35.0 ; ir ( thin film ) 1783 , 1784 ( c = o ) , 1124 ( s = o ) cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c25h23no5sna 472.1195 , found 472.1176 . propylamine ( 68 l , 0.83 mmol ) and 3,5-dimethoxybenzaldehyde ( 0.14 g , 0.84 mmol ) were dissolved in thf ( 8 ml ) . na2so4 ( 0.59 g , 4.2 mmol ) was added , and the mixture was allowed to stir for 1.5 h. sulfone anhydride 3a ( 0.20 g , 0.83 mmol ) was added , and the reaction was allowed to stir for an additional 1 h. the na2so4 was filtered off , and the solvent was removed in vacuo . the residue was redissolved in etoac , transferred to a separatory funnel , and washed with hcl ( 1.2 m or 10% ) and brine . the combined organic layers were dried over na2so4 , and the solvent was removed in vacuo . the resulting crude acid was dissolved in a mixture of toluene and methanol ( 13 ml:7 ml ) and cooled to 0 c . to the cold solution , tmschn2 ( 0.83 ml , 1.7 mmol , 2 m solution in hexanes ) was added dropwise over a period of 5 min , and the reaction was monitored by tlc for the disappearance of starting material . after 2 h the solvent was removed in vacuo , and the resulting material was purified by column chromatography ( 3060% etoac : hex ) to yield 11b ( 0.22 g , 57% over the two steps ) as an amorphous solid ( 50:50 etoac / hex , rf = 0.31 ) . h nmr ( 600 mhz , cdcl3 ) 8.017.83 ( m , 2h ) , 7.817.68 ( m , 1h ) , 7.687.52 ( m , 2h ) , 6.39 ( d , j = 2.3 hz , 1h ) , 6.22 ( s , 2h ) , 5.38 ( s , 1h ) , 3.74 ( s , 6h ) , 3.663.59 ( m , 1h ) , 3.55 ( d , j = 18.4 ( gem ) hz , 1h ) , 3.26 ( m , 3h ) , 3.14 ( d , j = 18.5 ( gem ) hz , 1h ) , 2.58 ( ddd , j = 13.8 , 8.7 , 5.2 hz , 1h ) , 1.651.32 ( m , 2h ) , 0.92 ( t , j = 7.3 hz , 3h ) ; c nmr ( 150 mhz , cdcl3 ) 169.9 , 164.7 , 160.9 , 137.3 , 135.3 , 134.9 , 130.4 , 129.2 , 100.8 , 76.7 , 64.3 , 55.4 , 52.8 , 43.2 , 35.8 , 20.2 ( 2c ) , 11.3 ; ir ( thin film ) 1748 , 1702 ( c = o ) , 1144 , 1457 ( s = o ) cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c23h27no7sna 484.1406 , found 484.1404 . n - benzylidenemethylamine ( 0.1 ml , 0.81 mmol ) was added to a round - bottom flask containing na2so4 ( 0.57 g , 4.0 mmol ) and thf ( 8 ml ) , and then sulfone anhydride 3a ( 0.19 g , 0.80 mmol ) was added . the reaction mixture was allowed to stir for 1.5 h , after which the na2so4 was removed by filtration and the solvent removed in vacuo . the residue was redissolved in etoac , transferred to a separatory funnel , and washed with hcl ( 1.2 m or 10% ) and brine . the combined organic layers were dried over na2so4 , and the solvent was removed in vacuo . the crude acid was redissolved in a mixture of toluene and methanol ( 13 ml:7 ml ) and cooled to 0 c . to the cold solution , tmschn2 ( 0.81 ml , 1.6 mmol , 2 m solution in hexanes ) was added dropwise over a period of 5 min , and the reaction was monitored by tlc for the disappearance of starting material . after 2 h the solvent was removed in vacuo , and the resulting material was purified by column chromatography ( 3060% etoac : hex ) to yield 11c ( 0.20 g , 67% over the two steps ) as an amorphous solid ( 50:50 etoac / hex , rf = 0.28 ) . h nmr ( 400 mhz , cdcl3 ) 7.88 ( dt , j = 8.6 , 2.2 hz , 2h ) , 7.727.63 ( m , 1h ) , 7.647.50 ( m , 2h ) , 7.367.23 ( m , 4h ) , 7.05 ( d , j = 7.2 hz , 2h ) , 5.31 ( s , 1h ) , 3.49 ( d , j = 18.4 ( gem ) hz , 1h ) , 3.15 ( d , j = 2.3 hz , 3h ) , 3.01 ( d , j = 18.5 ( gem ) hz , 1h ) , 2.58 ( s , 2h ) ; c nmr ( 100 mhz , cdcl3 ) 169.75 , 164.51 , 135.32 , 135.1 , 134.3 , 130.2 , 129.4 , 129.3 , 128.7 , 127.6 , 76.7 , 66.5 , 52.9 , 35.5 , 28.3 ; ir ( thin film ) 1736 , 1690 ( c = o ) , 1177 , 1360 ( s = o ) cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c19h19no5sna 396.0882 , found 396.0887 . ester 11a ( 0.104 g , 0.23 mmol ) was dissolved in anhydrous meoh ( 12 ml ) , and magnesium turnings ( 169 mg , 6.9 mmol , flame - dried under argon ) were added to the reaction mixture . the mixture was heated to 50 c for 15 min and then to 64 c for 1.5 h , cooled to rt , poured into an erlenmeyer flask containing 10% hcl ( 20 ml ) , and extracted with diethyl ether . the organic layer was dried over na2so4 , and the solvent was removed in vacuo . purification by flash chromatography ( 3050% etoac / hex ) yielded 12a as an oil ( 0.54 g , 76% ) . h nmr data for the major diastereomer ( 600 mhz , cdcl3 ) : 7.417.33 ( m , 4h ) , 7.307.23 ( m , 3h ) , 7.187.13 ( m , 1h ) , 7.057.01 ( m , 2h ) , 5.11 ( d , j = 14.7 hz , 1h ) , 4.634.56 ( d , j = 5.4 hz , 1h ) , 3.64 ( s , 3h ) , 3.48 ( d , j = 14.9 hz , 1h ) , 3.07 ( m , 1h ) , 2.942.75 ( m , 2h ) . the h nmr data are in accordance with the literature . ester 11b ( 75.4 mg , 0.163 mmol ) was dissolved in anhydrous meoh ( 12 ml ) , and magnesium turnings ( 162 mg , 6.67 mmol , flame - dried under argon ) were added to the reaction mixture . the mixture was heated to 50 c for 15 min and then to 64 c for 1.5 h , cooled to rt , poured into an erlenmeyer flask containing 10% hcl ( 30 ml ) , and extracted with diethyl ether . the organic layer was dried over na2so4 , and the solvent was removed in vacuo . purification by flash chromatography ( 3050% etoac / hex ) yielded 12b as an oil ( 0.35 g , 67% ) . h nmr ( 400 mhz , cdcl3 ) 6.42 ( t , j = 2.2 hz , 1h ) , 6.35 ( d , j = 2.3 hz , 2h ) , 4.83 ( d , j = 5.3 hz , 1h ) , 3.79 ( s , 6h ) , 3.74 ( s , 3h ) , 3.66 ( dt , j = 13.6 , 8.1 hz , 1h ) , 3.04 ( ddd , j = 9.6 , 7.0 , 5.2 hz , 1h ) , 2.84 ( dd , j = 17.2 , 9.8 hz , 1h ) , 2.76 ( d , j = 7.0 hz , 1h ) , 2.61 ( ddd , j = 13.6 , 8.3 , 5.2 hz , 1h ) , 1.541.37 ( m , 2h ) , 0.84 ( t , j = 7.4 hz , 3h ) ; c nmr ( 100 mhz , cdcl3 ) 173.1 , 172.7 , 161.5 , 142.0 , 104.5 , 100.1 , 64.2 , 55.5 , 52.6 , 45.9 , 42.5 , 33.8 , 20.2 , 11.3 ; hrms ( esi - tof ) m / z [ m + na ] calcd for c17h23no5na 344.1474 , found 344.1465 . to a 500 ml round - bottom flask were added benzenesulfinic acid sodium salt ( 5.1 g , 30.8 mmol ) , tert - butyl bromoacetate ( 3.8 ml , 25.6 mmol ) , and 170 ml of ethanol . the crude mixture was suspended in et2o ( 150 ml ) and washed with water ( 2 150 ml ) and brine ( 150 ml ) . the organic layer was dried over sodium sulfate and concentrated in vacuo to yield 8 ( 6.31 g , 96% ) as a clear oil . h nmr ( 300 mhz , cdcl3 ) 7.90 ( m , 2h ) , 7.68 ( m , 1h ) , 7.55 ( m , 2h ) , 4.03 ( s , 2h ) , 1.33 ( s , 9h ) ; c nmr ( 75 mhz , cdcl3 ) 161.2 , 138.8 , 134.1 , 129.1 , 128.5 , 83.6 , 62.0 , 27.6 . these data are in accordance with the literature . to a dry 250 ml round - bottom flask were added 8 ( 6.31 g , 24.6 mmol ) , cesium carbonate ( 400 mg , 1.23 mmol ) , tert - butyl acrylate ( 3.6 ml , 24.6 mmol ) , and 80 ml of acetonitrile . the reaction mixture was heated to 50 c overnight and then diluted with water and extracted with ethyl acetate ( 3 100 ml ) . the crude product was purified on silica gel ( 20:80 etoac / hex ) to yield 3ba ( 4.91 g , 52% ) as an amorphous solid . h nmr ( 300 mhz , cdcl3 ) 7.89 ( d , j = 7.3 hz , 2h ) , 7.68 ( m , 1h ) , 7.57 ( dd , j = 8.4 , 6.9 hz , 2h ) , 3.99 ( m , 1h ) , 2.24 ( m , 4h ) , 1.42 ( s , 9h ) , 1.35 ( s , 9h ) ; c nmr ( 75 mhz , cdcl3 ) 170.9 , 164.4 , 137.4 , 134.1 , 129.4 , 129.0 , 83.5 , 81.0 , 70.1 , 32.1 , 28.0 , 27.7 , 22.3 ; ir ( thin film ) 2979 , 1730 , 1309 , 1138 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c19h28o6sna 407.1504 , found 407.1502 . to a dry 250 ml round - bottom flask were added 8 ( 3.00 g , 11.7 mmol ) , cesium carbonate ( 382 mg , 1.17 mmol ) , tert - butyl crotonate ( 1.90 ml , 11.7 mmol ) , and 40 ml of acetonitrile . the reaction mixture was heated to reflux overnight and then diluted with water and extracted with ethyl acetate ( 3 100 ml ) . the crude product was purified on silica gel ( 20:80 etoac / hex ) to yield 3ca ( 3.75 g , 80% ) as an amorphous solid ( mixture of diastereomers ) . nmr data for one diastereomer : h nmr ( 600 mhz , cdcl3 ) 7.90 ( m , 3h ) , 7.64 ( m , 2h ) , 7.53 ( m , 3h ) , 4.10 ( d , j = 8.0 hz , 1h ) , 2.71 ( m , 2h ) , 2.43 ( m , 1h ) , 1.42 ( s , 16h , overlap with minor diastereomer ) , 1.27 ( s , 14h , overlap with minor diastereomer ) , 1.12 ( d , j = 6.7 hz , 3h ) ; c nmr ( 150 mhz , cdcl3 ) 170.7 , 164.4 , 138.4 , 134.0 , 129.2 , 128.9 , 83.3 , 80.8 , 73.7 , 39.9 , 29.6 , 28.1 , 27.6 , 17.1 ; ir ( thin film ) 2980 , 1729 , 1325 , 1135 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c20h30o6sna 421.1661 , found 421.1653 . to a dry 250 ml round - bottom flask were added 8 ( 3.00 g , 11.7 mmol ) , cesium carbonate ( 382 mg , 1.17 mmol ) , tert - butyl methacrylate ( 1.90 ml , 11.7 mmol ) , and 40 ml of acetonitrile . the reaction mixture was heated to reflux for 48 h and then diluted with water and extracted with ethyl acetate ( 3 100 ml ) . the crude product was purified on silica gel ( 20:80 etoac / hex ) to yield 3da ( 4.20 g , 90% ) as an amorphous solid ( mixture of diastereomers ) . nmr data for one diastereomer : h nmr ( 400 mhz , cdcl3 ) 7.89 ( m , 2h ) , 7.69 ( m , 1h ) , 7.58 ( m , 2h ) , 4.03 ( dd , j = 11.9 , 3.3 hz , 1h ) , 2.28 ( m , 2h ) , 1.99 ( m , 1h ) , 1.43 ( s , 9h ) , 1.37 ( s , 9h ) , 1.16 ( d , j = 6.6 hz , 3h ) ; c nmr ( 101 mhz , cdcl3 ) 174.2 , 164.7 , 137.6 , 134.3 , 129.5 , 129.2 , 83.6 , 81.1 , 69.6 , 38.2 , 30.6 , 28.2 , 27.8 , 18.4 ; ir ( thin film ) 2978 , 2936 , 1727 , 1325 , 1138 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c20h30o6sna 421.1661 , found 421.1662 . to a dry 250 ml flask were added 3ba ( 4.91 g , 12.7 mmol ) , ch2cl2 ( 50 ml ) , and trifluoroacetic acid ( 50 ml ) . the reaction mixture was stirred for 1 h and then concentrated in vacuo and azeotropically distilled with ch2cl2 ( 3 10 ml ) . the crude mixture was taken up in 45 ml of trifluoroacetic anhydride and stirred overnight . the reaction mixture was concentrated in vacuo and azeotroped with toluene ( 3 10 ml ) . filtering the off - white solid with diethyl ether yielded 3b ( 3.01 g , 93% ) as a white solid . mp 122.2122.8 c ; h nmr ( 600 mhz , cd3cn ) 7.97 ( m , 2h ) , 7.83 ( m , 1h ) , 7.70 ( m , 2h ) , 4.55 ( m , 1h ) , 3.06 ( m , 1h ) , 2.87 ( m , 1h ) , 2.50 ( m , 2h ) ; c nmr ( 150 mhz , cd3cn ) 165.3 , 160.7 , 137.3 , 135.0 , 129.5 , 129.0 , 63.4 , 27.1 , 17.1 ; ir ( thin film ) 3098 , 2929 , 1816 , 1746 1322 , 1152 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c11h10o5sna 277.0147 , found 277.0145 . to a dry 250 ml flask were added 3ca ( 3.73 g , 9.30 mmol ) , ch2cl2 ( 50 ml ) , and trifluoroacetic acid ( 50 ml ) . the reaction mixture was stirred for 1 h and then concentrated in vacuo and azeotropically distilled with ch2cl2 ( 3 10 ml ) . to the crude mixture was added acetic anhydride ( 33 ml ) , and the mixture was taken up in 100 ml of toluene and stirred overnight . the mixture was concentrated in vacuo and azeotroped with benzene ( 3 10 ml ) . trituration with diethyl ether afforded 3c ( 2.31 g , 92% ) as a solid ( mixture of diastereomers ) . nmr data for one diastereomer : h nmr ( 600 mhz , cd3cn ) 7.93 ( m , 2h ) , 7.84 ( m , 1h ) , 7.70 ( m , 2h ) , 4.40 ( m , 1h ) , 3.29 ( m , 1h ) , 3.03 ( m , 1h ) , 2.75 ( m , 1h ) , 1.18 ( m , 3h ) ; c nmr ( 151 mhz , cd3cn ) 164.8 , 160.4 , 135.2 , 129.6 , 129.3 , 128.9 , 69.6 , 33.9 , 24.4 , 19.4 ; ir ( thin film ) 2991 , 2957 , 1814 , 1758 , 1311 , 1146 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c12h12o5sna 291.0293 , found 291.0303 . to a dry 250 ml flask were added 3da ( 4.00 g , 10.0 mmol ) , ch2cl2 ( 50 ml ) , and trifluoroacetic acid ( 50 ml ) . the reaction mixture was stirred for 1 h and then concentrated in vacuo and azeotropically distilled with ch2cl2 ( 3 10 ml ) . to the crude mixture were added acetic anhydride ( 33 ml ) and 100 ml of toluene , and the resulting mixture was stirred overnight , concentrated in vacuo , and azeotroped with benzene ( 3 10 ml ) . trituration with diethyl ether afforded 3d ( 2.53 g , 94% ) as a solid ( mixture of diastereomers ) . nmr data for one diastereomer : h nmr ( 400 mhz , cdcl3 ) 7.90 ( m , 2h ) , 7.74 ( m , 1h ) , 7.62 ( m , 3h , overlap with minor diasteromer ) , 4.22 ( dd , j = 3.0 , 9.0 hz , 1h ) , 2.95 ( ddd , j = 2.1 , 6.1 , 15.2 hz , 1h ) , 2.762.58 ( m , 1h ) , 2.13 ( ddd , j = 6.2 , 13.2 , 15.2 hz , 1h ) , 1.44 ( s , 3h ) ; c nmr ( 100 mhz , cdcl3 ) 167.8 , 160.3 , 137.1 , 135.2 , 129.8 , 129.4 , 64.8 , 32.6 , 25.5 , 16.4 ; ir ( thin film ) 3066 , 2970 , 1803 , 1755 , 1310 , 1154 cm ; hrms ( esi - tof ) m / z [ m + h2o + na ] calcd for c12h14o6sna 309.0409 , found 309.0402 . to a dry 10 ml flask were added 2 ml of acetonitrile , triethyl orthoformate ( 0.11 ml , 0.63 mmol ) , amine ( 0.39 mmol ) , and aldehyde ( 0.39 mmol ) . anhydride 3b ( 0.39 mmol ) in 2 ml of acetonitrile was added , and the reaction mixture was stirred for 30 min at room temperature and then refluxed for 4 h. the mixture was cooled to room temperature and concentrated in vacuo . the product was purified by flash column chromatography ( conditions given for each compound ) . to a dry 10 ml flask was added 4 ml of acetonitrile , triethyl orthoformate ( 0.11 ml , 0.63 mmol ) , the prepared imine ( 0.39 mmol ) , and anhydride 3b d ( 0.39 mmol ) . the reaction mixture was stirred for 30 min at room temperature and then refluxed for 4 h. the mixture was cooled to room temperature and concentrated in vacuo . the residue was purified by flash column chromatography ( conditions given for each compound ) . general procedure c. the residue was purified by flash column chromatography ( 70:30 etoac / hex , rf = 0.40 ) to yield 13a as a solid ( 105 mg , 80% ) . mp 152.6153.1 c ; h nmr ( 300 mhz , cdcl3 ) 7.91 ( m , 2h ) , 7.69 ( m , 1h ) , 7.59 ( m , 2h ) , 7.32 ( m , 3h ) , 7.04 ( m , 2h ) , 5.10 ( s , 1h ) , 3.36 ( ddd , j = 5.0 , 4.9 , 2.3 hz , 1h ) , 2.84 ( s , 3h ) , 2.73 ( m , 1h ) , 2.45 ( ddd , j = 17.9 ( gem ) , 6.6 , 4.6 hz , 1h ) , 2.08 ( m , 2h ) ; c nmr ( 75 mhz , cdcl3 ) 169.3 , 139.0 , 137.3 , 134.4 , 129.6 , 129.3 , 128.6 , 128.4 , 126.0 , 65.0 , 60.7 , 34.4 , 27.9 , 17.8 ; ir ( thin film ) 3057 , 2930 , 1635 , 1302 , 1142 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c18h19no3sna 352.0983 , found 352.0973 . general procedure b. the residue was purified by flash column chromatography ( 60:40 etoac / hex , rf = 0.40 ) to yield 13b as a foam ( 117 mg , 84% ) . h nmr ( 300 mhz , cdcl3 ) 7.92 ( m , 2h ) , 7.747.64 ( m , 1h ) , 7.60 ( m , 2h ) , 7.30 ( m , 3h ) , 7.15 ( m , 2h ) , 5.40 ( s , 1h ) , 4.68 ( m , 1h ) , 3.28 ( m , 1h ) , 2.78 ( m , 1h ) , 2.49 ( ddd , j = 12.3 , 11.9 , 9.8 hz , 1h ) , 1.92 ( m , 2h ) , 1.31 ( d , j = 6.8 hz , 3h ) , 0.89 ( d , j = 7.0 hz , 3h ) ; c nmr ( 75 mhz , cdcl3 ) 169.7 , 141.6 , 137.5 , 134.3 , 129.6 , 129.0 , 128.6 , 128.0 , 126.0 , 65.0 , 53.8 , 47.6 , 28.5 , 20.3 , 20.1 , 17.5 ; ir ( thin film ) 2967 , 2945 , 1641 , 1303 , 1143 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c20h23no3sna 380.1296 , found 380.1306 . general procedure c. the residue was purified by flash column chromatography ( 70:30 etoac / hex , rf = 0.48 ) to yield 13c as a foam ( 130 mg , 85% ) . h nmr ( 600 mhz , cdcl3 ) 7.63 ( t , j = 7.4 hz , 1h ) , 7.39 ( m , 7h ) , 7.30 ( m , 3h ) , 7.25 ( t , j = 6.1 hz , 2h ) , 6.79 ( m , 2h ) , 5.76 ( d , j = 14.6 hz , 1h ) , 4.69 ( s , 1h ) , 3.22 ( d , j = 14.6 hz , 1h ) , 3.17 ( m , 1h ) , 3.07 ( m , 1h ) , 2.67 ( dd , j = 18.1 ( gem ) , 6.8 hz , 1h ) , 2.43 ( m , 1h ) , 2.18 ( m , 1h ) ; c nmr ( 150 mhz , cdcl3 ) 169.0 , 138.8 , 137.0 , 136.1 , 134.0 , 129.5 , 129.4 , 129.3 , 128.6 , 128.6 , 128.5 , 127.7 , 125.9 , 63.9 , 57.2 , 47.6 , 28.3 , 16.1 ; ir ( thin film ) 3059 , 2936 , 1642 , 1307 , 1144 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c24h23no3sna 428.1296 , found 428.1303 . general procedure c. the residue was purified by flash column chromatography ( 60:40 etoac / hex , rf = 0.40 ) to yield 13d as a foam ( 103 mg , 75% ) . h nmr ( 600 mhz , cdcl3 ) 7.99 ( m , 2h ) , 7.69 ( m , 1h ) , 7.59 ( m , 2h ) , 7.30 ( m , 3h ) , 6.98 ( dd , j = 7.3 , 2.0 hz , 2h ) , 5.40 ( d , j = 2.4 hz , 1h ) , 4.95 ( dd , j = 17.5 , 2.6 hz , 1h ) , 3.38 ( td , j = 5.0 , 2.5 hz , 1h ) , 3.30 ( dd , j = 17.5 , 2.5 hz , 1h ) , 2.81 ( ddd , j = 17.7 , 10.5 , 6.9 hz , 1h ) , 2.52 ( ddd , j = 18.0 ( gem ) , 6.7 , 4.3 hz , 1h ) , 2.28 ( m , 1h ) , 2.12 ( m , 2h ) ; c nmr ( 150 mhz , cdcl3 ) 168.7 , 138.4 , 137.3 , 134.4 , 129.5 , 129.3 , 128.9 , 128.6 , 126.2 , 77.7 , 73.1 , 64.6 , 57.9 , 34.3 , 28.3 , 17.3 ; ir ( thin film ) 3303 , 3055 , 1650 , 1267 , 1148 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c20h19no3sna 376.0983 , found 376.0977 . general procedure c. the residue was purified by flash column chromatography ( 60:40 etoac / hex , rf = 0.40 ) to yield 13e as a foam ( 124 mg , 89% ) . h nmr ( 600 mhz , cdcl3 ) 7.92 ( m , 2h ) , 7.71 ( m , 1h ) , 7.60 ( m hz , 2h ) , 7.29 ( m , 3h ) , 6.96 ( dd , j = 7.5 , 1.9 hz , 2h ) , 5.83 ( dddd , j = 17.7 , 10.5 , 8.2 , 4.3 hz , 1h ) , 5.21 ( m , 2h ) , 5.11 ( s , 1h ) , 4.86 ( ddd , j = 15.0 , 4.3 , 2.0 hz , 1h ) , 3.31 ( td , j = 4.7 , 1.8 hz , 1h ) , 3.002.82 ( m , 2h ) , 2.54 ( ddd , j = 18.0 ( gem ) , 6.8 , 3.7 hz , 1h ) , 2.19 ( m , 2h ) ; c nmr ( 151 mhz , cdcl3 ) 169.1 , 139.1 , 137.3 , 134.4 , 132.4 , 129.5 , 129.3 , 128.7 , 128.4 , 126.0 , 118.8 , 64.4 , 57.2 , 47.9 , 28.0 , 17.1 ; ir ( thin film ) 3054 , 2988 , 1641 , 1307 , 1148 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c20h21no3sna 378.1140 , found 378.1136 . general procedure c. the residue was purified by flash column chromatography ( 70:30 etoac / hex , rf = 0.40 ) to yield 13f as a foam ( 110 mg , 72% ) . h nmr ( 600 mhz , cdcl3 ) 7.92 ( m , 2h ) , 7.67 ( t , j = 7.5 hz , 1h ) , 7.56 ( m , 2h ) , 7.32 ( m , 2h ) , 7.27 ( m , 3h ) , 7.19 ( m , 5h ) , 5.54 ( s , 1h ) , 3.48 ( m , 1h ) , 2.95 ( m , 1h ) , 2.66 ( dt , j = 18.1 ( gem ) , 6.2 hz , 1h ) , 2.20 ( m , 2h ) ; c nmr ( 150 mhz , cdcl3 ) 169.8 , 142.3 , 139.7 , 137.2 , 134.4 , 129.6 , 129.2 , 129.1 , 128.6 , 128.3 , 127.3 , 127.0 , 126.2 , 64.9 , 62.2 , 28.8 , 18.2 ; ir ( thin film ) 3066 , 3008 , 2946 , 1654 , 1304 , 1143 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c23h21no3sna 414.1140 , found 414.1131 . general procedure c. the residue was purified by flash column chromatography ( 70:30 etoac / hex , rf = 0.46 ) to yield 13 g as a foam ( 126 mg , 91% ) . h nmr ( 600 mhz , cdcl3 ) 7.91 ( m , 2h ) , 7.69 ( m , 1h ) , 7.59 ( t , j = 7.8 hz , 2h ) , 7.31 ( dd , j = 8.4 , 6.6 hz , 2h ) , 7.25 ( m , 1h ) , 7.05 ( d , j = 7.4 hz , 2h ) , 5.22 ( s , 1h ) , 3.95 ( dtd , j = 13.6 , 6.8 , 3.3 hz , 1h ) , 3.32 ( td , j = 5.0 , 2.0 hz , 1h ) , 2.81 ( m , 1h ) , 2.43 ( m , 2h ) , 1.99 ( m , 2h ) , 1.61 ( m , 2h ) , 0.87 ( t , j = 7.4 hz , 3h ) ; c nmr ( 150 mhz , cdcl3 ) 169.2 , 139.7 , 137.4 , 134.3 , 129.6 , 129.2 , 128.5 , 128.3 , 126.1 , 64.7 , 57.9 , 48.0 , 28.0 , 20.3 , 17.6 , 11.3 ; ir ( thin film ) 2967 , 2929 , 2871 , 1639 , 1308 , 1147 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c20h23no3sna 380.1296 , found 380.1290 . general procedure b. the residue was purified by flash column chromatography ( 60:40 etoac / hex , rf = 0.50 ) to yield 13h as an orange amorphous solid ( 101 mg , 68% ) . h nmr ( 300 mhz , cdcl3 ) 7.93 ( dd , j = 7.2 , 1.6 hz , 2h ) , 7.70 ( m , 1h ) , 7.60 ( dd , j = 8.2 , 6.6 hz , 2h ) , 7.32 ( m , 5h ) , 6.37 ( dd , j = 16.0 , 1.2 hz , 1h ) , 6.07 ( dd , j = 15.8 , 6.4 hz , 1h ) , 4.94 ( dt , j = 6.6 , 1.6 hz , 1h ) , 4.66 ( m , 1h ) , 3.35 ( td , j = 6.4 , 2.0 hz , 1h ) , 2.68 ( dt , j = 17.2 , 7.0 hz , 1h ) , 2.40 ( dt , j = 17.2 , 7.1 hz , 1h ) , 2.09 ( dt , j = 14.4 , 7.2 hz , 1h ) , 1.99 ( dt , j = 14.4 , 7.2 hz , 1h ) , 1.33 ( d , j = 6.8 hz , 3h ) , 1.18 ( d , j = 6.9 hz , 3h ) ; c nmr ( 75 mhz , cdcl3 ) 169.7 , 137.6 , 135.4 , 134.2 , 132.0 , 129.8 , 129.6 , 128.8 , 128.6 , 128.4 , 126.5 , 63.5 , 51.8 , 47.3 , 29.1 , 20.7 , 20.2 , 18.78 ; ir ( thin film ) 3059 , 2975 , 1639 , 1446 , 1305 , 1145 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c22h25no3sna 406.1453 , found 406.1447 . general procedure b. the residue was purified by flash column chromatography ( 60:40 etoac / hex , rf = 0.42 ) to yield 13i as a foam ( 96 mg , 69% ) . h nmr ( 600 mhz , cdcl3 ) 7.92 ( d , j = 8.4 hz , 2h ) , 7.69 ( t , j = 7.4 hz , 1h ) , 7.60 ( t , j = 7.8 hz , 2h ) , 6.97 ( d , j = 8.6 hz , 2h ) , 6.84 ( m , 2h ) , 5.18 ( m , 1h ) , 3.93 ( m , 1h ) , 3.77 ( s , 3h ) , 3.30 ( m , 1h ) , 2.81 ( m , 1h ) , 2.45 ( m , 2h ) , 2.01 ( m , 2h ) , 1.61 ( m , 2h ) , 0.88 ( t , j = 7.5 hz , 3h ) ; c nmr ( 150 mhz , cdcl3 ) 169.2 , 159.5 , 134.2 , 131.5 , 129.5 , 128.6 , 127.3 , 114.5 , 105.0 , 64.9 , 57.5 , 55.3 , 48.0 , 28.1 , 20.3 , 17.6 , 11.3 ; ir ( thin film ) 2964 , 2920 , 1633 , 1250 , 1144 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c21h25no4sna 410.1402 , found 410.1400 . general procedure c. the residue was purified by flash column chromatography ( 60:40 etoac / hex , rf = 0.40 ) to yield 13j as a foam ( 133 mg , 87% ) . h nmr ( 600 mhz , cdcl3 ) 7.89 ( m , 2h ) , 7.69 ( m , 1h ) , 7.59 ( m , 2h ) , 7.28 ( m , 2h ) , 7.01 ( m , 2h ) , 5.21 ( s , 1h ) , 3.92 ( ddd , j = 13.6 , 9.0 , 7.4 hz , 1h ) , 3.27 ( td , j = 5.1 , 2.2 hz , 1h ) , 2.78 ( ddd , j = 17.4 , 10.1 , 6.8 hz , 1h ) , 2.41 ( m , 2h ) , 1.96 ( m , 2h ) , 1.59 ( m , 2h ) , 0.86 ( t , j = 7.4 hz , 3h ) ; c nmr ( 150 mhz , cdcl3 ) 169.2 , 138.3 , 137.2 , 134.4 , 134.2 , 129.6 , 129.4 , 128.5 , 127.5 , 64.6 , 57.5 , 48.0 , 28.1 , 20.3 , 17.9 , 11.3 ; ir ( thin film ) 2964 , 2937 , 1641 , 1306 , 1146 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c20h22clno3sna 414.0907 , found 414.0921 . general procedure c. the residue was purified by flash column chromatography ( 60:40 etoac / hex , rf = 0.42 ) to yield 13k as a foam ( 92 g , 61% ) . h nmr ( 600 mhz , cdcl3 ) 7.95 ( d , j = 8.4 hz , 2h ) , 7.70 ( t , j = 7.4 hz , 1h ) , 7.61 ( t , j = 7.9 hz , 2h ) , 7.24 ( m , 1h ) , 7.17 ( m , 2h ) , 7.05 ( d , j = 7.8 hz , 1h ) , 5.51 ( s , 1h ) , 3.97 ( m , 1h ) , 3.14 ( m , 1h ) , 2.94 ( m , 1h ) , 2.53 ( dd , j = 18.3 ( gem ) , 7.4 hz , 1h ) , 2.42 ( m , 1h ) , 2.34 ( m , 2h ) , 2.07 ( m , 1h ) , 1.98 ( m , 1h ) , 1.69 ( m , 2h ) , 1.14 ( t , j = 7.5 hz , 3h ) , 0.92 ( t , j = 7.4 hz , 3h ) ; c nmr ( 151 mhz , cdcl3 ) 168.9 , 141.1 , 137.6 , 136.1 , 134.3 , 129.63 , 129.60 , 128.63 , 128.60 , 126.5 , 126.1 , 62.5 , 54.2 , 48.2 , 27.3 , 24.3 , 20.4 , 16.3 , 15.4 , 11.5 ; ir ( thin film ) 3006 , 2966 , 1639 , 1300 , 1144 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c22h27no3sna 408.1609 , found 408.1620 . general procedure b. the residue was purified by flash column chromatography ( 80:20 etoac / hex , rf = 0.50 ) to yield 13l as a foam ( 104 mg , 64% ) . h nmr ( 600 mhz , cdcl3 ) 7.92 ( m , 2h ) , 7.69 ( t , j = 7.1 hz , 1h ) , 7.60 ( m , 2h ) , 6.32 ( s , 1h ) , 6.16 ( d , j = 2.1 hz , 2h ) , 5.14 ( d , j = 2.1 hz , 1h ) , 3.95 ( ddd , j = 13.4 , 9.6 , 6.5 hz , 1h ) , 3.72 ( s , 6h ) , 3.34 ( td , j = 4.8 , 1.9 hz , 1h ) , 2.79 ( m , 1h ) , 2.46 ( m , 2h ) , 2.03 ( m , 2h ) , 1.63 ( m , 2h ) , 0.89 ( t , j = 7.4 hz , 3h ) ; c nmr ( 150 mhz , cdcl3 ) 169.1 , 161.5 , 142.3 , 137.4 , 134.2 , 129.6 , 128.6 , 104.2 , 99.5 , 64.6 , 58.0 , 55.4 , 48.2 , 28.0 , 20.3 , 17.7 , 11.3 ; ir ( thin film ) 2962 , 2937 , 1641 , 1594 , 1306 , 1145 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c22h27no5sna 440.1508 , found 440.1517 . general procedure b. the residue was purified by flash column chromatography ( 70:30 etoac / hex , rf = 0.35 ) to yield 13 m as a foam ( 101 mg , 75% ) . h nmr ( 600 mhz , cdcl3 ) 7.88 ( d , j = 7.2 hz , 2h ) , 7.65 ( m , 1h ) , 7.56 ( t , j = 7.7 hz , 2h ) , 7.27 ( dd , j = 1.9 , 1.0 hz , 1h ) , 6.24 ( m , 1h ) , 6.10 ( m , 1h ) , 5.16 ( d , j = 2.9 hz , 1h ) , 3.78 ( ddd , j = 13.6 , 9.6 , 6.4 hz , 1h ) , 3.59 ( td , j = 6.1 , 3.1 hz , 1h ) , 2.71 ( m , 2h ) , 2.43 ( dt , j = 17.5 , 6.6 hz , 1h ) , 2.09 ( m , 2h ) , 1.58 ( m , 1h ) , 1.49 ( m , 1h ) , 0.85 ( t , j = 7.4 hz , 3h ) ; c nmr ( 150 mhz , cdcl3 ) 169.4 , 151.3 , 143.0 , 137.3 , 134.2 , 129.5 , 128.5 , 110.6 , 108.4 , 61.8 , 52.9 , 47.8 , 28.5 , 20.4 , 19.0 , 11.3 ; ir ( thin film ) 2965 , 2933 , 2875 , 1647 , 1503 , 1306 , 1148 cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c18h21no4sna 370.1089 , found 370.1076 . the residue was purified by flash column chromatography ( 70:30 etoac / hex , rf = 0.35 ) to yield 14 as a solid ( 111 mg , 87% ) . mp 127.4128.1 c ; h nmr ( 600 mhz , cdcl3 ) 7.99 ( m , 2h ) , 7.74 ( m , 1h ) , 7.65 ( m , 2h ) , 7.35 ( dd , j = 8.4 , 7.1 hz , 2h ) , 7.28 ( m , 1h ) , 7.05 ( d , j = 6.8 hz , 2h ) , 5.21 ( s , 1h ) , 3.45 ( d , j = 6.9 hz , 1h ) , 2.91 ( s , 3h ) , 2.53 ( m , 1h ) , 2.45 ( dd , j = 15.7 , 5.3 hz , 1h ) , 2.11 ( dd , j = 15.8 , 11.6 hz , 1h ) , 0.91 ( d , j = 6.7 hz , 3h ) ; c nmr ( 150 mhz , cdcl3 ) 171.1 , 139.3 , 137.0 , 134.5 , 129.6 , 129.4 , 129.1 , 127.9 , 125.3 , 73.7 , 60.3 , 38.1 , 34.4 , 29.1 , 22.4 ; ir ( thin film ) 2912 , 1658 , 1305 , 1130 cm ; hrms ( esi - tof ) m / z the residue was purified by flash column chromatography ( 80:20 etoac / hex , rf = 0.35 ) to yield 15 as a white solid ( 169 mg , 79% ) . mp 143.7144.3 c ; h nmr ( 600 mhz , cdcl3 ) 7.70 ( d , j = 7.5 hz , 2h ) , 7.50 ( t , j = 7.4 hz , 1h ) , 7.37 ( t , j = 7.7 hz , 2h ) , 5.89 ( s , 2h ) , 5.31 ( d , j = 9.5 hz , 1h ) , 4.22 ( ddd , j = 13.4 , 9.5 , 3.6 hz , 1h ) , 3.72 ( s , 3h ) , 3.71 ( s , 6h ) , 2.48 ( m , 4h , nch3 and 1 methylene h ) , 2.40 ( m , 1h ) , 1.84 ( m , 1h ) , 1.27 ( d , j = 6.9 hz , 3h ) ; c nmr ( 150 mhz , cdcl3 ) 173.1 , 161.4 , 158.6 , 137.4 , 133.4 , 128.50 , 128.48 , 106.8 , 90.6 , 61.9 , 55.6 , 55.2 , 51.9 , 35.0 , 31.8 , 29.3 , 17.1 ; ir ( thin film ) 3006 , 2939 , 1614 , 1286 , 1134 cm ; hrms ( esi - tof ) m / z [ m + h ] calcd for c22h28no6s 434.1637 , found 434.1649 . allylamine ( 0.29 ml , 3.8 mmol ) and trans - cinammaldehyde ( 0.53 ml , 4.2 mmol ) were added to a round - bottom flask containing thf ( 38 ml ) and na2so4 ( 3.3 g , 23.0 mmol ) , and the mixture was allowed to stir for 1 h. the anhydride ( 0.92 g , 3.8 mmol ) was added , and the resulting mixture was allowed to stir for 2 h. the na2so4 was filtered off , and the solvent was evaporated in vacuo . the residue was triturated with hexanes , transferred to a fritted funnel , and washed in portions with hexanes five times ( 5 ml ) and two times with a 9:1 mixture of cold hexanes and chcl3 ( 10 ml total ) , yielding 1.2 g of an off - white solid , which was 85% pure by nmr . a portion of the resulting acid ( 0.885 g , 2.2 mmol ) was redissolved in anhydrous toluene and methanol ( 60 ml:30 ml ) , and tmschn2 ( 2.2 ml , 4.4 mmol , 2 m in hexanes ) was added at 0 c dropwise over a period of 5 min . the mixture was allowed to stir for 1.5 h and monitored by tlc for disappearance of the starting material . the solvent was removed in vacuo , and the crude mixture was purified by flash column chromatography ( etoac / hex 3060% ) , yielding 17 ( 0.85 g , 92% ) as an amorphous solid ( 50:50 etoac / hex , rf = 0.44 ) . h nmr ( 600 mhz , cdcl3 ) 7.987.89 ( m , 2h ) , 7.817.56 ( m , 3h ) , 7.417.24 ( m , 5h ) , 6.54 ( d , j = 15.8 hz , 1h ) , 5.845.72 ( m , 2h ) , 5.345.25 ( m , 2h ) , 5.02 ( d , j = 8.4 hz , 1h ) , 4.40 ( ddt , j = 15.3 , 4.7 , 1.7 hz , 1h ) , 3.713.42 ( m , 3h ) , 3.443.27 ( m , 2h ) , 3.13 ( d , j = 18.2 ( gem ) hz , 1h ) ; c nmr ( 150 mhz , cdcl3 ) 169.4 , 165.3 , 136.7 , 135.8 , 135.2 , 135.0 , 131.7 , 130.6 , 129.3 , 128.9 , 128.9 , 126.8 , 121.9 , 119.4 , 76.1 , 62.0 , 53.4 , 43.8 , 35.6 . ir ( thin film ) 1736 , 1715 , 1694 ( c = o ) , 1309 , 1148 ( s = o ) cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c23h23no5sna 448.1195 , found 448.1195 . ester 17 ( 0.602 g , 1.41 mmol ) was dissolved in anhydrous benzene ( 114 ml ) , which was degassed by bubbling with argon for approximately 5 min . then grubbs second - generation catalyst ( 35 mg , 3 mol % ) was added , and the mixture was heated to reflux for 9 h. the solvent was evaporated in vacuo , and the residue was redissolved in etoac ( 30 ml ) . saturated nh4cl ( 10 ml ) was added , and the mixture was allowed to stir for 30 min . the mixture was transferred to a separtory funnel , and the organic layer was washed with brine , dried over na2so4 , and then purified ( etoac / hex 3060% ) , yielding 18 ( 0.39 g , 86% ) as an amorphous solid ( 50:50 etoac / hex , rf = 0.16 ) . h nmr ( 600 mhz , cdcl3 ) 8.027.93 ( m , 2h ) , 7.74 ( td , j = 7.4 , 1.3 hz , 1h ) , 7.61 ( t , j = 7.9 hz , 2h ) , 5.90 ( dq , j = 6.0 , 2.1 hz , 1h ) , 5.55 ( dq , j = 6.2 , 2.2 hz , 1h ) , 5.38 ( tt , j = 4.3 , 1.8 hz , 1h ) , 4.33 ( ddt , j = 15.7 , 4.2 , 2.1 hz , 1h ) , 3.783.67 ( m , 2h ) , 3.62 ( s , 3h ) , 3.00 ( d , j = 15.7 hz , 1h ) ; c nmr ( 150 mhz , cdcl3 ) 172.8 , 165.7 , 136.9 , 134.9 , 130.6 , 130.3 , 129.3 , 129.2 , 79.0 , 70.1 , 53.3 , 50.7 , 39.5 ; ir ( thin film ) 1735 , 1716 , 1687 ( c = o ) , 1311 , 1148 ( s = o ) cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c15h15no5sna 344.0569 , found 344.0568 . alkene 18 ( 0.25 g , 0.77 mmol ) was dissolved in anhydrous meoh ( 30 ml ) , and the reaction mixture was degassed by bubbling with argon for 5 min . then pd / c ( 45 mg ) was added and h2 was bubbled into the reaction flask , and the mixture was allowed to stir for 3 h ( monitored by tlc ) . the pd / c was filtered off and washed with etoac ( 3 10 ml ) . the solvent was removed in vacuo , yielding 19 ( 0.246 g , 99% ) as an amorphous solid ( 50:50 etoac / hex , rf = 0.1 ) . h nmr ( 600 mhz , cdcl3 ) 7.987.90 ( m , 2h ) , 7.797.70 ( m , 1h ) , 7.60 ( dd , j = 8.5 , 7.3 hz , 2h ) , 4.61 ( dd , j = 9.7 , 5.9 hz , 1h ) , 3.72 ( s , 3h ) , 3.623.49 ( m , 2h ) , 3.17 ( d , j = 17.1 hz , 1h ) , 3.153.05 ( m , 1h ) , 2.071.93 ( m , 2h ) , 1.91 ( dtd , j = 12.2 , 6.0 , 3.9 hz , 1h ) , 1.25 ( dq , j = 12.6 , 9.3 hz , 1h ) ; c nmr ( 150 mhz , cdcl3 ) 170.6 , 166.0 , 134.9 , 130.3 , 130.2 , 129.2 , 75.6 , 63.9 , 53.4 , 42.1 , 39.4 , 28.5 , 25.8 ; ir ( thin film ) 1734 , 1686 ( c = o ) , 1146 ( s = o ) cm ; hrms ( esi - tof ) m / z [ m + na ] calcd for c15h17no5sna 346.0725 , found 346.0730 . to sulfone 19 ( 0.10 g , 0.31 mmol ) dissolved in 3 ml of chcl3 was added dbu ( 70 l , 0.46 mmol ) , and the reaction mixture was stirred overnight . the mixture was concentrated in vacuo and purified by flash chromatography ( 70:30 etoac / hex , rf = 0.40 ) to yield 20 as a colorless oil ( 0.047 g , 84% ) . h nmr ( 600 mhz , cdcl3 ) 3.72 ( s , 3h ) , 3.58 ( t , j = 7.1 hz , 2h ) , 3.52 ( m , 2h ) , 2.93 ( m , 2h ) , 2.40 ( p , j = 7.4 hz , 2h ) ; c nmr ( 150 mhz , cdcl3 ) 172.8 , 164.0 , 161.6 , 98.6 , 51.0 , 41.5 , 41.2 , 26.6 , 25.6 . these data ester 18 ( 50 mg , 0.16 mmol ) was dissolved in 2 ml of chcl3 , and dbu ( 36 l , 0.24 mmol ) was added . after 90 min , the solvent evaporated in vacuo , and the crude mixture was purified by flash chromatography ( 20:80 etoac / hex , rf = 0.45 ) , yielding 21 as an oil ( 12 mg , 41% ) . h nmr ( 600 mhz , cdcl3 ) 7.04 ( d , j = 3.4 hz , 1h ) , 6.47 ( t , j = 3.5 hz , 1h ) , 6.17 ( d , j = 3.1 hz , 1h ) , 4.20 ( dd , j = 9 , 3 hz , 1h ) , 3.78 ( s , 3h ) , 3.48 ( dd , j = 19.8 ( gem ) , 3.6 hz , 1h ) , 3.19 ( dd , j = 18.6 ( gem ) , 9 hz , 1h ) ; c nmr ( 150 mhz , cdcl3 ) 170.6 , 169.6 , 136.1 , 119.2 , 112.2 , 106.6 , 52.9 , 38.1 , 38.0 . alkene 20 ( 47 mg , 0.25 mmol ) was dissolved in 2.2 ml of methanol under an atmosphere of argon , and 10% pd / c ( 14 mg ) was added to the vial . the flask was purged with h2 gas via a balloon , and the reaction mixture was stirred under an atmosphere of h2 overnight . the reaction vessel was purged with argon , and then the mixture was filtered through celite and concentrated to yield 22 as an oil ( 45 mg , 99% ) . h nmr ( 600 mhz , cdcl3 ) 4.09 ( ddd , j = 9.7 , 9.1 , 3.8 hz , 1h ) , 3.69 ( s , 3h ) , 3.57 ( q , j = 9.3 hz , 1h ) , 3.38 ( td , j = 8.5 , 4.8 hz , 1h ) , 3.03 ( t , j = 11.7 hz , 1h ) , 2.78 ( d , j = 8.9 hz , 2h ) , 2.05 ( q , j = 9.5 hz , 1h ) , 1.95 ( m , 1h ) , 1.82 ( m , 1h ) , 1.24 ( m , 1h ) ; c nmr ( 150 mhz , cdcl3 ) 174.0 , 172.2 , 62.7 , 51.9 , 41.8 , 39.9 , 36.0 , 27.4 , 26.0 . these data

sulfone - substituted - and -lactams have been prepared in a single step with high diastereoselectivity . sulfonylglutaric anhydrides produce intermediates that readily decarboxylate to provide -lactams with high diastereoselectivity . substituents at the 3- or 4-position of the glutaric anhydride induce high levels of stereocontrol . sulfonylsuccinic anhydrides produce intermediate carboxylic acids that can be trapped as methyl esters or allowed to decarboxylate under mild conditions . this method has been applied to a short synthesis of the pyrrolizidine alkaloid ( )-isoretronecanol .

pubmed

the morbidity of breast cancer is gradually increasing , and a growing number of young people are now at risk for this disease . in china , breast cancer continues to be the leading cause of death among women . with the improvement in diagnosis and development of drugs and radiotherapy technology . therefore , early detection of the tumor and accurate analysis of molecular biology characteristics are still of great importance . microrna ( mirna ) is a non - coding protein and single - stranded small rna . it mainly inhibits translation or induces the degradation of some related mrnas through target mrna recognition , leading to changes in objective mrna at the protein expression level . recent studies showed that some mirnas have functions similar to that of oncogenes or anti - oncogenes , which may play an important role in the invasion and development of breast cancer . mir-125a , mir-206 , and mir-145 are similar to anti - oncogenes , and they are related to the occurrence of tumors . thus , mir-206 plays a key role in adjustment during breast cancer proliferation . according to the research of scott and gk , mir-125a can affect the protein levels of her-2 and her-3 , thereby inhibiting the metastatic and invasive abilities of breast cancer cells . moreover , lee showed that mir-145 expression increases after mir-145 precursors transfect breast cancer cell lines , but can dose - dependently inhibit cell growth . mir-21 and mir-10b are similar to oncogenes , and related to the invasion and metastasis of tumors . in vitro and in vivo experiments showed that eliminating mir-21 gene fragments can inhibit the proliferation and invasion of mcf-7 and mda - mb-231 cells . in the 4t1 rat breast tumor metastasis model , mir-10b can effectively inhibit the metastasis of malignant breast cancer cells with high specificity . the possible mechanism for this effect may be the inhibition of the entry of 4ti tumor cells into blood vessels . however , the specific roles of mir-21 and mir-10b in the occurrence and development of breast cancer remain unclear . the expression of mir-21 , mir-10b , mir-125a , mir-206 , and mir-145 in breast cancer tissues and para - carcinoma tissues was examined . the expression of estrogen receptor ( er ) and progesterone receptor ( pr ) in breast cancer was analyzed by immunohistochemistry , whereas that of her-2 and topoisomerase 2a ( top 2a ) was analyzed by fluorescence in situ hybridization ( fish ) . the relationship between the expression levels of mirnas and breast cancer clinical pathology was analyzed . thus , this research aimed to investigate the function of mirnas in the invasion and development of breast cancer . cancer tissues were obtained from these patients , who were pathologically confirmed to have invasive ductal carcinoma from january 1 2013 to december 1 2013 in the tumor hospital of the second affiliated hospital of xjtu . all patients were female , with an age range of 4262 years ( mean age , 51 years ) . these patients underwent modified radical mastectomy or reserved radical mastectomy , and had complete clinical pathology data . the diameter of tumors ranged from 1.6 cm to 4.5 cm ( mean diameter , 2.2 cm ) . one sample was cancer tissues , and the other sample was para - carcinoma normal gland tissues , which were more than 3 cm away from cancer tissues . the samples were immediately flushed with 0.9% rna- and enzyme - free nacl , rapidly immersed in rna enzyme inhibitor , and incubated at 4c overnight . the protective liquid was removed from the samples , which were stored in a refrigerator at 80c . the patients were not treated before the operation , and neither neoadjuvant chemotherapy nor endocrine therapy was conducted . based on histological classification by the who ( 2003 ) standards , m - mlv reverse transcriptase , rna enzyme inhibitor , oligo dt , dntps , kod - plus , and sybr mastermix were obtained from takara ( japan ) , and primers were from shanghai shenggong company ( china ) . breast cancer her-2 , neu ( 17q12 ) , top 2a ( 17q21 ) , and csp 17 multicolor detection kits were purchased from guangzhou biping medical technology company . total rna ( 500 ng ) was used for rt by mirna stem - loop rt primers . the reaction was conducted in a 10-l system at the following reaction conditions : 16c for 30 min , 42c for 15 min , and 85c for 5 s. real - time qpcr was performed with a 20-l reaction system . the mirna reaction detection system included 1 l of rt products , 1 sybr green i mastermix , 0.5 ixmol / l mirna - specific f - primer and 0.5 ixmol / l universal r - primer . the conditions of immediate quantitative pcr were as follows : 95c for 10 min , followed by 40 cycles of 95c for 15 s and 60c for 1 min . the circulation times in which real - time qrt - pcr reached the set - up threshold in every reaction tube ( ct data ) were recorded . according to some related studies , the 2 method was used to express the gene expression of tumor tissues relative to that of the paired normal tissues . moreover , ct=(ct mirna - ct u6 ) tumor ( ct mirna - ct u6 ) was normal . samples were fixed in formalin , embedded in paraffin , and sliced into 4-m - thick sections . the results show positive expression of ep and er at 10% , and the cancer cell nucleus was brown . samples were fixed in formalin , embedded in paraffin , and sliced into 4-m - thick sections . sections were initially observed under the 40 objective with a suitable filter , and counted under the 100 objective . the signals of gene - specific probe ( gsp ) her-2 ( red ) , gsp top 2a ( green ) , and centromeric probe ( csp ) 17 ( cyan ) were counted in 60 nuclei at the clear tumor areas . the sum of signals of gsp her-2 , gsp top 2a , and csp 17 were recorded , and the data of gsp her-2/csp 17 and gsp top 2a / csp 17 were calculated . the signals of her and top 2a were analyzed according to the following criteria : a ) the signal of gsp her-2 and top 2a ( red , green ) combined to bunches ; b ) gsp her-2 and top 2a / csp 17 ( red , green / cyan ) 2.2 and 2 ; c ) gsp her-2 and top 2a / csp 17 ( red , green / cyan ) < 1.8 and 0.8 ; and d ) gsp her-2 and top 2a / csp 17 ( red , green / cyan ) between 1.82.2 and 0.82.0 . if a or if d , new signal data should be counted in 20 other nuclei , or another researcher should recalculate the data . researchers should indicate the consequence if the recalculated data were at the critical value . under situations with weak signal or high background , signal judgment may be disturbed , so the experiment was repeated using another tissue slice . differences in expression were analyzed by spss 13.0 . given the limited number of samples in this study , the data demonstrate an abnormal distribution . all mirna expression data are shown as the median and interquartile range ( iqr ) . the nonparametric wilcoxon test was used to rank matched - pair data , and the mann - whitney u test was used to compare data between the 2 groups . cancer tissues were obtained from these patients , who were pathologically confirmed to have invasive ductal carcinoma from january 1 2013 to december 1 2013 in the tumor hospital of the second affiliated hospital of xjtu . all patients were female , with an age range of 4262 years ( mean age , 51 years ) . these patients underwent modified radical mastectomy or reserved radical mastectomy , and had complete clinical pathology data . the diameter of tumors ranged from 1.6 cm to 4.5 cm ( mean diameter , 2.2 cm ) . one sample was cancer tissues , and the other sample was para - carcinoma normal gland tissues , which were more than 3 cm away from cancer tissues . the samples were immediately flushed with 0.9% rna- and enzyme - free nacl , rapidly immersed in rna enzyme inhibitor , and incubated at 4c overnight . the protective liquid was removed from the samples , which were stored in a refrigerator at 80c . the patients were not treated before the operation , and neither neoadjuvant chemotherapy nor endocrine therapy was conducted . based on histological classification by the who ( 2003 ) standards , m - mlv reverse transcriptase , rna enzyme inhibitor , oligo dt , dntps , kod - plus , and sybr mastermix were obtained from takara ( japan ) , and primers were from shanghai shenggong company ( china ) . breast cancer her-2 , neu ( 17q12 ) , top 2a ( 17q21 ) , and csp 17 multicolor detection kits were purchased from guangzhou biping medical technology company . total rna ( 500 ng ) was used for rt by mirna stem - loop rt primers . the reaction was conducted in a 10-l system at the following reaction conditions : 16c for 30 min , 42c for 15 min , and 85c for 5 s. real - time qpcr was performed with a 20-l reaction system . the mirna reaction detection system included 1 l of rt products , 1 sybr green i mastermix , 0.5 ixmol / l mirna - specific f - primer and 0.5 ixmol / l universal r - primer . the conditions of immediate quantitative pcr were as follows : 95c for 10 min , followed by 40 cycles of 95c for 15 s and 60c for 1 min . the circulation times in which real - time qrt - pcr reached the set - up threshold in every reaction tube ( ct data ) were recorded . according to some related studies , the 2 method was used to express the gene expression of tumor tissues relative to that of the paired normal tissues . moreover , ct=(ct mirna - ct u6 ) tumor ( ct mirna - ct u6 ) was normal . samples were fixed in formalin , embedded in paraffin , and sliced into 4-m - thick sections . the results show positive expression of ep and er at 10% , and the cancer cell nucleus was brown . samples were fixed in formalin , embedded in paraffin , and sliced into 4-m - thick sections . sections were initially observed under the 40 objective with a suitable filter , and counted under the 100 objective . the signals of gene - specific probe ( gsp ) her-2 ( red ) , gsp top 2a ( green ) , and centromeric probe ( csp ) 17 ( cyan ) were counted in 60 nuclei at the clear tumor areas . the sum of signals of gsp her-2 , gsp top 2a , and csp 17 were recorded , and the data of gsp her-2/csp 17 and gsp top 2a / csp 17 were calculated . the signals of her and top 2a were analyzed according to the following criteria : a ) the signal of gsp her-2 and top 2a ( red , green ) combined to bunches ; b ) gsp her-2 and top 2a / csp 17 ( red , green / cyan ) 2.2 and 2 ; c ) gsp her-2 and top 2a / csp 17 ( red , green / cyan ) < 1.8 and 0.8 ; and d ) gsp her-2 and top 2a / csp 17 ( red , green / cyan ) between 1.82.2 and 0.82.0 . if a or d , new signal data should be counted in 20 other nuclei , or another researcher should recalculate the data . researchers should indicate the consequence if the recalculated data were at the critical value . under situations with weak signal or high background , signal judgment may be disturbed , so the experiment was repeated using another tissue slice . differences in expression were analyzed by spss 13.0 . given the limited number of samples in this study , the data demonstrate an abnormal distribution . all mirna expression data are shown as the median and interquartile range ( iqr ) . the nonparametric wilcoxon test was used to rank matched - pair data , and the mann - whitney u test was used to compare data between the 2 groups . among the 35 samples , the relative quantity ( 2 ) of mir-125a was below 1 ( 80% ) in 28 cases and above 1 ( 20% ) in 7 cases . the relative quantity ( 2 ) of mir-206 was below 1 ( 86% ) in 30 cases of breast cancer and above 1 ( 14% ) in 5 cases . the relative quantity ( 2 ) of mir-145 was below 1 ( 60% ) in 21 cases of breast cancer and above 1 ( 40% ) in 14 cases . the data ranged from 0.13 to 7.73 and the iqr was 0.511.72 . the median was 0.78 , which is 0.78 times that of normal tissue . the relative quantity ( 2 ) of mir-21 was above 1 ( 94% ) in 33 cases of breast cancer and below 1 ( 6% ) in 2 cases . the relative quantity ( 2 ) of mir-10b was above 1 ( 63% ) in 22 cases of breast cancer and below 1 ( 37% ) in 13 cases . the difference was statistically significant ( p<0.05 ) . among the 35 patients , 16 cases ( 45.7% ) exhibited her-2 gene amplification , which was demonstrated as a red cluster or multiple red points , whereas 18 cases ( 51.4% ) exhibited top 2a gene amplification , which was demonstrated as a green cluster or multiple green points ( table 1 ) . negative expression of her-2 and top 2a was observed in the 17 chromosome ( figure 1 ) . the expression levels of mir-125a , mir-206 , and mir-145 in breast cancer tissues were all lower than those in para - carcinoma tissues . low expression of mir-125a in the metastatic lymph node group and high negative expression of her-2 and top 2a in patients were unrelated to the tumor size , patient age , menstruation , negative er , and pr expression ( p<0.10 ) . mir-206 was highly expressed in patients with small tumors , negative er , negative pr , and negative her-2 ( p<0.10 ) . thus , mir-206 was irrelevant to patient age , menstruation , metastatic lymph node , and top 2a expression ( table 2 ) . mir-145 was negatively correlated with the tumor size and lymph node metastasis ( p<0.05 ) , and had no correlation with patient age , menstruation , and pr expression . however , mir-145 was correlated with negative er , negative her-2 , and negative top 2a ( p<0.05 ) ( table 3 ) . the expression of mir-21 and mir-10b in breast cancer tissues was obviously higher than that in para - carcinoma tissues . mir-21 exhibited high expression in older , postmenopausal patients with various symptoms , such as lymph node metastasis and positive her-2 ( p<0.10 ) , but it was not related to the tumor size and expression levels of top 2a , er , and pr . the expression of mir-10b was related to the tumor size , lymph node metastasis , and positive top-2a ( p<0.05 ) , but it was not associated with age , menstruation , er , pr , and her-2 ( table 3 ) . among the 35 samples , the relative quantity ( 2 ) of mir-125a was below 1 ( 80% ) in 28 cases and above 1 ( 20% ) in 7 cases . the relative quantity ( 2 ) of mir-206 was below 1 ( 86% ) in 30 cases of breast cancer and above 1 ( 14% ) in 5 cases . the relative quantity ( 2 ) of mir-145 was below 1 ( 60% ) in 21 cases of breast cancer and above 1 ( 40% ) in 14 cases . the data ranged from 0.13 to 7.73 and the iqr was 0.511.72 . the median was 0.78 , which is 0.78 times that of normal tissue . the relative quantity ( 2 ) of mir-21 was above 1 ( 94% ) in 33 cases of breast cancer and below 1 ( 6% ) in 2 cases . the relative quantity ( 2 ) of mir-10b was above 1 ( 63% ) in 22 cases of breast cancer and below 1 ( 37% ) in 13 cases . among the 35 patients , 16 cases ( 45.7% ) exhibited her-2 gene amplification , which was demonstrated as a red cluster or multiple red points , whereas 18 cases ( 51.4% ) exhibited top 2a gene amplification , which was demonstrated as a green cluster or multiple green points ( table 1 ) . negative expression of her-2 and top 2a was observed in the 17 chromosome ( figure 1 ) . the expression levels of mir-125a , mir-206 , and mir-145 in breast cancer tissues were all lower than those in para - carcinoma tissues . low expression of mir-125a in the metastatic lymph node group and high negative expression of her-2 and top 2a in patients were unrelated to the tumor size , patient age , menstruation , negative er , and pr expression ( p<0.10 ) . mir-206 was highly expressed in patients with small tumors , negative er , negative pr , and negative her-2 ( p<0.10 ) . thus , mir-206 was irrelevant to patient age , menstruation , metastatic lymph node , and top 2a expression ( table 2 ) . mir-145 was negatively correlated with the tumor size and lymph node metastasis ( p<0.05 ) , and had no correlation with patient age , menstruation , and pr expression . however , mir-145 was correlated with negative er , negative her-2 , and negative top 2a ( p<0.05 ) ( table 3 ) . the expression of mir-21 and mir-10b in breast cancer tissues was obviously higher than that in para - carcinoma tissues . mir-21 exhibited high expression in older , postmenopausal patients with various symptoms , such as lymph node metastasis and positive her-2 ( p<0.10 ) , but it was not related to the tumor size and expression levels of top 2a , er , and pr . the expression of mir-10b was related to the tumor size , lymph node metastasis , and positive top-2a ( p<0.05 ) , but it was not associated with age , menstruation , er , pr , and her-2 ( table 3 ) . recent studies have already proved that the expression of mirnas changes is closely related to various types of human cancer . however , the functions of mirnas are complicated because they can effectively regulate and control hundreds of genes , including oncogenes and anti - oncogenes . studies indicated that mir-125a ( has - mir-125a ) is located at chromosome 19q13.41 , including 2 mirnas that are in the same precursor ( mir-125a.5p and mir-125a.3p ) , and has the same origin as lin-4 in nematodes . mir-125a can be detected as a phylogenetic mutation , which is associated with the occurrence of breast cancer . mir-125a can also inhibit tumors by directly lowering the mrna and protein levels of some genes , such as erbb2 and rna binding protein hur . mir-206 is located at human chromosome 6p12.2 , and is considered to be a type of mirna related to skeletal muscles and the myocardium . it can boost muscle differentiation by lowering the p180 subunit of dna polymerase and muscle transcription factors ( e.g. , is13 and myor ) . mir-206 can also inhibit the proliferation of breast cancer cells , mainly by lowering the mrna and protein expression levels of er and er co - regulatory proteins via the egf / mapk pathway [ lowering steroid acceptor nuclear co - activators ( e.g. , src-1 and src-3 ) and transcription factor gata-3 ] . mir-206 can also act on the protein cx43 to inhibit the invasion and metastasis of cancer cells . mir-206 can result in mutations at the rs6920648 locus , and is associated with the existence of breast cancer . the expression of mir-145 decreases in many types of tumor tissues , including breast cancer , colon cancer , and lung cancer . this phenomenon might be related to several factors , such as the location of mir-145 , ability of mir-145 to easily cause deficiency in tumor cells , and genetic changes in the receptor s surface . similarly , mir-145 can inhibit hyperplasia in tumor cells by affecting target genes , such as c - myc , irs-1 , oct4 , sox2 , and klf4 . mir-21 is an mirna located in the fra17b fragile area of chromosome 17q23.2 , and has an independent transcription unit . moreover , mir-21 controls the expression of many anti - oncogenes , including tpm1 , programmed cell death 4 , maspin , and bcl-2 , to prompt the metastasis and hyperplasia of breast cancer cells . mir-10b is located between the hoxd4 and hoxd8 genes of 2p31.1 , and its structure is 5-uacccuguagaaccgaauuugug-3. mir-10b is related to many types of tumors . adjusting the twist - mir-10b - hoxd 10-rhoc pathway is the main mechanism by which mir-10b inhibits the metastasis of breast cancer cells . mir-10b also facilitates the metastasis of breast cancer cells by regulating the expression of the target genes upar and syndecan-1 . early diagnosis is essential to prevent and cure tumors . in this study , the relative transcript levels of mir-21 and mir-10b in patients tumor tissues were higher than those in para - carcinoma normal tissues . by contrast , the relative transcript levels of mir-145 , mir-125a , and mir-206 in patients tumor tissues were apparently lower than those in para - carcinoma normal tissues . according to the literature [ 1214 ] , the relative transcript levels of mir-145 , mir-125a , and mir-206 in serum are also visibly abnormal compared with those in serum from healthy females . therefore , these mirnas can be used as markers of breast cancer for early diagnosis . our results show that mir-145 and breast cancer tumor size , as well as mir-145 and lymph node metastasis , was negatively correlated in 35 cases of breast cancer . although mir-125a and mir-206 were expressed at low levels in the large mass group and lymph node metastasis group , statistical analysis showed no significant correlation between the expression of mir-125a and mir-206 and breast tumor size , as well as between the expression of mir-125a and mir-206 and lymph node metastasis . we are currently further expanding the number of samples for analysis to determine the intrinsic correlations . preliminary results show that mir-145 , mir-125a , and mir-206 were similar to tumor suppressor genes , and may be involved in the suppression of breast cancer invasion and metastasis ; these findings are consistent with those reported in the literature . as indicated in the literature , mir-10b expression may indicate the invasion and development of breast cancer . combined with the findings of lee , the results show that mir-21 may be correlated with old patients with lymph node metastasis . hormone receptor - positive breast cancer , her-2-positive breast cancer , and triple - negative breast cancer ( tnbc ) ] based on er , pr , and her-2 expression . her-2-positive breast cancer is highly malignant , and can reappear and transfer easily . herceptin - targeted treatment has made significant progress for this type of cancer . lee showed that mir-21 is related to the metastasis of breast cancer lymph nodes and positive her-2 expression . moreover , mir-21 is an independent prognostic factor of breast cancer . combined with cong s research , the results showed that mir-21 is over - expressed in breast cancer cells resistant to trastuzumab , which causes the loss of pten expression . a previous study showed that mir-125a is negatively associated with her-2 , indicating that the her-2 gene may be under the regulation and control of mir-125a ; therefore , mir-125a can distinctly inhibit the expression of oncogenes her-2 and her-3 . mir-125a expression is up - regulated when the hdac inhibitor entinostat is used to treat her-2-positive breast cancer cells , and has been shown to be an important mechanism of the functions of entinostat . thus , mir-21 and mir-125a are significant to developing new anti - her-2 drugs and overcoming herceptin resistance in drugs . tnbc is characterized by high malignancy , high possibility of reappearance and metastasis , and poor treatment methods . in this study , mir-145 and mir-206 were found to be related to negative er and negative her-2 ( p<0.05 ) . mir-145 was highly expressed in negative pr ( p>0.05 ) , whereas mir-206 was negatively correlated with negative pr ( p<0.10 ) . showed that the metastasis of cancer cells could be inhibited by leading mir-145 and mir-206 into metastatic breast cancer cells . mir-145 and mir-206 are expected to be the new target spots in tnbc treatment . besides inhibiting tnbc development , mir-206 can also inhibit positive er lines , and is considered as the switch of breast cancer when it is transferred to the base type from lumen - a . top 2a codes dna topoisomerase ii , and is one of the components of the nuclear matrix . top 2a can adjust the copy , transcription , recombination , and repair of dna , which is not only a hyperplasia index of tumor cells , but also a treatment target of anthracycline chemotherapy drugs . in breast cancer tissues , tumors with high expression of top 2a this situation can be improved after using anthracycline alone or anthracycline combination chemotherapy . in this research , mir-21 was found to be positively related to top 2a , whereas mir-125a and mir-145 were negatively associated with top 2a . these findings show that mir-145 and mir-125a might inhibit the top 2a gene , and were connected with anthracycline drug resistance . therefore , mir-21 , mir-125a , and mir-145 may contribute to the chemotherapy effect of epirubicin , and are important in guiding the development of chemotherapy for breast cancer . therefore , mir-125a , mir-206 , mir-145 , mir-21 , and mir-10b are highly expected to be novel tumor markers for the diagnosis and prognosis of breast cancer . mir-21 and mir-125a could contribute to the treatment of her-2-positive breast cancer cells , whereas mir-145 and mir-206 could accelerate the development of treatment methods for tnbc . however , the detailed mechanism and whether these mirnas can be used as treatment targets in clinical applications should be verified in future investigations .

backgroundthis study aimed to investigate the expression and significance of 5 types of mirnas in breast cancer to provide a theoretical and practical foundation for using these mirnas in the diagnosis and treatment of breast cancer , thereby improving medical services.material/methodsstem-loop real - time rt - pcr was used to detect the expression levels of mir-145 , mir-21 , mir-10b , mir-125a , and mir-206 in 35 cases of breast cancer and adjacent normal breast tissues , and to analyze the relationship of mirnas expression with clinicopathological features of breast cancer . the expression levels of estrogen receptor ( er ) and progesterone receptor ( pr ) were examined by immunohistochemistry . fluorescence in situ hybridization was used for the detection of her-2 and top 2a.resultsthe expression levels of mir-145 , mir-125a , and mir-206 in breast cancer were lower than those in adjacent normal tissues . mir-145 was negatively correlated with tumor size , lymph node metastasis , er , her-2 , and top 2a ( p<0.05 ) , regardless of age , menstruation , and pr . mir-125a was correlated with negative node status , negative her-2 status ( p<0.05 ) , whereas tumor size , age , menstruation , er , and pr were independent factors . mir-206 expression was correlated with negative er status , negative pr status , and negative her-2 status ( p<0.05 ) , regardless of age , menstruation , lymph node metastasis , and top 2a . mir-21 and mir-10b expression in breast cancer tissues was significantly higher than that in adjacent tissues ( p<0.05 ) . mir-21 in post - menstrual patients with lymph node metastasis was highly expressed ( p<0.05 ) , and had no correlations with tumor size , er , pr , and top 2a expression . mir-10b expression was positively correlated with breast cancer tumor size , lymph node metastasis , and top 2a status ( p<0.05 ) , but had no correlations with age , menstruation , er , pr , and her-2.conclusionsmir-145 , mir-21 , mir-10b , mir-125a , and mir-206 may play important roles in breast cancer development and invasion .

pubmed

however , transplantation is limited not only by organ shortage but also by its unfavorable long - term success due to chronic allograft injury ( cai ) [ 1 , 2 ] . chronic allograft vasculopathy ( cav ) , glomerulosclerosis , and interstitial fibrosis and tubular atrophy ( ifta ) are the dominating histopathological findings in cai [ 1 , 2 ] . its pathogenesis is still poorly understood , but acute rejection episodes were defined as an important risk factor and experimental data suggest that they play an important pathogenetic role [ 15 ] . transplantation of kidneys in fischer-344 ( f344 ) to lewis rat strain combination is a well - established experimental model for human cai . we and others thoroughly characterized a variant of this model , where immunosuppression is omitted [ 710 ] . this model allows investigation of a severe but reversible acute rejection episode , which peaks 9 days after allogenic transplantation and precedes the development of cai . acute rejection of f344 to lewis renal allografts is characterized by a strong mononuclear infiltration of the allograft interstitium . at the same time , numerous leukocytes accumulate in the arterial , capillary , and venous blood vessels . after resolution of acute rejection , the number of intravascular leukocytes decreases but remains chronically elevated compared to healthy kidneys . a majority of blood leukocytes in day 9 allografts are monocytes displaying a unique state of partial activation regarding cell surface antigen and cytokine expression . these cells interact with graft endothelial cells and might trigger cav , which is characterized by intimal hyperplasia . to identify factors contributing to the pathogenesis of cai during reversible acute rejection , we compared the transcriptome of mononuclear leukocytes isolated from renal allografts to isografts 9 days after transplantation . in this gene array experiment , tissue transglutaminase ( transglutaminase 2 , tgm2 ) was found among the genes , which were upregulated by mononuclear leukocytes accumulating in the lumina of allograft blood vessels . transglutaminases catalyze posttranscriptional modifications of proteins , resulting in cross - linking of proteins such as cytoskeleton or extracellular matrix ( ecm ) , as well as in modifications via amine incorporation into proteins and via deamidation [ 11 , 12 ] . these modifications can have drastic consequences including increased mechanical stability , formation of autoantigens , and profound changes in protein function [ 11 , 13 , 14 ] . among the 9 transglutaminases present in humans , tgm2 is most intensively investigated and a bewildering functional diversity is described . it acts as classical ca - dependent transglutaminase , as g - protein , as disulfide isomerase , and as protein kinase [ 16 , 17 ] . in addition , it interacts with a plethora of other proteins and is involved in numerous cellular processes such as apoptosis and cell survival , phagocytosis , cell adhesion and migration , and cell signaling . tgm2 is ubiquitously expressed and localizes to virtually all compartments of the cell including the cytoplasm , the nucleus , and mitochondria . in addition , tgm2 can be attached to the surface and is secreted into the extracellular space , where it can cross - link itself to extracellular proteins [ 15 , 18 ] . we are interested in tgm2 in the context of reversible acute rejection and subsequent chronic deterioration of renal allografts . tgm2 might exert essential functions involved in rejection such as antigen presentation and t cell activation via dendritic cells , activation of the nfb pathway , and enabling the expression of proinflammatory factors , leukocyte adhesion to vascular endothelial cells , and leukocyte migration . during resolution of acute rejection , its role in apoptosis , phagocytosis , and release of tgf- may be of importance [ 21 , 22 ] . finally , during the development of cai , tgm2 might protect graft blood vessels as already evidenced in the context of atherosclerosis . alternatively , tgm2 could contribute to vascular remodeling by increasing vascular stiffness and by promoting epithelial / endothelial to mesenchymal transition [ 24 , 25 ] , which was suggested to contribute to ifta . in this study , we investigate tgm2 mrna and protein expression by mononuclear blood leukocytes isolated from lewis to lewis isografts and f344 to lewis allografts and by respective renal tissue tgm2 expressions compared to activated caspase-3 , a protease critically involved in apoptosis . leukocytes and grafts are studied during acute rejection on day 9 after transplantation and after resolution of acute rejection on day 42 . furthermore , allograft recipients were treated with cystamine during the first month after transplantation to assess the role of early extracellular transglutaminase activity in the pathogenesis of cai . male lewis ( rt1 ) ( janvier , st . berthevin , france ) and f344 ( rt1 ) ( harlan winkelmann , borchen , germany ) rats weighing 270300 g were used for transplantation . animals were kept under conventional conditions and received humane care according to the german law on the protection of animals and the principles of laboratory animal care formulated by the national society for medical research as well as the nih guide for the care and use of laboratory animals . experiments were approved by the local authorities ( permit number gi 20/10 number 23/2008 ; gi 20/27 number 51/2010 ) . kidneys were transplanted orthotopically to totally nephrectomized lewis recipients as described previously , except that the ureter was anastomosed end to end . for allogenic transplantation , f344 rats and , for isogenic transplantation , 30 g ampicillin after surgery ( ratiopharm , ulm , germany ) ; no immunosuppressant was given . total ischemic times remained below 30 min . on day 9 or 42 after transplantation application of 60 mg / kg sodium pentobarbital ( narcoren , merial , hallbergmoos , germany ) and grafts were removed , cut in small pieces , and snap frozen in liquid nitrogen until use . allograft recipients were treated with the transglutaminase inhibitor cystamine dihydrochloride ( sigma - aldrich , taufkirchen , germany ) for 28 days starting at the day of transplantation . cystamine was diluted in sterile saline and delivered continuously using subcutaneously implanted osmotic minipumps ( 2ml4 alzet , cupertino , ca ) at a concentration of 12 mg per day . animals were sacrificed 12 weeks after transplantation ; grafts were removed , fixed in 4% buffered paraformaldehyde , and embedded in paraffin . to study renal function , isolation of mononuclear blood cells was described previously in detail [ 28 , 29 ] . injection of 200 u of heparin ( liquemin n 5000 , roche , basel , switzerland ) , and kidneys were intensively perfused with cold ca- and mg - free phosphate buffered saline ( paa , pasching , austria ) , supplemented with 2.7 mm edta and 0.1% bovine serum albumin ( serva , heidelberg , germany ) . to deplete erythrocytes and granulocytes total rna was isolated from 30 mg kidney tissue or from 5 10 mononuclear leukocytes harvested from the blood vessels of control kidneys , isografts , or allografts on day 9 or 42 after transplantation . rna extraction was performed using the rneasy mini kit ( qiagen , hilden , germany ) according to the instructions of the supplier . one g of total rna was reversely transcribed using the m - mlv h reverse transcriptase and 1 g of random hexamer primers ( promega , mannheim , germany ) . the reaction was carried out at 40c for 1 h. real - time rt - pcr was used to assess the mrna gene expression of tgm2 . reactions were performed in an abi 7900 sequence detection system ( applied biosystems , foster city , ca ) using platinum sybr green qpcr super mix - udg ( invitrogen , karlsruhe , germany ) . the relative gene expression values were calculated by the equation 2 , where ct is the difference in ct values between the porphobilinogen deaminase ( pbgd ) housekeeping gene and tgm2 . data for each experimental group are expressed in relation to the expression in the healthy control , which was set to 1 arbitrary unit . all primers for real - time rt - pcr were synthesized by mwg biotech ( ebersberg , germany ) . primer sequences are as follows : for tgm2 , 5-cca gcg tgg aca gac tta ca-3 ( sense ) , 5-ctg ctc cac atc gtc aga ca-3(antisense ) and for pbgd , 5-ggc gca gct aca gag aaa gt-3(sense ) , 5-agc cag gat aat ggc act ga-3(antisense ) . pcr conditions included denaturation for 5 min at 95c , followed by 45 cycles of 20 s at 95c , 20 s at 60c , and 10 s at 72c . to confirm the production of a single amplicon with the expected molecular mass , in addition , each product was sequenced ( seqlab , gttingen , germany ) to confirm the specificity of the pcr . a negative control , where cdna was omitted , was included in every run . in negative controls , protein concentrations were determined using micro bca protein assay kit ( pierce biotechnology , rockford , il ) . equal amounts of protein ( 40 g of tissue extract or 8 g of cellular extract ) were resolved on 8% or 15% reducing sds - polyacrylamide gels and transferred onto polyvinylidene difluoride membranes ( millipore , billerica , ma ) . membranes were blocked in 1x roti - block solution ( roth , karlsruhe , germany ) diluted in 50 mm tris - hcl , ph 7.6 , and 0.9% nacl for 60 min at rt and then incubated overnight at 4c with mouse monoclonal antibodies ( mabs ) to tgm2 ( clone tg100 ) ( thermo fisher scientific , fremont , ca ) diluted 1 : 4000 or 1 : 6000 for cellular extract or kidney tissue , respectively . optionally , membranes were incubated overnight with rabbit anti - active caspase-3 abs ( abcam , cambridge , uk ) diluted 1 : 1000 in roti - block solution . to ensure equal protein loading , membranes were blocked with 5% milk and incubated with mabs to gapdh ( novus biologicals , littleton , co ) , 1 : 20000 . bound antibodies were visualized with horseradish peroxidase - conjugated secondary abs ( dako , glostrup , denmark ) , 1 : 5000 , using the chemiluminescent reagent lumi - light western blotting substrate ( roche , mannheim , germany ) . densitometric analyses were performed using a digital gel documentation system ( biozym , hessisch oldendorf , germany ) . all data of individual samples were divided by the values obtained for gapdh on the same blot . the mean of the tgm2/gapdh or active caspase-3/gapdh ratio of the controls was set to 1 arbitrary unit and each individual value including control values was calculated accordingly . histological sections were stained with acidic orcein 12 weeks after transplantation to visualize the internal and external elastic lamina of arteries and to evaluate a possible effect of cystamine on arterial remodelling . at least 10 arteries of the muscular type were investigated per section . to estimate the relative thickness of arterial media and intima , the ratio of the total vessel diameter , including media and intima , and the diameter of the lumen of the artery were determined . additionally , the percentage of arteries exhibiting intimal hyperplasia was analyzed . histological sections were stained with azocarmine / aniline blue ( azan ) 12 weeks after transplantation and tunica adventitia was measured . to estimate relative thickness of the tunica adventitia , the diameter of outer arterial diameter ( including tunica adventitia , media , intima , and lumen ) was determined and divided by diameter of inner arterial diameter ( including media , intima , and lumen ) . sections of 6 m were dewaxed and rehydrated . to unmask tgm2 immunoreactivity in the fixed tissue , antigen retrieval was performed in 0.01 m sodium citrate buffer ( ph 6.0 ) for 15 min at 120c in a steamer . sections were pretreated with protease xiv ( sigma - aldrich ) for 15 min at rt for detection of cd163 or active caspase-3 . after washing in pbs , ph 7.2 , the paraffin sections were incubated for 30 min at rt with pbs supplemented with 1% bsa and 0.1% nan3 . thereafter , mouse mabs to tgm2 diluted 1 : 1500 or ed2 ( anti - cd163 , serotec , oxford , uk ) abs diluted 1 : 200 or polyclonal rabbit abs to active caspase-3 ( abcam ) diluted 1 : 100 in pbs supplemented with 1% bsa ( serva ) were applied and incubated at 4c overnight . bound , primary tgm2 abs were detected using rabbit anti - mouse immunoglobulin and goat anti - rabbit hrp labeled abs ( dako ) and 3,3-diaminobenzidine ( dab ) ( sigma - aldrich ) . to amplify the signal for ed2 abs , additionally , anti - rabbit envision peroxidase system ( dako ) was applied according to the manufacturer 's instructions . to detect abs bound to active caspase-3 , anti - rabbit envision peroxidase system ( dako ) was used . sections were weakly stained with hemalum and coverslipped with pertex ( medite , burgdorf , germany ) . to detect tgm2-positive monocytes / macrophages , sections were first stained with mabs to tgm2 as described , followed by protease xiv treatment for 15 min at rt and overnight incubation at 4c with mab ed1 directed to a cd68-like antigen ( serotec ) diluted 1 : 500 in pbs supplemented with 1% bsa and 0.1% nan3 . to detect bound primary abs , rabbit anti - mouse immunoglobulin and alkaline phosphatase anti - alkaline phosphatase ( apaap ) ( both from dako ) were used in combination with fast blue ( sigma - aldrich ) as chromogen . sections were evaluated with an olympus bx51 microscope and analysis software ( olympus , hamburg , germany ) . data were analyzed , where applicable , by nonparametric kruskal - wallis test followed by the mann - whitney rank sum test using spss software ( spss software , munich , germany ) . renal transplantation in the f344 to lewis rat strain combination results in accumulation of mononuclear leukocytes in allograft blood vessels [ 9 , 31 ] . to isolate these cells , kidneys were intensively perfused on days 9 and 42 after transplantation . in agreement with the previously published data , we observed a strong increase in the number of mononuclear leukocytes accumulating in the vasculature of allografts on day 9 compared to isografts . furthermore , the number of intravascular leukocytes was reduced on day 42 ; however , it remained still increased compared to day 42 isografts . the difference in cell number was also observed between perfusates from control kidneys and isografts on days 9 and 42 ( figure 1 ) . the cellular composition of intravascular leukocytes obtained by perfusion on day 9 and day 42 was published before [ 9 , 31 ] . the mrna expression of tgm2 was analyzed by real - time rt - pcr in mononuclear leukocytes isolated from renal blood vessels of control animals and isogenic and allogenic renal transplants . real - time rt - pcr led to a single amplicon of the expected molecular mass in all samples analyzed . in negative controls , where cdna was omitted , interestingly , an elevated level of tgm2 mrna was detected in cells collected from allografts on day 9 , as well as on day 42 after transplantation , when compared to the respective isografts ( figure 2(a ) ) . tgm2 mrna expression was also increased in day 9 and day 42 allograft tissue compared to respective isografts . in contrast to graft blood leukocytes , where the expression was stronger on day 9 compared to day 42 , tgm2 tissue expression did not differ between day 9 and day 42 ( figure 2(b ) ) . protein expression of tgm2 was investigated by immunoblotting of lysates of mononuclear intravascular leukocytes isolated from control animals and isogenic or allogenic renal transplants ( figures 3(a ) and 4(a ) ) as well as of total tissue lysates ( figures 3(c ) and 4(c ) ) . expression of tgm2 was detected in all groups investigated as a major band with expected molecular mass of ~70 kda . on day 42 densitometrical analyses revealed elevated levels of tgm2 expression in blood mononuclear leukocytes isolated from isografts on day 9 compared to cells from control kidneys . furthermore , an elevated expression level of tgm2 was detected in perfusates from allografts on day 9 compared to respective isograft samples ( figure 3(b ) ) . in contrast to the results from real - time rt - pcr , no difference in the expression of tgm2 was detected in perfusates on day 42 after transplantation ( figure 4(b ) ) as well as in total kidney tissue on day 9 and day 42 after transplantation ( figures 3(d ) and 4(d ) ) . so far , the expression of tgm2 was analyzed in the total intravascular population of mononuclear leukocytes . to examine tgm2 expression by graft monocytes , we performed immunohistochemical staining on paraffin sections from control kidneys , isografts , and allografts explanted on day 9 after transplantation ( figures 5(a)5(d ) ) . in line with previously published data describing accumulation of blood leukocytes , tgm2-positive cells were most abundant in the vasculature of day 9 allografts ( figure 5(c ) ) . in addition to intravascular leukocytes , blood plasma exhibited tgm2 immunoreactivity , predominantly in allografts . to identify monocytes expressing tgm2 , double - staining experiments with mab ed1 detecting cd68-like antigen were performed ( figure 5(d ) ) . in isograft tissue , tgm2 immunoreactivity was detected in the vascular endothelium of some arteries , veins , and capillaries . in the media of arteries , smooth muscle cells were immunopositive in the adventitia extracellular material and cells with a fibroblast - like shape . in the renal parenchyma , renal tubules , most probably proximal tubules , collecting ducts , and the outer layer of bowman 's capsule were stained with antibodies to tgm2 . the staining was identical to the pattern seen in isografts but it was more intense all over . in the leukocytic infiltrate , no conspicuous additional staining was detected ( data not shown ) . tgm2 was described as a marker for m2 macrophages ; we investigated if graft monocytes also express the classical m2 cell surface marker cd163 . to detect cd163-positive cells , immunohistochemical staining with mab ed2 as expected , we detected a very low number of positive cells in isografts ( figure 5(e ) ) . in contrast , cd163-positive cells were more frequent in allografts ( figure 5(f ) ) . in both groups analyzed , no cd163-positive cells were detected in the lumina of blood vessels . therefore , we analyzed if caspase-3 , a key protease directly involved in apoptosis , is also expressed by intravascular graft leukocytes . to examine activation of caspase-3 during reversible acute rejection , detection of active caspase-3 in renal mononuclear graft leukocytes as well as in graft tissue on days 9 and 42 after transplantation was performed by immunoblotting ( figures 6(a ) , 6(c ) , 7(a ) , and 7(c ) ) . the appearance of the active p19 ( ~19 kda ) and p17 ( ~17 kda ) fragments of caspase-3 was detected . densitometric analyses revealed increased levels of active caspase-3 in mononuclear blood leukocytes from allografts on day 9 ( figure 6(b ) ) . active caspase-3 was also detected in perfusates from day 42 isografts and allografts , but no difference was observed among them ( figures 7(a ) and 7(b ) ) . also , in allograft tissue , elevated levels of active caspase-3 were measured on days 9 and 42 compared to respective isografts ( figures 6(c ) and 7(c ) ) . furthermore , these results were corroborated by immunohistochemical staining of graft tissue on day 9 after transplantation . active caspase-3-positive cells were detected in the graft blood vessels predominantly in allografts veins , whereas caspase-3 immunoreactivity was less prominent in leukocytes accumulating in arteries ( figure 6(d ) ) . to examine the role of tgm2 in the pathogenesis of cai , renal allograft recipients were treated with cystamine , a general inhibitor of tgm , or placebo . creatinine clearance was monitored 4 , 8 , and 12 weeks after transplantation to determine renal function . no differences were observed between placebo- ( 3.4 1.5 at 4 weeks ; 5.1 0.7 at 8 weeks ; 3.0 0.7 at 12 weeks ; n = 6 per group ; data are given as mean standard deviation , ml / min ) and cystamine - treated animals ( 3.9 1.2 at 4 weeks ; 4.4 0.6 at 8 weeks ; 3.5 0.5 at 12 weeks ; n = 6 per group ; data are given as mean standard deviation , ml / min ) independent of the time point investigated . at the end of the study , at 12 weeks after transplantation , animals were sacrificed and relative thickness of arteries was quantified on sections stained with acidic orcein . no significant difference was observed between both experimental groups ( figures 8(a ) , 8(c ) , and 8(d ) ) . furthermore , arteries with intimal hyperplasia were detected in the sham - treated group , whereas cystamine treatment had no effect on intimal remodeling ( figures 8(b ) , 8(c ) , and 8(d ) ) . additionally , histological sections were stained with azocarmine / aniline blue ( azan ) 12 weeks after transplantation to detect interstitial fibrosis . we demonstrate in this study that tgm2 mrna and protein are overexpressed by mononuclear leukocytes , predominantly by activated monocytes , which accumulate in the vascular bed of rat renal allografts ( f344 to lewis ) on day 9 after transplantation . after resolution of acute rejection on day 42 , tgm2 mrna levels are lower but still moderately increased compared to respective isografts , which is not reflected in increased protein levels . tgm2 mrna expression was also induced in graft tissue upon allogenic transplantation . on the protein level , however , no quantitative changes were detected . we focused on the well - characterized population of mononuclear leukocytes , which accumulate in large numbers in the blood vessels of experimental allografts during acute rejection . the number of leukocytes , which were isolated from control kidneys , isografts , and allografts 9 and 42 days after transplantation by intensively perfusing renal blood vessels , nicely corroborated data published previously [ 9 , 31 ] . the acute rejection episode , which peaks at around day 9 after transplantation , is of major interest for the understanding of cai , since clinical and experimental data suggest that acute rejection is an important triggering factor [ 15 ] . as cav , a hallmark of cai , is characterized by arterial intimal hyperplasia , leukocytes interacting with allogenic endothelial cells were suggested to play a pivotal pathogenic role . the number of intravascular leukocytes decreases after reversion of acute rejection in the f344 to lewis rat strain combination but remains elevated for several weeks . first hints regarding the overexpression of tgm2 by mononuclear leukocytes isolated from the blood vessels of allografts on day 9 after transplantation came from gene array data . these data were confirmed by real - time rt - pcr and immunoblotting using antibodies , which detected a major band of the expected molecular mass . immunohistochemistry with the same antibodies revealed that intravascular leukocytes in renal allografts were strongly tgm2-immunoreactive . on tissue sections double - stained with mab ed1 directed to a cd-68-like antigen , an established marker for rat monocytes / macrophages , we identified graft monocytes as strongly tgm2-immunopositive . it might be argued that the increase in tgm2 expression in mononuclear blood leukocytes from day 9 allografts is due to the increase in the proportion of monocytes , which is indeed seen . however , isograft blood leukocytes contain about 50% monocytes and allografts about 70% , whereas tgm2 mrna expression levels increase by about 10-fold , suggesting that the increased proportion of monocytes is not the only explanation for the observed increase in tgm2 mrna levels . interactions of monocytes with graft endothelial cells , which is a prerequisite for intravascular accumulations , may induce tgm2 expression as described previously for monocytes adhering to endothelial cells in vitro . in addition , proinflammatory cytokines produced during acute rejection are known inducers of tgm2 expression [ 14 , 15 , 33 , 34 ] . in graft tissue , which was investigated for comparison , mrna levels were elevated on both days 9 and 42 . tgm2 protein was detected by immunoblotting but , in contrast to tgm2 mrna , no significant increase in protein signals was seen . however , some weak additional bands of lower molecular mass that were only detected in allograft perfusates and tissue would be compatible with an accelerated degradation of tgm2 in allografts . another explanation for the discrepancy between mrna and protein is based on reports in which tgm2 acts as a substrate of itself and forms insoluble cross - linked complexes , which are not detected in conventional immunoblots . for the detection of tgm2 covalently bound to extracellular matrix , unfixed cryostat sections should be stained in future studies . in line with this idea and with our mrna data , tissue sections of renal day the renal expression pattern of tgm2 was in line with other publications on rodent and human tgm2 expression . recently , shrestha et al . reported upregulation of tgm2 mrna , protein , and transglutaminase activity in cai kidneys . their model also is based on the f344 to lewis rat strain combination but , in contrast to our model , includes a 10-day course of suboptimal immunosuppression , which interferes with early acute rejection , followed by delayed contralateral nephrectomy . our study and the study by these authors complement each other , as they predominantly investigate end - stage cai , whereas we focus on the early phase posttransplantation . evidence increased mrna levels of tgm2 as well as increased tgm2 immunopositivity in allograft tissue sections compared to isografts . of note , these authors also demonstrate that , in addition to blood leukocytes investigated only in our study , renal tubules and glomeruli also overexpress tgm2 in allografts . during the pathogenesis of cai , urinary secretion of the transglutaminase cross - link product (-glutamyl)-lysine is increased . in end - stage cai , this product is detected more abundantly in allografts compared to isografts . from both studies , however , it is difficult to conclude if tgm2 is a marker of cai reflecting changes in the cellular composition of the graft or actively contributes to the pathogenesis of cai as shown for diverse models of experimental renal fibrosis [ 3740 ] . tgm2 was recently described to be a reliable marker of m2 macrophages , an anti - inflammatory subpopulation of differentiated macrophages . to the best of our knowledge , tgm2 expression by blood monocytes in vivo has not been described before , although , similarly to macrophages , blood monocytes can be activated and express cytokine patterns and cell surface molecules resembling proinflammatory m1 macrophages and anti - inflammatory m2 macrophages . we previously demonstrated that blood monocytes of f344 to lewis renal allografts are activated , acquire an intermediate state of differentiation , and express both cytokines , typical for m1 and for m2 macrophages , during acute rejection . we do not know , however , if this population is a mixture of m1- and m2-like monocytes or if individual monocytes are characterized by an intermediate phenotype . to further characterize these monocytes , we investigated expression of cd163 , a typical m2 cell surface marker , which was not detected on graft monocytes during the peak of acute rejection . hence , we conclude that tgm2 can be expressed by activated monocytes but we can not decide if , similarly to macrophages , tgm2 expression is confined to m2-like monocytes . the functional relevance of tgm2 expression by m2 macrophages , however , is still elusive and similarly its role in monocytes is unclear . we described before that monocytes isolated from the blood vessels of renal allografts during fatal acute rejection undergo apoptosis when cultivated ex vivo . as tgm2 is more strongly expressed in apoptotic cells [ 11 , 13 , 44 ] , we investigated if levels of activated caspase-3 correlate with expression of tgm2 in perfusate cells . indeed , a stronger induction of activated caspase-3 was seen in leukocytes isolated from day 9 allografts compared to respective isografts . this suggests that tgm2 expression by blood mononuclear cells might be linked to apoptosis . in line with the idea that tgm2 enhances apoptosis depending on a prolonged contact with endothelial cells in the inflamed allograft , activation of caspase-3 was more visible in veins compared to arteries . tgm2 plays a dual role in apoptosis : expression of tgm2 can induce apoptosis but it also stabilizes the cytoskeleton of dying cells , which prevents cell rupture and release of proinflammatory cytoplasmic components [ 13 , 4446 ] . as a large number of monocytes accumulate in allograft vessels , their apoptosis might be a tremendous anti - inflammatory stimulus capable of reverting rejection . this issue certainly deserves further investigation . in the tissue of day 9 and day 42 allografts , immunohistochemistry , however , evidenced that tgm2 not only is present in the cytoplasm of graft blood monocytes but was also detected in the surrounding blood plasma . due to rejection - associated vascular damage and the high number of leukocytes accumulating in the blood vessels of allografts , we assume that blood flow slowed down considerably in allografts and that endothelial cells and underlying extracellular matrix are in intensive and prolonged contact with products secreted locally . in the context of hypertension , tgm2 and other active transglutaminases were shown to contribute to the pathogenesis of vascular remodeling [ 4750 ] . active transglutaminases were suggested to cross - link extracellular matrix proteins and to result in an increased rigidity of vascular walls , which precedes inward remodeling of arteries [ 4750 ] . in line with this idea , patients suffering from celiac disease , which typically form autoantibodies to tgm2 , are less likely to have a diagnosis of hypertension . it is not known if tgm2 plays a similar role in the pathogenesis of cav . we performed animal experiments to investigate this hypothesis and chronically applied biologically meaningful concentrations of cystamine , an inhibitor of transglutaminase activity , for 4 weeks after transplantation . however , our hypothesis was not supported . within 84 days after surgery , allografts developed first hallmarks of cai , such as ifta , cav , and renal dysfunction , irrespective of cystamine treatment . we can , however , not exclude that extracellular transglutaminase activity is involved in both remodeling and re - remodeling of graft arteries as a protective effect of tgm2 was reported for experimental atherosclerosis [ 23 , 53 , 54 ] . the experimental rat model is characterized by a minor mismatch in the mhc class-1 locus and does not include immunosuppression , which is not typical for clinical transplantation . in spite of these disadvantages , it is a favorable , experimental model to investigate an acute rejection episode preceding cai . we investigate neither transglutaminase activity in the graft nor excretion of its products , and animal experiments were performed using a single dose of transglutaminase inhibitor , which was applied for just one month . we ignore if treatment with other concentrations of cystamine or later time - points would lead to allograft protection . to evaluate the effect of cystamine , we focus on renal function and graft remodeling within 84 days after transplantation . we do not know if , after longer periods of time , differences would have developed among the experimental groups . in addition , cystamine is a weak and rather unspecific inhibitor of tgm2 as it also inhibits other members of the transglutaminase family as well as certain proteases . more specific inhibitors for tgm2 are commercially available . finally , apart from its role in apoptosis , there is still a plethora of potential functions of tgm2 [ 11 , 15 ] , some of which are enumerated in the introduction , that are awaiting investigation in the context of acute and chronic allograft rejection . only a part of these functions are inhibited by exogenous cystamine . in conclusion , this is the first study to demonstrate expression of tgm2 by monocytes activated in vivo during a reversible acute rejection episode . potentially , the function of monocytic tgm2 is induction of apoptosis , which in turn might contribute to the reversion of acute rejection in this experimental model . our data do not support but also do not falsify the hypothesis that tgm2 expressed by graft monocytes during reversible acute rejection contributes to an early induction of cai .

acute rejection is a major risk factor for chronic allograft injury ( cai ) . blood leukocytes interacting with allograft endothelial cells during acute rejection were suggested to contribute to the still enigmatic pathogenesis of cai . we hypothesize that tissue transglutaminase ( tgm2 ) , a multifunctional protein and established marker of m2 macrophages , is involved in acute and chronic graft rejection . we focus on leukocytes accumulating in blood vessels of rat renal allografts ( fischer-344 to lewis ) , an established model for reversible acute rejection and cai . monocytes in graft blood vessels overexpress tgm2 when acute rejection peaks on day 9 after transplantation . concomitantly , caspase-3 is activated , suggesting that tgm2 expression is linked to apoptosis . after resolution of acute rejection on day 42 , leukocytic tgm2 levels are lower and activated caspase-3 does not differ among isografts and allografts . cystamine was applied for 4 weeks after transplantation to inhibit extracellular transglutaminase activity , which did , however , not reduce cai in the long run . in conclusion , this is the first report on tgm2 expression by monocytes in vivo . tgm2 may be involved in leukocytic apoptosis and thus in reversion of acute rejection . however , our data do not support a role of extracellular transglutaminase activity as a factor triggering cai during self - limiting acute rejection .

pubmed

endothelial cells ( huvec passage 46 ; clonetics , walkersville , md ) were grown to confluence , then stimulated with plasminogen activation inhibitor-1 ( pai-1 , 10 ng / ml ; american diagnostica inc . , supernatant was collected 3 hours after stimulation and centrifuged ( 145 g , 8 minutes ) to remove cell debris . the supernatant was subsequently ultracentrifuged ( 100,000 g , 6 minutes , 4 degrees ) and pelleted . emps were re - suspended in phosphate buffered saline ( pbs ) and stored at 80c . flow cytometric analysis was used to characterize and quantify emps based on cd31- and annexin v - positivity occurring in a size gate less than 1 m . this size gate was set on forward versus side scatter using latex standard beads measuring 0.84 m ( spherotech fp-0856 - 2 ) . enumeration was completed against a known concentration of 7.6 m polystyrene beads placed within the sample ( spherotech pps-4 ) . the flow rate used for analysis was 12 l / min ( figs . 1 and 2 ) . 7.6 m latex beads are shown in gate p1 and allowed for enumeration of emps . within gate p2 , events positive for annexin v ( p3 ) and cd31 ( p4 ) were enumerated against p1 to quantify emp levels . the same settings were used to quantify emps in perfusate samples . as a separate and confirmatory measure , transmission electron microscopy ( tem ) . a 25-l drop of emp in solution was pipetted onto a formvar plastic coated grid ( ted pella , inc . ) and allowed to air dry for 1 hour . the microparticle grid was examined and photographed by tem with a jeol 100 cx microscope ( fig . 3 ) . emp size distribution as measured by tem ( 10,000magnification ) accompanied by a representative micrograph . emps ( 30010 ) were introduced to an iv bag ( model patient ) containing 300 ml of 0.9 ns ( model patient ) and manually agitated for 3 minutes . the iv bag was connected to a primed cvvh circuit containing 170 ml of 0.9 ns and mixed ( ~65010 emp / ml final mixed perfusate concentration ) as shown in fig . , the perfusate was circulated through the cvvh filter ( nxstage purema , 200 m pore size , 35 m wall thickness , rate 250 ml / hour ) for a period of 70 minutes . 0.5 ml aliquots were removed for flow cytometric analysis at 510 minute intervals for 70 minutes of dialysis . emp levels were recorded and expressed as a fraction of starting concentration after mixing occurred . three runs were completed and the fraction of emps versus time was plotted with standard error of the mean ( sem ) . at the end of 4 hours filtration , . a 300 ml iv bag of 0.9 ns served as the model patient and was spiked with emps . the circuit contained 170 ml 0.9 ns and ran counter - current against 1 l of 0.9 ns dialysate . after mixing , 0.5 ml aliquots were drawn from the patient for analysis as dialysis was completed via the filter . endothelial cells ( huvec passage 46 ; clonetics , walkersville , md ) were grown to confluence , then stimulated with plasminogen activation inhibitor-1 ( pai-1 , 10 ng / ml ; american diagnostica inc . , supernatant was collected 3 hours after stimulation and centrifuged ( 145 g , 8 minutes ) to remove cell debris . the supernatant was subsequently ultracentrifuged ( 100,000 g , 6 minutes , 4 degrees ) and pelleted . emps were re - suspended in phosphate buffered saline ( pbs ) and stored at 80c . flow cytometric analysis was used to characterize and quantify emps based on cd31- and annexin v - positivity occurring in a size gate less than 1 m . this size gate was set on forward versus side scatter using latex standard beads measuring 0.84 m ( spherotech fp-0856 - 2 ) . enumeration was completed against a known concentration of 7.6 m polystyrene beads placed within the sample ( spherotech pps-4 ) . the flow rate used for analysis was 12 l / min ( figs . 1 and 2 ) . 7.6 m latex beads are shown in gate p1 and allowed for enumeration of emps . within gate p2 , events positive for annexin v ( p3 ) and cd31 ( p4 ) were enumerated against p1 to quantify emp levels . the same settings were used to quantify emps in perfusate samples . as a separate and confirmatory measure , transmission electron microscopy ( tem ) . a 25-l drop of emp in solution was pipetted onto a formvar plastic coated grid ( ted pella , inc . ) and allowed to air dry for 1 hour . the microparticle grid was examined and photographed by tem with a jeol 100 cx microscope ( fig . 3 ) . emp size distribution as measured by tem ( 10,000magnification ) accompanied by a representative micrograph . emps ( 30010 ) were introduced to an iv bag ( model patient ) containing 300 ml of 0.9 ns ( model patient ) and manually agitated for 3 minutes . the iv bag was connected to a primed cvvh circuit containing 170 ml of 0.9 ns and mixed ( ~65010 emp / ml final mixed perfusate concentration ) as shown in fig . , the perfusate was circulated through the cvvh filter ( nxstage purema , 200 m pore size , 35 m wall thickness , rate 250 ml / hour ) for a period of 70 minutes . 0.5 ml aliquots were removed for flow cytometric analysis at 510 minute intervals for 70 minutes of dialysis . emp levels were recorded and expressed as a fraction of starting concentration after mixing occurred . three runs were completed and the fraction of emps versus time was plotted with standard error of the mean ( sem ) . at the end of 4 hours filtration , a 300 ml iv bag of 0.9 ns served as the model patient and was spiked with emps . the circuit contained 170 ml 0.9 ns and ran counter - current against 1 l of 0.9 ns dialysate . after mixing , 0.5 ml aliquots were drawn from the patient for analysis as dialysis was completed via the filter . the decline in emp levels due to dialysis is rapid with 50% of clearance being seen in the first 30 minutes . within 40 minutes , the circulating emp levels were reduced to 29% of starting concentration and this level stayed relatively constant over the duration of the study . to determine the fate of the cleared emps , we measured emp levels in the dialysate . the dialysate contained 5.7% of the original emp concentration ( ~65010 emp / ml ) at the end of 4 hours of uninterrupted dialysis . emps levels expressed as a fraction of initial concentration by time and averaged over 3 experiments . the data presented here demonstrate that counter - current dialysis using a standard clinical cvvh filter and under clinical conditions removes emps from a modelled patient 's circulation . given our previous evidence of emp - induced ali , these findings support the idea of cvvh as a meaningful therapeutic intervention in ali due to clearance of emps from the patient circulation . the experimental conditions achieve the same plasma level of emps as seen in human clinical disease ( 110 per ml ) . while this work demonstrates that a standard 200-m cvvh circuit and filter entraps emps , there are significant limitations to the model presented . the observed emp clearance is likely a saturable phenomenon , which would explain the relatively constant emp concentration from 40 to 70 minutes and low dialysate emp levels . the relative volume of human circulation ( ~5 l ) to cvvh circuit ( ~200 ml ) is much higher in the clinical condition as compared to our model ( 300 and 170 ml , respectively ) . while this would not affect a steady state of dialysis , a saturable filter emp - binding additionally , emps may continue to be generated in a patient and a non - saturable clearance method would be required to achieve a lower steady state emp concentration . convection and pressure gradient forces were not modelled , as these interventions require replacement fluid and this would confound emp measurement . however , if emp charge and size are amenable , forced hindered diffusion via the application of convective and pressure forces coupled with replacement by emp - free fluid would likely sustain clearance . in conjunction with particle trapping by the filter , the first order kinetics of emp clearance could be prolonged at a minimum . additionally , emp interactions in vivo with plasma oncotic elements ( circulating cells , albumin , etc . ) we do not feel that such elements have a major impact upon the steady state of emps in vivo , since characterized emps in human disease states using flow cytometry have identified the 0.510 per ml threshold injury value using flow cytometry of submicron events occurring in platelet - free plasma ( 54 ) . thus , the absence of these elements in our model is representative of the steady state of unbound plasma emps present in human disease . the low dialysate emp level and plateauing circulation emp level beyond 40 minutes suggest that emp sequestration occurs within the cvvh filter or tubing and that this is a saturable phenomenon . frequent changing of the cvvh filter is a logical step that may overcome this limitation . additionally , more efficient and selective emps clearance can be realized by conjugating endothelium - specific antibodies to commercially available specialty cvvh filters ( aethlon adapt ) . this approach would provide more selective binding sites and higher affinity for circulating emps . by combining a selective filter with convection and pressure gradients , emp clearance could likely be substantially prolonged . clinical data generally support the protective role of cvvh in ali management . a clinical randomized trial and several animal trials demonstrate that the achievement of negative fluid balance is associated with improved outcomes from ali ( 6265)(67,68 ) . in addition to its salutary effects on emp levels , early cvvh may prevent or minimize volume overload that is commonly observed with the resuscitation phase of critical illness , particularly in persons suffering concomitant acute kidney injury ( aki ) . thus , cvvh could be used as an effective tool targeting both the molecular machinery and iatrogenic components of ali . as a translatable next step , we plan to assay patients pre- and post - dialysis to observe emp concentration effects . ultimately , the application of dialysis to rodent models of emp - induced ali would examine efficacy in on - going diseases and emp thresholds associated with worse injuries . while the utility of ultrafiltration can be tested in an animal model , diffusion appears to be enough to remove emps due to their small size : standard dialysis alone was effective in this in vitro model . the authors have not received any funding or benefits from industry or elsewhere to conduct this study .

backgroundendothelium - derived microparticles ( emps ) are submicron vesicles released from the plasma membrane of endothelial cells in response to injury , apoptosis or activation . we have previously demonstrated emp - induced acute lung injury ( ali ) in animal models and endothelial barrier dysfunction in vitro . current treatment options for ali are limited and consist of supportive therapies . we hypothesize that standard clinical continuous venovenous hemofiltration ( cvvh ) reduces serum emp levels and may be adapted as a potential therapeutic intervention.materials and methodsemps were generated from plasminogen activation inhibitor-1 ( pai-1)-stimulated human umbilical vein endothelial cells ( huvecs ) . flow cytometric analysis was used to characterize emps as cd31- and annexin v - positive events in a submicron size gate . enumeration was completed against a known concentration of latex beads . ultimately , a concentration of ~650,000 emp / ml perfusate fluid ( total 470 ml ) was circulated through a standard cvvh filter ( pore size 200 m , flow rate 250 ml / hr ) for a period of 70 minutes . 0.5 ml aliquots were removed at 5- to 10-minute intervals for flow cytometric analysis . emp concentration in the dialysate was measured at the end of 4 hours to better understand the fate of emps.resultsa progressive decrease in circulating emp concentration was noted using standard cvvh at 250 ml / hr ( a clinical standard rate ) from a 470 ml volume modelling a patient 's circulation . a 50% reduction was noted within the first 30 minutes . emps entering the dialysate after 4 hours were 5.7% of the emp original concentration.conclusionthese data demonstrate that standard cvvh can remove emps from circulation in a circuit modelling a patient . an animal model of hemofiltration with induction of emp release is required to test the therapeutic potential of this finding and potential of application in early treatment of ali .

pubmed

patients with frequent angina ( 1 episode per week ) report greater physical limitations and decreased quality of life compared with patients with minimal angina.1 the goals of chronic stable angina management are not only to control symptoms , but also to reduce the risk of adverse clinical outcomes.26 as symptom control impacts the patient s quality of life , maximum consideration should be given to the array of available management options that aim to reduce angina symptoms . for many patients , this would include a physical activity program aimed at improving exercise tolerance in accordance with current secondary prevention guidelines . several studies have shown that optimal medical therapy , along with aggressive risk factor modification , is as effective as revascularization in reducing the risk of adverse coronary events.79 optimal medical therapy includes anti - anginal or anti - ischemic as well as disease- modifying therapy.8,10 the anti - ischemic agents include short- and long - acting nitrates , beta - blockers , and calcium channel blockers.3,4,11 these agents reduce angina symptoms and prolong exercise duration and/ or time to st - segment depression.3,11 frequently , a combination of drugs is necessary for optimal symptom control.3 nitrates work primarily by venodilation and afford rapid relief of angina.12 a number of placebo - controlled studies have shown that the sublingual application of nitroglycerin increases exercise duration in patients with stable angina.1317 published data also suggest that residual and recurrent angina is frequently experienced by patients who have already undergone either a percutaneous or surgical revascularization procedure , and these patients require continued medical therapy with multiple anti - anginal agents , including short- and long - acting nitrates , for control of their symptoms.8,18,19 increased physical exercise is also important in patients with stable angina to reduce the risk of cardiovascular events , and current recommendations call for the use of sublingual nitroglycerin as part of optimal medical therapy.5,20 this randomized , double - blind , placebo - controlled , crossover , dose - ranging multicenter study in patients with chronic stable angina evaluated the effect of a sublingual nitroglycerin spray formulation on time to onset of angina during exercise , total exercise duration , and time to exercise - induced myocardial ischemia , as evidenced by electrocardiographic st - segment depression . brief results were previously published in german.21 the goal of the present article is to more fully describe the study methodology , patient characteristics , and detailed results in the context of current optimal medical therapy . this study was carried out at four centers in the united states and conducted in accordance with the international conference on harmonisation guidelines for good clinical practice and food and drug administration guidelines . all patients provided written informed consent at the screening visit , prior to starting any study - related procedures . adults ( 18 years of age ) with coronary artery disease as documented by a 50% reduction in arterial lumen diameter in 1 or more major coronary arteries or their primary branches , a previously documented myocardial infarction , or reversible perfusion defect on stress thallium-201 scintigraphy and a diagnosis of chronic stable exertional angina that had been made at least 2 months prior to screening were eligible to be screened . in the 2 months prior to study entry , patients were required to have been on a stable medical treatment regimen for their angina . patients with unstable angina , myocardial infarction , coronary angioplasty , or cardiac surgery within 3 months of screening were excluded . additional exclusion criteria included a history of congestive heart failure ( nyha iii or iv ) , significant disease of the cardiac conduction system , uncontrolled hypertension or hypotension , any physical conditions known to affect st - segment morphology , or severe organ damage or disease of any system . patients were not permitted to receive concomitant therapy with long - acting nitrates or digitalis preparations during the study . in addition , participation in an investigational drug study within 1 month prior to the screening visit or while participating in the study was prohibited . patients with clinically significant deviations from normal in either the physical exam or lab parameters and patients with peripheral arterial disease that limited their exercise capacity were also excluded . according to the approved protocol , each center was to enroll a minimum of 12 patients to ensure evaluable outcomes for 40 patients at the end of study . following an initial screening visit , eligible patients returned for a baseline exercise tolerance test ( ettb ) according to a standard bruce protocol.22 of note , exercise testing is associated with minimal complications ; death and myocardial infarction occur at a rate of 1 per 2,500 tests performed.15,23 patients who developed angina within 39 minutes of commencing exercise were eligible to continue in the study . following a 90-minute rest period , these patients were assessed for nitrate response . patients who were able to exercise for 60 seconds longer in this ett ( ettres ) than in the ettb were eligible to continue in the study . at their next visit , patients completed a confirmatory ett ( ettconf ) to establish reproducibility ( within 20% ) of the time to development of moderate angina when compared to the ettb . in order to reduce baseline variability , study - related sublingual nitroglycerin was discontinued 6 hours prior to testing , while other anti - anginal medications were discontinued approximately 24 hours prior to each visit . after baseline and confirmatory etts the goal of this crossover study design with each patient serving as his / her own control was to minimize the impact of any training effect on the mean values for each treatment dose . the crossover period consisted of 5 additional visits within 14 days , at which patients were administered placebo or sublingual nitroglycerin spray ( 0.2 mg , 0.4 mg , 0.8 mg , or 1.6 mg ; nitrolingual pumpspray , g. pohl - boskamp gmbh & co. kg , germany ) . in order to minimize the impact of any training effect , patients completed a control ett ( ettc ) and an investigational ett ( ettinv ) at each visit . ettc studies were carried out at approximately the same time of day as the introductory ett studies ( ettb and ettconf ) . following a 90-minute rest period , primary efficacy endpoints were time to onset of angina symptoms and time to development of moderate angina ( defined as the level of activity at which the patient would normally stop activity ) . the secondary endpoint was time to 1-mm st - segment depression ( myocardial ischemia ) . to minimize the impact of any training effect , efficacy endpoints are described as the mean difference between ettinv and ettc at each visit . the difference between the confirmation and baseline etts was then subtracted from this value to further reduce the impact of any training effect . safety parameters included evaluation of all changes in physical examination , vital signs , ecg , and laboratory evaluations . this study was carried out at four centers in the united states and conducted in accordance with the international conference on harmonisation guidelines for good clinical practice and food and drug administration guidelines . all patients provided written informed consent at the screening visit , prior to starting any study - related procedures . adults ( 18 years of age ) with coronary artery disease as documented by a 50% reduction in arterial lumen diameter in 1 or more major coronary arteries or their primary branches , a previously documented myocardial infarction , or reversible perfusion defect on stress thallium-201 scintigraphy and a diagnosis of chronic stable exertional angina that had been made at least 2 months prior to screening were eligible to be screened . in the 2 months prior to study entry , patients were required to have been on a stable medical treatment regimen for their angina . patients with unstable angina , myocardial infarction , coronary angioplasty , or cardiac surgery within 3 months of screening were excluded . additional exclusion criteria included a history of congestive heart failure ( nyha iii or iv ) , significant disease of the cardiac conduction system , uncontrolled hypertension or hypotension , any physical conditions known to affect st - segment morphology , or severe organ damage or disease of any system . patients were not permitted to receive concomitant therapy with long - acting nitrates or digitalis preparations during the study . in addition , participation in an investigational drug study within 1 month prior to the screening visit or while participating in the study was prohibited . patients with clinically significant deviations from normal in either the physical exam or lab parameters and patients with peripheral arterial disease that limited their exercise capacity were also excluded . according to the approved protocol , each center was to enroll a minimum of 12 patients to ensure evaluable outcomes for 40 patients at the end of study . following an initial screening visit , eligible patients returned for a baseline exercise tolerance test ( ettb ) according to a standard bruce protocol.22 of note , exercise testing is associated with minimal complications ; death and myocardial infarction occur at a rate of 1 per 2,500 tests performed.15,23 patients who developed angina within 39 minutes of commencing exercise were eligible to continue in the study . following a 90-minute rest period , these patients were assessed for nitrate response . patients who were able to exercise for 60 seconds longer in this ett ( ettres ) than in the ettb were eligible to continue in the study . at their next visit , patients completed a confirmatory ett ( ettconf ) to establish reproducibility ( within 20% ) of the time to development of moderate angina when compared to the ettb . in order to reduce baseline variability , study - related sublingual nitroglycerin was discontinued 6 hours prior to testing , while other anti - anginal medications were discontinued approximately 24 hours prior to each visit . after baseline and confirmatory etts the goal of this crossover study design with each patient serving as his / her own control was to minimize the impact of any training effect on the mean values for each treatment dose . the crossover period consisted of 5 additional visits within 14 days , at which patients were administered placebo or sublingual nitroglycerin spray ( 0.2 mg , 0.4 mg , 0.8 mg , or 1.6 mg ; nitrolingual pumpspray , g. pohl - boskamp gmbh & co. kg , germany ) . in order to minimize the impact of any training effect , patients completed a control ett ( ettc ) and an investigational ett ( ettinv ) at each visit . ettc studies were carried out at approximately the same time of day as the introductory ett studies ( ettb and ettconf ) . following a 90-minute rest period , primary efficacy endpoints were time to onset of angina symptoms and time to development of moderate angina ( defined as the level of activity at which the patient would normally stop activity ) . the secondary endpoint was time to 1-mm st - segment depression ( myocardial ischemia ) . to minimize the impact of any training effect , efficacy endpoints are described as the mean difference between ettinv and ettc at each visit . the difference between the confirmation and baseline etts was then subtracted from this value to further reduce the impact of any training effect . safety parameters included evaluation of all changes in physical examination , vital signs , ecg , and laboratory evaluations . of the 61 patients assessed for eligibility , 10 patients did not meet the protocol criteria and were therefore excluded . a total of 51 patients performed the baseline exercise test and qualified for continuation . following a 90-minute rest period , patients underwent a subsequent stress test after receiving 0.4 mg of open - label sublingual nitroglycerin spray . all 51 patients showed an improvement in exercise duration of 60 seconds or greater ( range 1.024.7 minutes ) . a total of 51 patients between the ages of 34 and 77 years continued in the double - blind phase and were randomized to 1 of 5 treatment sequences . two patients did not complete the study ( 1 patient was lost to follow - up and 1 patient discontinued prematurely due to ventricular tachycardia / fibrillation requiring cardioversion after the control exercise test at visit 5 ; this event was not considered to be treatment related ) . overall patient characteristics including angina and cardiovascular event history , along with concomitant medications , are described in table 1 . a dose - dependent increase in time to onset of moderate angina the time to onset of angina was significantly higher in all treatment groups ( 0.2 mg , 0.4 mg , 0.8 mg , and 1.6 mg ) than in the placebo group ( table 2 , fig . the increase in time to onset of angina and to onset of moderate angina was most pronounced in the 1.6-mg group , with dose linearity between 0.4- and 1.6-mg doses of nitroglycerin spray . the occurrence of a minimum 1.0-mm st - segment depression was later in all treatment groups ( 0.2 mg , 0.4 mg , 0.8 mg , 1.6 mg ) compared with the placebo group ( table 2 , fig . of 51 patients , 12 ( 24% ) reported adverse events at the qualification visit ( open - label sublingual nitroglycerin response test ) . adverse events considered related to treatment were reported for 2 ( 4% ) , 4 ( 8% ) , 8 ( 16% ) , 9 ( 18% ) , and 13 ( 26% ) patients receiving placebo and 0.2 mg , 0.4 mg , 0.8 mg , and 1.6 mg nitroglycerin spray , respectively . the most frequently reported adverse events of this nature were headache and dizziness , all of which were mild to moderate in severity . the incidence of treatment - related adverse events was dose dependent , with the incidence of headache increasing from 0 with placebo to 3 ( 6% ) , 5 ( 10% ) , 6 ( 12% ) , and 8 ( 16% ) after 0.2 mg , 0.4 mg , 0.8 mg , and 1.6 mg nitroglycerin spray , respectively . no serious adverse events related to treatment were reported . per protocol , patients with systolic blood pressure ( sbp ) less than 100 mmhg were excluded from the study . symptomatic hypotension was not reported as an adverse event , however sbp decreased to a level of 100 mmhg or less in 1 ( 2% ) , 0 ( 0% ) , 1 ( 2% ) , 0 ( 0% ) , and 3 ( 5% ) patients after receiving placebo and 0.2 mg , 0.4 mg , 0.8 mg , and 1.6 mg nitroglycerin spray in the double - blind phase . of the 61 patients assessed for eligibility , 10 patients did not meet the protocol criteria and were therefore excluded . a total of 51 patients performed the baseline exercise test and qualified for continuation . following a 90-minute rest period , patients underwent a subsequent stress test after receiving 0.4 mg of open - label sublingual nitroglycerin spray . all 51 patients showed an improvement in exercise duration of 60 seconds or greater ( range 1.024.7 minutes ) . a total of 51 patients between the ages of 34 and 77 years continued in the double - blind phase and were randomized to 1 of 5 treatment sequences . two patients did not complete the study ( 1 patient was lost to follow - up and 1 patient discontinued prematurely due to ventricular tachycardia / fibrillation requiring cardioversion after the control exercise test at visit 5 ; this event was not considered to be treatment related ) . overall patient characteristics including angina and cardiovascular event history , along with concomitant medications , are described in table 1 . a dose - dependent increase in time to onset of moderate angina was seen following administration of nitroglycerin spray ( table 2 , fig . the time to onset of angina was significantly higher in all treatment groups ( 0.2 mg , 0.4 mg , 0.8 mg , and 1.6 mg ) than in the placebo group ( table 2 , fig . the increase in time to onset of angina and to onset of moderate angina was most pronounced in the 1.6-mg group , with dose linearity between 0.4- and 1.6-mg doses of nitroglycerin spray . the occurrence of a minimum 1.0-mm st - segment depression was later in all treatment groups ( 0.2 mg , 0.4 mg , 0.8 mg , 1.6 mg ) compared with the placebo group ( table 2 , fig . overall , nitroglycerin spray was generally well tolerated . of 51 patients , 12 ( 24% ) reported adverse events at the qualification visit ( open - label sublingual nitroglycerin response test ) . adverse events considered related to treatment were reported for 2 ( 4% ) , 4 ( 8% ) , 8 ( 16% ) , 9 ( 18% ) , and 13 ( 26% ) patients receiving placebo and 0.2 mg , 0.4 mg , 0.8 mg , and 1.6 mg nitroglycerin spray , respectively . the most frequently reported adverse events of this nature were headache and dizziness , all of which were mild to moderate in severity . the incidence of treatment - related adverse events was dose dependent , with the incidence of headache increasing from 0 with placebo to 3 ( 6% ) , 5 ( 10% ) , 6 ( 12% ) , and 8 ( 16% ) after 0.2 mg , 0.4 mg , 0.8 mg , and 1.6 mg nitroglycerin spray , respectively . no serious adverse events related to treatment were reported . per protocol , patients with systolic blood pressure ( sbp ) less than 100 mmhg were excluded from the study . symptomatic hypotension was not reported as an adverse event , however sbp decreased to a level of 100 mmhg or less in 1 ( 2% ) , 0 ( 0% ) , 1 ( 2% ) , 0 ( 0% ) , and 3 ( 5% ) patients after receiving placebo and 0.2 mg , 0.4 mg , 0.8 mg , and 1.6 mg nitroglycerin spray in the double - blind phase . lifestyle modifications , including supervised exercise therapy , play an important role in the success of optimal medical therapy.2427 however , patients with stable angina generally have impaired exercise tolerance and reduced daily physical activity.28 sublingual nitroglycerin increases physical exercise tolerance which , in turn , helps to increase the amount of exertion possible prior to the onset of angina.12,13 sublingual forms of nitroglycerin are readily absorbed through mucous membranes , and their effect is prompt , reliable , and more effective than other forms of nitroglycerin , including ointments , transdermal patches , and sustained - release preparations.11,29 the results of this study are in line with prior findings and demonstrate a significant improvement in exercise tolerance with sublingual nitroglycerin spray in patients with stable angina . although sublingual nitroglycerin spray was generally well tolerated , an increase of treatment - related adverse events was observed with higher dose levels ( 26% of patients with 1.6 mg nitroglycerin spray ) . one important issue that is not specifically addressed in this study is the clinical relevance of the nearly 1-minute improvement in exercise duration with sublingual nitroglycerin spray over placebo . it must be recognized that this improvement was experienced by patients who were exercising at a higher work load ( higher speed and incline during the treadmill test ) after receiving the study medication . as most real - world patients have a much slower walking pace , this finding indicates that prophylactic nitroglycerin spray might allow much greater increases in walking times during normal daily activities . although this outcome was not formally evaluated in the present study or discussed in previous publications to our knowledge , patients have often reported a marked increase in angina - free walking time and exercise duration after taking prophylactic sublingual nitroglycerin . since this study of sublingual nitroglycerin spray was conducted , there have been significant changes in optimal medical therapy for stable coronary artery disease . this is particularly apparent in the greater use of beta - blockers compared with calcium channel blockers and long - acting nitrates to treat angina , and the routine use of daily aspirin and a statin to reduce serious cardiovascular outcomes.3,8 although beta - blockers are currently considered a standard treatment for stable angina , patients receiving these medications may continue to experience angina.30 however , patients with a history of beta - blocker use who are appropriately supplemented with sublingual nitroglycerin or long - acting nitrates show significant improvements in angina - free exercise duration and total walking time compared with placebo.3,5,29 the american college of cardiology and american heart association guidelines for the management of patients with chronic stable angina emphasize the initial use of medical therapy.5,20,31 these guidelines include the recommendation for all patients to undergo a risk assessment with a physical activity history and/or an exercise test to guide prognosis and prescription . ett is associated with a very low rate of serious adverse events such as death ( 0.5 per 10,000 tests ) , myocardial infarction ( 3.58 per 10,000 tests ) , or serious arrhythmias ( 4.78 per 10,000 tests).15 guideline - compliant medical therapy improves clinical outcome in many patients with stable angina , as evidenced by an observational study in which increasingly guideline - compliant therapy was associated with a reduction in death and myocardial infarction in patients with stable angina during 1 year of follow - up.32 indeed , implementation of a quality improvement program in utah to enhance the prescription of appropriate discharge medications among patients with cardiovascular disease was associated with improvements in cardiovascular readmission rates and reductions in mortality.33 a number of studies have compared the effect of optimal medical therapy versus revascularization on symptoms and outcomes in patients with coronary artery disease and stable angina . in general , these studies have shown that optimal medical therapy is effective in controlling symptoms and , along with risk factor modification , can reduce the risk of adverse cardiac events.8,34 moreover , these studies have shown that there is no significant difference in serious adverse clinical events , such as myocardial infarction and death , between patients who have received medical therapy and those who have undergone revascularization.2,5,8 the clinical outcomes utilizing revascularization and aggressive drug evaluation ( courage ) trial , which compared the efficacy of percutaneous coronary intervention ( pci ) plus optimal medical therapy versus optimal medical therapy alone , demonstrated similar rates of death and nonfatal myocardial infarction in both patient groups over a follow - up period of 55 months.8 moreover , both groups demonstrated a substantial reduction in angina symptoms , with 74% and 72% of patients in the pci and optimal medical therapy groups , respectively , angina - free after 5 years of follow - up.8 further analysis of data from the courage study found that both groups of patients had marked improvements in health status , including reductions in angina symptoms , and improved quality of life . although the pci group demonstrated significantly greater health benefits in the short term , these benefits disappeared after longer - term follow - up , and by 36 months there was no significant difference in health status between the 2 groups.9 the bypass angioplasty revascularization investigation 2 diabetes trial in patients with diabetes , coronary artery disease , and classic angina compared the effects of revascularization with medical therapy alone on clinical outcomes . during the 5-year follow - up , no difference was found between the medical therapy and revascularization groups on the risk of all - cause death , myocardial infarction , or stroke.34 furthermore , a meta - analysis of 11 randomized trials comparing pci with conservative treatment in patients with stable coronary artery disease found no significant difference between the 2 treatment strategies with regard to overall mortality , cardiac death , or myocardial infarction.7 current recommendations state that patients with stable angina may defer surgical intervention without additional risk to allow for determination of response to optimal medical therapy.5,11 despite the published outcome data and current guideline recommendations , many patients are still referred for revascularization , mainly due to a belief that these procedures reduce the risk of myocardial infarction or death more effectively than optimal medical therapy alone.2,3,8,35 additional studies assessing the long - term benefits of exercise in the context of medical therapy compared with revascularization outcomes could help to further inform the clinical decision making process . in this study , sublingual nitroglycerin spray was shown to increase time to onset of moderate angina during exercise . this improvement in physical exercise tolerance may be of great clinical relevance , as the prophylactic use of sublingual nitroglycerin spray allows patients to face exertion with confidence and permits exercise for a longer amount of time . these results support the use of sublingual nitroglycerin spray as nitrate therapy in patients with stable angina who are being managed with optimal medical therapy . this approach may also be beneficial for patients who have persistent angina post - revascularization . further studies are needed to evaluate the long - term beneficial effects of using routine prophylactic nitroglycerin spray in patients with stable angina to increase exercise duration and improve quality of life and well - being .

backgroundsublingual nitroglycerin increases exercise duration in patients with stable angina . brief results from this study were published previously in german . here , we more fully describe the study methodology , patient characteristics , and detailed results.methodsthis double - blind , crossover study enrolled 51 patients with stable angina . patients were randomized to 1 of 5 treatment sequences and were administered placebo or nitroglycerin spray ( 0.2 mg , 0.4 mg , 0.8 mg , or 1.6 mg ) . patients carried out 1 control exercise tolerance test ( ett ) and 1 investigational ett at each visit.resultsdose-dependent increases in time to onset of angina , time to onset of moderate angina , and the occurrence of a minimum 1.0-mm st - segment depression were seen following administration of nitroglycerin spray.conclusionsthese results support the use of sublingual nitroglycerin spray in patients with stable angina who are being managed with medical therapy and in patients who have persistent angina post - revascularization .

pubmed

the residual and resistant microorganisms have been recognized as the basic reason for failures in root canal treatments . fungi , chemoorganotrophic eukaryotic microorganisms , may play a key part in periradicular diseases . while they have been found sporadically in initial root canal infections , failure of some endodontic treatments may therefore be explained by the presence of candida albicans in the oral flora . it is reported that in 20% of endodontic treatment failures , c. albicans is present . candida species , which have a tendency to invade dentin , are regarded as dentinophilic microorganisms . the presence of c. albicans in endodontic treatment failures may be the result of coronal leakage after , or contamination during , the endodontic treatment . several factors , such as an ability to survive despite a lack of nutrients or resistance to intracanalicular treatment materials , may result in c. albicans remaining in the root canal system . c. albicans has been found in some failed endodontic treatments ; a phenomenon that might be explained by the invasive nature of this fungus or its resistance to some intracanalicular medications , e.g. , calcium hydroxide . if repeated treatment has either failed or is unfeasible , the only alternative is a root - end resection . apical sealing , avoiding contamination , and reducing the number of microorganisms , including bacteria and fungi , influence the success rate of such treatment . therefore , the antibacterial and antifungal properties of root - end filling materials are important to the outcome of these procedures . mineral trioxide aggregate ( mta ) was introduced in 1993 and , after different examinations , approved , in 1998 , by the us food and drug administration as an endodontic biomaterial , used in root - end filling , vital pulp capping , apexification , and root or furcal perforation repair.[58 ] mta is marketed as gray and white . however , because of the tooth - discoloring properties of gray mta , white mta has been pushed to replace it . mta has a number of advantages , including excellent sealing ability , biocompatibility , high alkalinity , radiopacity , antimicrobial effects , and resistance to fluid and blood contamination.[911 ] in spite of having disadvantages , such as delayed setting time , poor handling properties , and high price , mta , because of its physical and chemical properties , has become the gold standard of root - end filling materials . mta - angelus particles are not as homogenous as proroot mta . in comparison to proroot mta , mta - angelus shows several advantages , such as good marginal adaptation , good sealing , sealing ability in mineralized tissues with complete closure , and has been reported to be inflammation - free in most cases.[1517 ] mta - angelus has an excellent antibacterial effect and a strong fungicidal effect against c. albicans . while both the white and gray mta - angelus have arsenic in their structures , white mta - angelus has the benefit of containing arsenic levels below the limit set by the iso 9917 - 1 standard . a limited number of researches have compared the antifungal effects of proroot mta and mta - angelus . however , such studies have been conducted using agar diffusion test ( adt ) , a method that does not seem appropriate because of low solubility and diffusibility of mta . the aim of this in vitro study was to evaluate and compare the antifungal properties of fresh and set proroot mta and mta - angelus , in concentrations of 50 mg / ml and 100 mg / ml , against c. albicans at 1- , 24- , and 48-hour time points . the null hypothesis of this study was that there is no difference in the in vitro antifungal properties of these two materials . the antifungal activity of proroot mta ( dentsply tulsa dental , tulsa , usa ) as well as mta - angelus ( angelus , londrina , brazil ) was evaluated against c. albicans . the culture of sabouraud dextrose agar ( merck , darmstadt , germany ) was prepared according to the manufacturer 's instructions with a definite ratio of powder to liquid . samples of live c. albicans , ( atcc 10231 ) provided by scientific research organization , ( tehran , iran ) were subcultured on the sabouraud dextrose agar plates . the agar plates , containing the fungus , were then maintained at 37c . times and conditions for the culture of c. albicans in this study were determined following the nccls standards ( national committee on clinical laboratory standards ) . a total of 50 culture wells were divided into four experimental groups ( freshly mixed mta , freshly mixed mta - angelus , 24-h set mta , 24-h set mta - angelus ) and two control groups ( positive control , negative control ) . the root canal filling materials were prepared according to their instructions and then mixed with the appropriate volume of sterile water to reach the concentrations of 50mg / ml or 100mg / ml . for the set groups , the mixture was prepared and left for 24 hours . each group consisted of 10 wells , and the control groups consisted of five wells each . suspensions were then made by the addition of several fungal colonies from the surface of the sabouraud dextrose agar plates to sabouraud dextrose broth media . a final concentration of 10 cfu / ml ( colony forming unit / milliliter ) was achieved , as recommended by the nccls , via microscopic cell counting . wells with 1 ml of c. albicans suspension mixed and 1 ml of sabouraud dextrose broth media without root end - filling materials served as positive control and wells with 1 ml of sabouraud dextrose broth media without c. albicans served as negative control . all wells were incubated at 37c and evaluated for 1 , 24 , and 48 hours by an independent observer by assessing the colonies of c. albicans in each well . this assessment was based on the turbidity of the suspension in the tubes . at each time point , 0.02 ml of each suspension was cultured on sabouraud dextrose agar plate to confirm c. albicans growth . after the culture of 0.02ml of the treated suspension on the agar plates , the fungal growth was assessed at 1- , 24- , and 48-hour time points . while fungal growth was not observed in any of the negative control groups , after 1 hour , both the mta and mta - angelus were incapable of killing c. albicans . the results were the same for both freshly mixed and set samples at both concentrations ( 50 and 100 mg / ml ) . increasing the incubation time to 24 hours and both materials were fungicidal for c. albicans at both concentrations of 50 and 100 mg / ml , and in both states of fresh and set [ table 1 ] . comparison of proroot mta and mta - angelus in concentration of 50 mg / ml and 100 mg / ml at 1 , 24 , and 48-h time intervals there was no significant difference between using the two materials at either of the concentrations or either of the 1-hour , 24-hour , or 48-hour time points ( p<0.05 ) . in this study , the antifungal properties of proroot mta and mta - angelus against c. albicans in two concentrations and in fresh and set situations were compared by using tube - dilution test . because the two materials elicited the same reaction against c. albicans in different conditions , the null hypothesis that there is no difference in the in vitro properties of these two materials has to be accepted . the most common method for assessing the antifungal or antibacterial activity of root - end filling materials however , several parameters , such as lack of standardization of inoculum density , adequate culture medium , agar viscosity , plate storage condition , size and number of specimens per plate , time and temperature of incubation , and reading point of the inhibition zones , can affect the results of the adt . the method used in this in vitro study was the tube - dilution susceptibility test ; an efficient method for evaluating antifungal characteristics of materials . in this technique , direct contact between c. albicans and the experimental materials was allowed , allowing a more realistic interaction . because of possible changes in the chemical structure of the products , root - end filling materials should be tested in two situations ; one where the material is used immediately after mixing , and another , where it is used after having been allowed to reach its final chemical composition , or set state . in this study , proroot mta and mta - angelus were tested in freshly mixed and set states , to mimic clinical use of the materials . after setting , consequently , the perceived difference in antifungal patterns of the materials may be a result of the differences in the degree of setting . however , in our study , the results were similar in the two different states for all tested materials . based on nccls standards , the results of this study showed that c. albicans was not affected by the freshly mixed and set , proroot mta and mta - angelus , at 1 hour . however , these materials were effective in eliminating c. albicans by the 24-h time point . some studies have reported the inhibitory effect of proroot mta and mta - angelus on c. albicans by determining the diameter of the inhibition zones in millimeters , and have resulted conclusions similar to this study . the presence of calcium hydroxide , produced by a hydration reaction from the mixing of mta with water , and an increase in ph , as a result of the release of hydroxyl ions , is probably the mechanism of the antimicrobial activity of mta - angelus . they suggested that the presence of calcium hydroxide is capable of , initially , reducing and , ultimately , eliminating the fungus . it is important to mention that the results presented in this study were obtained under in vitro conditions , and may not accurately describe in vivo activity . further studies are necessary to investigate the effect of these materials on bacteria and fungi in clinical application . this study shows that mta - angelus has an effective antifungal property comparable to proroot mta at concentrations of 50 and 100 mg / ml .

background : fungi may play a key part in periradicular diseases . the aim of this study was to evaluate and compare the antifungal properties of two root - end filling materials , proroot mineral trioxide aggregate ( mta ) and mta - angelus , against candida albicans using tube - dilution test.materials and methods : the antifungal properties of proroot mta and mta - angelus against c. albicans was assessed at 1 , 24 , and 48 hours following administration of two concentrations of the antifungal agents ( 50 and 100 mg / ml ) . a total of 50 culture wells were divided into four experimental groups ( freshly mixed mta , freshly mixed mta - angelus , 24-h set mta , and 24-h set mta - angelus ) and two control groups . each well was prepared for one specific agent with a specific concentration . for the set groups , the mixture was prepared and left for 24 hours . one milliliter of suspension of fungal colonies with concentration of 104 cfu / ml was then added to the mixtures in each well . all wells were incubated at 37c and assessed at 1 , 24 , and 48 hours . this observation was based on the turbidity of the suspension in the tubes . at each time point , 0.02 ml of each suspension was cultured on a sabouraud dextrose agar plate to confirm c. albicans growth . the results were analyzed using kruskal - wallis test.results:although all fresh and set samples were incapable of killing c. albicans at 1 hour , they demonstrated fungicidal ability on agar plates at 24 and 48-hour time points.conclusion:mta-angelus proved to be an effective antifungal agent compared to proroot mta at concentrations of 50 mg / ml and 100 mg / ml .

pubmed

deficits of motor and sensory functions after stroke on the side contralateral to the damaged hemisphere are often evident1 , whereas the ipsilateral side may be primarily regarded as normal or unaffected . however , there is increasing evidence of the presence of subtle motor deficits in motor performance on the ipsilateral side as well2 , 3 . ipsilateral motor deficits emerge during the acute phase and demonstrate chronic persistence3,4,5 ; the reasons for ipsilateral motor deficits are still unclear . clinical assessment tools may not be sufficient for differentiating ipsilateral motor deficits ; however , deficits in dexterous motor and coordination function on the ipsilateral side have been identified in laboratory testing2 , 6,7,8 . however , kinematic deficits in the ipsilateral upper limb in performance of various specific motor tasks requiring dexterity and coordination , such as a tracking task , a goal - direction movement , and a tapping task , have been found in recent studies2 , 6 , 8 , 11 . on the basis of these observations , several possible mechanisms for ipsilateral motor deficits have been suggested , such as disrupted counterbalance of each hemisphere , dysfunction of the uncrossed corticospinal track , or the different roles of both sides in hemispheric functions6 , 7 , 11,12,13,14,15 . as mentioned above , patients with brain damage suffer from ipsilateral motor deficits in performance of the ipsilateral upper limb . until now , most studies of ipsilateral deficits in stroke patients have concentrated on the motor , rather than sensory deficits . therefore , the purpose of the current study was to investigate the presence of motor and sensory deficits in the ipsilateral upper limb , and to examine the correlation between the two variables in patients with stroke using a tracking and reposition sense test . fifty hemiparetic stroke patients ( 25 patients with right brain injury and 25 patients with left brain injury ) referred to a local rehabilitation hospital were consecutively recruited in the order of their registration . the inclusion criteria were ; first ever stroke confirmed by medical history and brain mri ; right handed individual verified by the edinburg handedness inventory ; no symptoms of unilateral neglect or hemianopsia ; no cognitive problem ( mini - mental state examination>24 points ) ; no apraxic behavior ( ideomotor apraxia score developed by ambosoni et al . ( > 11 points)16 ; and no musculoskeletal dysfunction in the unaffected upper limb . we recruited 40 sex- and age - matched normal control subjects . to control the known effects of hand asymmetry , the accuracy and proprioceptive tests were performed by the 20 control subjects using their dominant right hand , and the remaining subjects used their non - dominant left hand . all subjects gave their written informed consent prior to participation , and this study was approved by the local ethics committee . tracking and joint position sense tests were conducted for the hand ipsilateral to the damaged hemisphere of the patients , and with the corresponding hand of the same side of the control subjects . all subjects were seated in front of a table , with the forearm comfortably supported and the elbow flexed at 90. a plastic frame with an embedded potentiometer was used to measure the accuracy of movement and proprioceptive sense in the metacarpophalangeal ( mp ) joints . the potentiometer detected flexion / extension motion of the mp joint , and transmitted the analog signal to a computer with analog - to - digital data acquisition software , that sampled the signal at a frequency of 200 hz . in the tracking task , the subject was instructed to track the red target sine wave displayed for 15 seconds on the computer screen as accurately as possible . the response sine wave made by the subject was displayed as a black solid line , which tracked up as the mp joint was extended , and tracked down as the mp joint was flexed . accuracy of the motor performance was analyzed by an accuracy index ( ai ) , which was normalized to the range of motion of the mp joint of each individual subject , and takes into account the differences between subjects in the excursion of the target and response waves17.ai = 100(p e)/p where e is the root mean square ( rms ) error between the target line and the response line , and p is the size of the subject s target pattern , calculated as the rms difference between the sine wave and the midline dividing the upper and lower phases of the sine wave . the degree of p is determined by the scale of the vertical axis of the range of subject s mp joint motion . prior to the evaluation , three practice trials were provided after one demonstration , using sine waves which were different from the sine waves used in the actual test to prevent a learning effect . the joint position sense was evaluated on the mp joint ipsilateral to the damaged brain hemisphere of the patients , and the joint on the corresponding side of the control subjects . in addition , the same experimental apparatus and environment used for the performance of the tracking task were used . the subjects were instructed to actively reproduce the position of the mp joint which was passively positioned by the examiner . three different passively - positioned angles were randomly presented , in terms of 50% , 70% , and 90% flexion of the total range of motion of the mp joint . the mean value of three trials of the joint reposition errors between the passively - positioned angles and the actively - positioned angles was calculated . the test was performed to analyze the differences in sex distribution between the patient and control groups . the independent t - test was performed to determine the significance of differences in age and accuracy of the tracking task / joint position sense . in addition , correlation between the ai and joint position sense was investigated using pearson s correlation coefficient . chicago , il , usa ) was used for the statistical analysis of all data , and statistical significance was accepted for p values < 0.05 . no significant differences were observed between the two groups in terms of distribution of sex and age . the meanssd of the accuracy index and reposition error score of both groups are shown in table 2 . in terms of motor function , the stroke group showed a lower accuracy index in the mp joint than the control group . a higher score of reposition errors in the joint reposition test in relation to sensory function was observed in the stroke group , compared to the control group . the results of the statistical analysis indicate that both measures in the stroke group were significantly different from the control group ( p<0.05 ) . ipsilateral sensory deficits showed significant correlation with motor deficits ( r= 0.549 ) ( p<0.001 ) ( table 2 ) . in the current study , we attempted to assess motor function using a tracking task for visuomotor coordination , and proprioceptive sense using a joint reposition test for the ipsilateral upper limb . our findings reveal a lower accuracy index in the tracking task , and higher error scores in the joint reposition test in the stroke group , compared to sex- and age - matched normal subjects . in addition , there was a negative correlation between the motor and sensory deficits , indicating stroke patients would have difficulty in performing complicated motor tasks requiring delicate sensoriomotor functions using pure integrity of movement accuracy and proprioceptive sense . our present results are in accordance with those of several previous studies2 , 8 , suggesting the presence of motor deficits in upper limbs ipsilateral to the damaged hemisphere in the visuomotor tracking task . a possible mechanism for the motor dysfunction in the ipsilateral hemisphere of stroke patients has been suggested by previous studies , which reported bilateral hemisphere activation when normal subjects executed a unilateral upper limb task18,19,20,21 . if functional integrity of both the right and left brain cortex is necessary for normal motor control of the upper limb , it is expected that the ipsilateral upper limb would be affected after stroke . in the present study , ipsilateral sensory deficits related to proprioceptive sense were also observed . according to our findings , ipsilateral sensory deficits may be connected with bilateral hemisphere activation during performance of motor tasks . the primary sensory cortex ( s1 ) conveys efferent projection to the posterior parietal cortex ( brodmann s area 5 and 7 ) , which is connected bilaterally through the corpus callsosum . therefore , as suggested by our results , it is possible that disturbance of transcallosal transfer after unilateral brain damage may lead to ipsilateral sensory deficits . in addition , there is a close relationship between sensory and motor function , because the posterior parietal cortex is connected with the frontal motor areas . thus , the posterior parietal cortex would have an effect on the initial movement and sensory feedback during performance of a complex motor task22 . on this basis , the correlation shown in our study between ipsilateral motor deficits and sensory deficits can be explained . these findings imply that interest in the ipsilateral side of stroke patients should focus on ipsilateral sensory deficits as well as ipsilateral motor deficits . motor deficits of the ipsilateral limbs of individuals with stroke have been reported in many studies ; besides , our study showed sensory deficits related to proprioceptive sense . on the basis of these results , we think that the difficultly stroke patients experience in task performance using the ipsilateral upper limb may be affected by both motor and sensory deficits . studies on recovery of motor deficits on the ipsilateral side after stroke are in progress . jung et al.14 reported that motor deficits in the ipsilateral upper limb show maximal recovery within one month after onset of stroke , but the deficits do not completely recover . thus , it will be necessary to study the recovery of ipsilateral sensory deficits after stroke onset , and we will be investigating this . we acknowledge that our study had some limitations , in that the effects of specific lesion location and the extent of the damage were not identified . therefore , future studies will be required in order to determine more detailed mechanisms of other movement and sensory deficits , other than proprioceptive sense , in the ipsilateral upper limb of patients with unilateral brain injury .

[ purpose ] previous studies have reported on motor deficits in the ipsilateral upper limbs ( ul ) of a damaged brain hemisphere in motor tasks . however , little is known about sensory deficits on the ipsilateral side . therefore , we investigated whether both motor and sensory function of the ipsilateral ul are affected in patients with stroke . [ subjects and methods ] fifty patients with unilateral stroke and 40 age- and sex- matched normal subjects participated in this study . subjects were evaluated on performance of a tracking task for motor function , and by the joint reposition test for integrity of proprioceptive sense in the ipsilateral ul . [ result ] the comparison of the stroke group and the control group showed significant differences in performance of the tracking task and the joint reposition test . the accuracy index for the tracking task showed significant correlation with the error score for the joint reposition test in the stroke group . [ conclusion ] these results suggest that the ipsilateral ul of stroke patients has impairment in sensory function which is related to proprioceptive sense , along with motor deficits . therefore , we think that the difficulty stroke patients experience with motor tasks for the ipsilateral ul is induced by diminished integrity of sensorimotor function due to both sensory and motor deficits .

pubmed

the specific targeting of nanoparticles to target cells in an organ of interest has been a long - term goal of many laboratories interested in using nanoparticles to mediate the functional delivery of biopharmaceutical agents ( including nucleic acids ) to defined target cells . one of the main problems in using targeting ligands in conjunction with nanoparticles is the competition between specific - binding mediated cell entry and non - specific enhanced cell uptake mechanisms ( andreu et al , 2008 ) . previously , we have observed that non - specific effects can be completely overwhelming ( waterhouse et al , 2005 ) , an observation that has led to a series of further studies ( wang et al , 2009 ) intended to determine and characterize those factors that might influence non - specific enhanced cell uptake mechanisms such as nanoparticle size and heterogeneity , surface hydrophilicity and charge , not to forget other factors such as ligand conformation , points of attachment and surface density . recently , we described how ph - triggered , pegylated sirna nanoparticles ( also known as ph - triggered sirna - abc nanoparticles according to our recently described abcd nanoparticle structural paradigm ( kostarelos and miller , 2005 ; thanou et al , 2007 ; miller , 2008a ; miller , 2008b ) . these nanoparticles with a surface - covering of 5mol% polyethylene glycol 2000 ( peg ) were able to mediate the passive targeting of sirna to liver cells in vivo , thereby providing proof of concept for an effective rnai therapeutic approach to the treatment of hepatitis b virus infections ( sifectplus nanoparticles ) ( carmona et al , 2009 ) . in addition , pegylated sirna nanoparticles enabled for long - term circulation ( ltc sirna - abc nanoparticles ; lesirna nanoparticles ) were shown to do the same to tumour cells in vivo , thereby providing proof of concept for an effective rnai therapeutic approach to cancer treatment ( kenny et al , 2011 ) . in the case of the ph - triggered , pegylated sirna nanoparticles , the peg coating required for the biological stabilization of these nanoparticles was introduced by a purpose - designed post - coupling methodology . here , we report how this methodology has now been adapted to introduce integrin - targeting peptide motifs onto liposome and nanoparticle surfaces and so enable integrin receptor - mediated cellular uptake of agents to cells . there have been some spectacular data obtained in recent years using v6 integrin - receptor binding motifs for receptor - mediated delivery of different imaging agents to cells in vivo ( dicara et al , 2007 ; hausner et al , 2007 ; dicara et al , 2008 ; hausner et al , 2009 ) . we have described previously the use of integrin - receptor targeting peptides to promote functional gene delivery to cells ( harbottle et al , 1998 ; cooper et al , 1999 ) . here , we now report on the use of an alternative cyclic pentapeptide construct that makes use of a single d - tyrosyl amino acid residue to present the arginine - glycine - aspartate ( rgd ) amino acid residue triad in an " active " conformation capable of mediating intracellular delivery through v3/5 receptor binding and internalization ( chen et al , 2004 ) . arginine - glycine - glutamate ( rge ) controls are also reported in order to provide the means to demonstrate pure integrin - receptor mediated cellular uptake by rgd presenting imaging nanoparticles . dioleoyl l--phosphatidylethanolamine ( dope ) 1 , dimyristoyl l--phosphatidylcholine ( dmpc ) 2 ( sigma ) , dope - lissamine - rhodamine b ( dope - rhoda ) 3 were obtained from avanti polar lipids ( usa ) ( figure 1 ) . general synthetic procedures were performed as described previously ( carmona et al , 2009 ; mvel et al , 2010 ) . syntheses of n - cholesteryloxycarbonyl-3,7-diazanonane-1,9-diamine ( cdan ) 4 and cholesteryl - aminoxy ( ca ) lipid 5 were performed as described previously ( keller et al , 2003 ; oliver et al , 2004 ; carmona et al , 2009 ) ( figure 1 ) . the synthesis of other compounds necessary for our experiments were performed as described ( scheme 1 ) . hplc purification of final products required the use of vydac c-4 reversed phase preparative column with 1ml / min flow rate : mobile phases as follows used trifluoroacetic acid ( tfa ) ; a : h2o ( 0.1% , v / v , tfa ) ; b : mecn ( 0.1% , v / v , tfa ) ; c : meoh ( 0.1% , v / v , tfa ) . program set at : 0 - 15.0min ( 100% , a ) , 15.1 - 25.0min ( 0 - 100% , b ) , 25.1 - 45min ( 100% , c ) , 45.1 - 55min ( 100% , a ) . elution parameters : rgd - peg - cho 13a rt 15.2min , rge - peg - cho 13b rt 14.8min , prnhco - peg - cho 14 rt 15.8min , ca lipid 5 rt 23.8min , rgd - peg - ca 15a product rt 24.1min , rge - peg - ca 15b product rt 23.8min , and prnhco - peg - ca 16 product rt 26.3min ; mass spectrometry : m / z ( esi ) 1878 ( m - h , rgd - peg - cho 13a ) , 1917 ( mna , rge - peg - cho 13b ) , 1326 ( mh , prnhco - peg - cho 14 ) , 2406 ( mh , rgd - peg - ca 15a ) , 2443 ( mna , rge - peg - ca 15b ) , 1869 ( mna , prnhco - peg - ca 16 ) ( figure 1 ) . main lipids were used to prepare liposomes cl1 and cl2 and hence 10mol% pegylated bcd1 and 1 - 5mol% pegylated bcd2 nanoparticles . syntheses of -terminally modified - peg - chos : i ) a ) side chain protected , fluorenylmethyloxycarbonyl ( fmoc ) amino acid residue n - terminal coupling of l - arg(npbf ) , l - lys(boc ) , d - tyr(otbu ) and finally l - asp(otbu ) [ or l - glu(otbu ) ] to glycyl-2-chlorotrityl resin 6 using 2-(1h - benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate ( hbtu ) with di - isopropylethylamine ( dipea ) to mediate residue coupling and 2% ( v / v ) each of 1,8-diazabicyclo[5.4.0]undec-7-ene ( dbu ) and piperidine in dimethylformamide ( dmf ) for fmoc deprotection between rounds of peptide bond formation : b ) resin release with 20% ( v / v ) each of acetic acid and trifluoroethanol ( tfe ) in ch2cl2 , 31 - 78% ; ii ) a ) 3eqv diphenylphosphoryl azide ( dppa ) in dry dmf , 5 eqv nahco3 , 0c ambient temperature , ph 8.5 , 74 - 80% : b ) 2.5% ( v / v ) each h2o and triisopropylsilane ( tis ) in trifluoroacetic acid ( tfa ) , 80 - 100% ; iii ) a ) ho - c2h4-oh , al2o3 , ccl4 , reflux , 27% : b ) pentafluorophenol ( pfpoh ) , n , n'-dicyclohexylcarbodiimide ( dcc ) , etoac , 71% ; iv ) a ) amino - peg - propionic acid , 4 eqv triethylamine ( tea ) , chcl3 , reflux , 84% : b ) 1.2 eqv pfpoh , 1.2 eqv dcc , etoac , 76% ; v ) 0.2 m na2hpo4 , 0.1 m naoh , 69 - 100% ; vi ) 10% ( v / v ) tfa in h2o , 100% ; vii ) a ) propylamine ( prnh2 ) , 4 eqv tea , chcl3 , reflux , 100% : b ) 10% ( v / v ) tfa in h2o , 51% ( v / v ) . designated lipids were dissolved in chloroform at 5mg / ml and then appropriate aliquots were combined in a presilanized round - bottom flask ( 5ml ) . in each case , the organic solvent was evaporated to dryness to form an even thin lipid film that was further purged with a stream of argon gas to remove residual traces of the organic solvent . the lipid film was hydrated using double distilled h2o ( ddh2o ) to give a multilamellar liposome suspension that was subsequently subjected to sonication in a water bath at 40c for 30min ( sonomatic water bath , langford ultrasonics , 33 khz ultrasound frequency ) . post - sonication , small unilamellar vesicles ( 50 - 80 nm ) ( b - component , cl1 or cl2 ) were diluted to 1.5mg / ml and incubated at room temperature for 15min . thereafter , appropriate aliquots of -terminally modified - peg - cho 13a , 13b or 14 ( 1mg / ml in water , cd molecules ) were introduced for post - coupling such that their final composition was between 1 and 10mol% of total lipid . the ph of solution was monitored by ph boy ( camlab ltd , cambridgeshire , uk ) and adjusted to ph 4 if required by addition of small aliquots of aqueous solutions of naoh ( 0.99 m ) or hcl ( 0.99 m ) . on completion of the reaction ( approximately 16 - 24hr ) ( as judged by hplc analysis ) , the solution ph was accurately readjusted to ph 7 resulting in 10mol% pegylated bcd1 and 1 - 5mol% pegylated bcd2 nanoparticles , respectively , ready for use . 1 - 5mol% pegylated abcd2 nanoparticles were prepared by combining 1 - 5mol% pegylated bcd2 nanoparticles with various volumes of aqueous 4 mm hepes , ph 7.0 - 7.4 , sirna solution ( 50m a - component ) under heavy vortex conditions ( final [ sirna ] typically 100g / ml ; 7m ) using a lipid : sirna ratio of 12:1 ( w / w ) that correlates with a lipid - nucleic acid n / p charge ratio of approximately 2 . lipid compositions of two liposome formulations prepared in double distilled water with final diameters of 50 - 80 nm . for lipid structures see figure 1 . these experiments were performed on a facs calibur instrument ( bd biosciences ) . in each set of indicated experiments 1 , 2 or 5mol% pegylated abcd2 nano - particles ( 14060 nm ) were prepared with sirna ( [ sirna ] 80pmoles / well ) and uptake studies performed with huvec cells previously grown to 80% confluent at 37c , 10% co2 . dioleoyl l--phosphatidylethanolamine ( dope ) 1 , dimyristoyl l--phosphatidylcholine ( dmpc ) 2 ( sigma ) , dope - lissamine - rhodamine b ( dope - rhoda ) 3 were obtained from avanti polar lipids ( usa ) ( figure 1 ) . general synthetic procedures were performed as described previously ( carmona et al , 2009 ; mvel et al , 2010 ) . syntheses of n - cholesteryloxycarbonyl-3,7-diazanonane-1,9-diamine ( cdan ) 4 and cholesteryl - aminoxy ( ca ) lipid 5 were performed as described previously ( keller et al , 2003 ; oliver et al , 2004 ; carmona et al , 2009 ) ( figure 1 ) . the synthesis of other compounds necessary for our experiments were performed as described ( scheme 1 ) . hplc purification of final products required the use of vydac c-4 reversed phase preparative column with 1ml / min flow rate : mobile phases as follows used trifluoroacetic acid ( tfa ) ; a : h2o ( 0.1% , v / v , tfa ) ; b : mecn ( 0.1% , v / v , tfa ) ; c : meoh ( 0.1% , v / v , tfa ) . program set at : 0 - 15.0min ( 100% , a ) , 15.1 - 25.0min ( 0 - 100% , b ) , 25.1 - 45min ( 100% , c ) , 45.1 - 55min ( 100% , a ) . elution parameters : rgd - peg - cho 13a rt 15.2min , rge - peg - cho 13b rt 14.8min , prnhco - peg - cho 14 rt 15.8min , ca lipid 5 rt 23.8min , rgd - peg - ca 15a product rt 24.1min , rge - peg - ca 15b product rt 23.8min , and prnhco - peg - ca 16 product rt 26.3min ; mass spectrometry : m / z ( esi ) 1878 ( m - h , rgd - peg - cho 13a ) , 1917 ( mna , rge - peg - cho 13b ) , 1326 ( mh , prnhco - peg - cho 14 ) , 2406 ( mh , rgd - peg - ca 15a ) , 2443 ( mna , rge - peg - ca 15b ) , 1869 ( mna , prnhco - peg - ca 16 ) ( figure 1 ) . main lipids were used to prepare liposomes cl1 and cl2 and hence 10mol% pegylated bcd1 and 1 - 5mol% pegylated bcd2 nanoparticles . syntheses of -terminally modified - peg - chos : i ) a ) side chain protected , fluorenylmethyloxycarbonyl ( fmoc ) amino acid residue n - terminal coupling of l - arg(npbf ) , l - lys(boc ) , d - tyr(otbu ) and finally l - asp(otbu ) [ or l - glu(otbu ) ] to glycyl-2-chlorotrityl resin 6 using 2-(1h - benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate ( hbtu ) with di - isopropylethylamine ( dipea ) to mediate residue coupling and 2% ( v / v ) each of 1,8-diazabicyclo[5.4.0]undec-7-ene ( dbu ) and piperidine in dimethylformamide ( dmf ) for fmoc deprotection between rounds of peptide bond formation : b ) resin release with 20% ( v / v ) each of acetic acid and trifluoroethanol ( tfe ) in ch2cl2 , 31 - 78% ; ii ) a ) 3eqv diphenylphosphoryl azide ( dppa ) in dry dmf , 5 eqv nahco3 , 0c ambient temperature , ph 8.5 , 74 - 80% : b ) 2.5% ( v / v ) each h2o and triisopropylsilane ( tis ) in trifluoroacetic acid ( tfa ) , 80 - 100% ; iii ) a ) ho - c2h4-oh , al2o3 , ccl4 , reflux , 27% : b ) pentafluorophenol ( pfpoh ) , n , n'-dicyclohexylcarbodiimide ( dcc ) , etoac , 71% ; iv ) a ) amino - peg - propionic acid , 4 eqv triethylamine ( tea ) , chcl3 , reflux , 84% : b ) 1.2 eqv pfpoh , 1.2 eqv dcc , etoac , 76% ; v ) 0.2 m na2hpo4 , 0.1 m naoh , 69 - 100% ; vi ) 10% ( v / v ) tfa in h2o , 100% ; vii ) a ) propylamine ( prnh2 ) , 4 eqv tea , chcl3 , reflux , 100% : b ) 10% ( v / v ) tfa in h2o , 51% ( v / v ) . designated lipids were dissolved in chloroform at 5mg / ml and then appropriate aliquots were combined in a presilanized round - bottom flask ( 5ml ) . in each case , the organic solvent was evaporated to dryness to form an even thin lipid film that was further purged with a stream of argon gas to remove residual traces of the organic solvent . the lipid film was hydrated using double distilled h2o ( ddh2o ) to give a multilamellar liposome suspension that was subsequently subjected to sonication in a water bath at 40c for 30min ( sonomatic water bath , langford ultrasonics , 33 khz ultrasound frequency ) . post - sonication , small unilamellar vesicles ( 50 - 80 nm ) ( b - component , cl1 or cl2 ) were diluted to 1.5mg / ml and incubated at room temperature for 15min . thereafter , appropriate aliquots of -terminally modified - peg - cho 13a , 13b or 14 ( 1mg / ml in water , cd molecules ) were introduced for post - coupling such that their final composition was between 1 and 10mol% of total lipid . the ph of solution was monitored by ph boy ( camlab ltd , cambridgeshire , uk ) and adjusted to ph 4 if required by addition of small aliquots of aqueous solutions of naoh ( 0.99 m ) or hcl ( 0.99 m ) . on completion of the reaction ( approximately 16 - 24hr ) ( as judged by hplc analysis ) , the solution ph was accurately readjusted to ph 7 resulting in 10mol% pegylated bcd1 and 1 - 5mol% pegylated bcd2 nanoparticles , respectively , ready for use . 1 - 5mol% pegylated abcd2 nanoparticles were prepared by combining 1 - 5mol% pegylated bcd2 nanoparticles with various volumes of aqueous 4 mm hepes , ph 7.0 - 7.4 , sirna solution ( 50m a - component ) under heavy vortex conditions ( final [ sirna ] typically 100g / ml ; 7m ) using a lipid : sirna ratio of 12:1 ( w / w ) that correlates with a lipid - nucleic acid n / p charge ratio of approximately 2 . lipid compositions of two liposome formulations prepared in double distilled water with final diameters of 50 - 80 nm . for lipid structures see figure 1 . these experiments were performed on a facs calibur instrument ( bd biosciences ) . in each set of indicated experiments 1 , 2 or 5mol% pegylated abcd2 nano - particles ( 14060 nm ) were prepared with sirna ( [ sirna ] 80pmoles / well ) and uptake studies performed with huvec cells previously grown to 80% confluent at 37c , 10% co2 . post - coupling represents an optimal way to attach a biological targeting ligand of interest to a nanoparticle surface with controlled mol% attachment and ligand orientation ( scheme 2 ) . our approach to the development of a post - coupling methodology has been to learn from the chemistry employed to create ph - triggered , pegylated sirna nanoparticles ( as mentioned in the introduction ) in order to design - hybrid targeting ligands ( l ) that comprise a peptide targeting moiety conjugated to a peg extension chain enabled for postcoupling to a nanoparticle surface ( scheme 2 ) . rgd peptides and corresponding rge control peptides were prepared as follows ( scheme 1 ) . starting with glycyl-2-chlorotrityl solid - phase peptide resin 6 , the desired pentapeptide rgd - targeting moiety was prepared in an open chain , protected form 7 by solid - peptide synthesis , then subject to cyclization and deprotection to give cyclic pentapeptide 8a . a control cyclic - rge pentapeptide 8b ( with aspartate [ d ] replaced by glutamate [ e ] ) was prepared in an equivalent manner . thereafter , the aldehyde functionality of 4-carboxybenzaldehyde 9 was acetal protected and the carboxy function was activated for coupling by means of pentafluorophenol ( pfpoh ) esterification giving 10 . reagent 10 was then coupled to amino - peg - propionic acid and the free carboxylate activated once again for coupling by means of pfpoh esterification giving heterofunctional peg moiety 11 . the coupling of 11 to 8a or 8b resulted in rgd acetal 12a and rge acetal 12b respectively . routine acetal deprotection yielded the desired products rgd - peg - cho 13a and control rge - peg - cho 13b ( scheme 1 ) . an alternative control aldehyde was obtained through combination of heterofunctional peg moiety 11 with propylamine followed by acetal deprotection resulting in the non - peptide control product prnhco - peg - cho 14 ( scheme 1 ) . all three -terminally modified - peg - cho compounds prepared as described above were used in subsequent experiments described below . using all three -terminally modified - peg - cho compounds in turn , coupling reactions were performed individually with neutral cholesteryl - aminoxy lipid ( ca ) 5 ( figure 1 ) and then with neutral liposomes ( equivalent in composition to cl1 , table 1 ) . reactions were monitored by hplc and mass spectrometry in water at ph4 and shown to reach completion after 16 - 24h stirring at ambient temperature giving 15a , 15b , or 16 as appropriate . liposome - coupling experiments were then performed under similar conditions in order to couple rgd - peg - cho 13a ( or control aldehydes rge - peg - cho 13b and prnhco - peg - cho 14 ) to alternative liposome formulations cl1 or cl2 ( table 1 , figure 1 ) , making use of ca lipid 5 once again in order to present the aminoxy functional groups required for conjugation of liposome surfaces with each -terminally modified - peg - cho compound in turn ( scheme 2 ) . upon conclusion of these liposome - coupling reactions , ph was raised to 7 in order to stabilize the resulting oxime bonds and the resulting pegylated liposomes . resulting ligand - pegylated liposomes ( approx 100 nm in diameter ) were described as belonging to either bcd1 or bcd2 nanoparticle families depending upon whether the parent liposome formulation used in preparation was cl1 or cl2 , respectively . initially bcd1 family members rgd - bcd1 , rge - bcd1 and prnhco - bcd1 were prepared with 1mol% of dope - rhoda 3 for fluorescence microscopy cell uptake studies . the results demonstrate that only 10mol% pegylated rgd - bcd1 nanoparticles were capable of delivering measurable and observable levels of fluorescent - label to integrin - rich human umbilical vein endothelial cells ( huvec ) ( figure 2a ) . this is very interesting in view of the physical properties of bcd1 nanoparticles , namely approx 100 nm in diameter , 10mol% pegylation with peg , and a -potential of essentially zero mv . the lack of charge originates from the fact that the majority lipid components needed to prepare cl1 liposomes were neutral while the minority ca lipid 5 component possesses an aminoxy functional group unprotonated at ph 7 due to its pka value of about 4.5 ( compared with the pka values of more typical amine functional groups that are found around 10 ) ( castro , 1990 ) . we would argue that such nanoparticle physical properties could be an ideal starting point from which to optimize receptor specific cellular uptake of nanoparticles relative to non - specific enhanced cell uptake . subsequently , bcd2 family members rgd - bcd2 , rge - bcd2 and prnhco - bcd2 were prepared with 1.0 mol% of dope - rhoda 3 and up to 5mol% of each -terminally modified - peg - cho for fluorescence - activated cell sorting ( facs ) studies . in addition these cationic liposome systems were further combined with a non - functional nucleic acid ( small interfering rna [ sirna ] ) ( a component ) in order to neutralize the positive charge and convert the bcd2 family members in to a varieties of corresponding low charge abcd2 nanoparticles with lipid : sirna the physical properties of these nanoparticles were diameters of approximately 140 nm , up to 5mol% pegylation with peg , and a -potential of less than 10mv . in these cases , facs studies were performed with low charge 1- , 2- and 5mol% pegylated rgd - abcd2 , rge - abcd2 and prnhco - abcd2 nanoparticles . in each case , the discrimination between rgd presenting and control nanoparticles was still clearly present and was found to increase as a function of the mol% pegylation coverage with peg ( figure 2b ) . the fact that the discrimination was not as substantial as observed above ( figure 2a ) appears to be due to the lower mol% levels of pegylation coverage and the presence of residual nanoparticle charge . therefore , control of both pegylation levels ( plus other peg - structure related criteria ) and residual charges are essential in order to optimize receptor mediated cell uptake in preference to non - specific enhanced cell uptake mechanisms . schematic illustration of nanoparticle formulations involving the inclusion of an aminoxy coupling lipid by a formulation procedure known as premodification followed by the post coupling of -terminally ligand ( l)-modified peg aldehydes ( l - peg - cho ) . biological receptor mediated functional delivery of imaging agents is well established ( dicara et al , 2007 ; dicara et al , 2008 ; hausner et al . 2007 ; hausner et al , 2009 ) , even of genes ( harbottle et al , 1998 ; cooper et al , 1999 ) . however , receptor - mediated delivery can not be taken as a given following the covalent attachment of a biological targeting ligand to a nanoparticle ( waterhouse et al , 2005 ; andreu et al , 2008 ) . therefore , in our view , receptor - mediated delivery phenomena should never be treated with the casual assumption that receptor - mediation is inevitable once an appropriate receptor - specific ligand has been mounted on a nanoparticle surface . any given receptor - specific ligand should in fact be subject to a standard set of trial experiments to ensure that the biophysical properties of a nanoparticle platform and the presentation of the receptor - specific ligand are optimized for bonefide receptor - mediated delivery of the corresponding nanoparticle . in this paper we describe just such a set of trial experiments ( figure 2 ) with sufficient controls to demonstrate clearly that specific receptor - mediated uptake by cells has been enabled over background . accordingly , we would like to propose that both studies involving first the delivery of an imaging agent and then second a potential agent of pharmaceutical interest ( api ) , should be standard assays to perform and demonstrate before all else that a nanoparticle - attached ligand is truly a receptor - specific ligand in the context of the nanoparticle platform to which the ligand is covalently attached . if this is not shown , then nanoparticle biophysical properties , ligand attachment and orientation , and mol% ligand presentation should all be systematically altered until specific receptor - mediated cell uptake can be observed substantially over and above any non - specific enhanced cell uptake background ( kamaly et al , 2009 ; kamaly et al , 2010 ) . fluorescence microscopy of huvec cells , previously grown to 80% confluent at 37c with 10% ( v / v ) co2 , treated with 10mol% pegylated bcd1 nanoparticles prepared from rgd - peg - cho 13a or rge - peg - cho 13b . cells were in contact with nanoparticles for at least 4hr prior to mounting and observation of rhodamine fluorescence under a nikon 600 fluorescence microscope . cell uptake was determined after 4hr using rhodamine fluorescence and detected using the fl-2 channel . ab corresponds with simple sirna - lipoplex control nanoparticles , formulated from cl2 cationic liposomes and an equivalent final [ sirna ] . in our case here , we can say with confidence that our post - coupling chemistry and methodology for the attachment of integrin - targeting rgd ligands has resulted in the successful formation of integrin - targeted imaging nanoparticles that can also mediate integrin - specific delivery of an api such as sirna to v3/5 integrin - receptor presenting cells . therefore , this post - coupling chemistry and premodification - postcoupling methodology could be applicable to other nanoparticle platforms with equal success provided that the biophysical properties of the nanoparticle platform also conform to the following biophysical parameters : nanoparticle dimensions of approx 100 nm in diameter nanoparticle -potential values that converge on neutral ( 0 mv ) nanoparticle ligand surface coverage of approx 2 mol% ( or higher ) further research with this and other nanoparticle systems will now be needed to demonstrate if these three nanoparticle " rules " for receptor - mediated cell entry are indeed general rules or simply guidelines for receptor - mediated cellular uptake of nanoparticle systems by corresponding receptor expressing cells in vitro , ex vivo and/or in vivo . the data described here represent the completion of a first study involving the preparation of ligand - mounted pegylated nanoparticles constructed by a bespoke pre - modification postcoupling methodology . data suggest for the first time in our hands that this methodology may be used to ensure that receptor mediated cell uptake of attached nanoparticles can be " engineered " to dominate non - specific enhanced cell uptake mechanisms .

a key goal of our research is the targeted delivery of functional biopharmaceutical agents of interest , such as small interfering rna ( sirna ) , to selected cells by means of receptor - mediated nanoparticle technologies . recently , we described how ph - triggered , pegylated sirna - nanoparticles ( ph triggered sirna - abc nanoparticles ) were able to mediate the passive targeting of sirna to liver cells in vivo . in addition , pegylated sirna nanoparticles enabled for long - term circulation ( ltc sirna - abc nanoparticles , lesirna nanoparticles ) were shown to do the same to tumour cells in vivo . further gains in the efficiency of sirna delivery are expected to require active targeting with nanoparticles targeted for delivery and cellular uptake by means of attached biological ligands . here we report on the development of a new synthetic chemistry and a bioconjugation methodology that allows for the controlled formulation of pegylated nanoparticles which surface - present integrin - targeting peptides unambiguously and so enable integrin receptor - mediated cellular uptake . furthermore , we present delivery data that provide a clear preliminary demonstration of physical principles that we propose should underpin successful , bonefide receptor - mediated targeted delivery of therapeutic and/or imaging agents to cells .

pubmed

the millennium cohort is the largest prospective study using primary data collection ever undertaken by the department of defense ( dod ) . the study began in 2001 with the objective to collect and evaluate data on health , behavioral risk factors , and occupational characteristics related to military service that may be associated with adverse health outcomes ( 14 ) . this report includes participants from the first enrollment cycle that spanned 20012003 and comprised 77,047 participants ( 36% of those able to be contacted ) . approximately 71% of those participants enrolled at baseline responded to follow - up surveys conducted in 2004 and 2007 . the surveys were administered via paper and the internet and included questions on self - reported provider - diagnosed medical conditions , mental health symptoms , physical and functional status , alcohol and tobacco use , occupational status , military exposures , sleep patterns , and demographic information . this study was approved by the naval health research center institutional review board , and informed consent was obtained from all study participants . this research has been conducted in compliance with all applicable federal regulations governing the protection of human subjects in research ( protocol nhrc.2000.0007 ) . of the 77,047 participants enrolled at baseline , follow - up surveys were completed by 55,021 participants at year 3 and 54,790 at year 6 . since incident diabetes was the outcome of interest , eligible participants included those who were followed until either the outcome was reported to have occurred at year 3 or 6 , or until their last completed follow - up if the outcome was not reported . exclusions included participants reporting provider - diagnosed diabetes at baseline , missing outcome or exposure data , or if dod outpatient or inpatient medical encounter data contained codes for type 1 diabetes ( n = 72 ) ( icd-9 codes 250.x1 and 250.x3 ) . the primary data sources were the self - administered millennium cohort surveys and dod electronic personnel files , managed by the defense manpower data center ( dmdc , seaside , ca ) . for these analyses , electronic records from dmdc provided information on age , sex , race / ethnicity , education , pay grade , service branch , service component , military occupation , dates of separation , and deployment dates . dmdc information was supplemented with self - reported data to reduce missing values when necessary . three sleep variables were included in the analyses : 1 ) trouble sleeping , 2 ) sleep duration , and 3 ) sleep apnea . trouble sleeping was assessed using the sleep items on the prime - md patient health questionnaire ( phq ) and the ptsd checklist civilian version ( pcl - c ) ( 1720 ) . the phq asks , over the last 4 weeks , how often have you experienced trouble falling asleep or staying asleep ? the pcl - c asks , in the past month , have you had trouble falling asleep or staying asleep ? participants with trouble sleeping were defined as those who responded moderately or above on the pcl - c sleep item or several days or longer on the phq sleep item . sleep duration was reported as whole number of hours slept in an average 24-h period over the past month . participants with sleep apnea were identified by self - report as being told by a doctor or other health professional they had sleep apnea . ptsd was assessed using the pcl - c ( 17 ) , a 17-item self - report screening tool for ptsd symptoms during the past 30 days on a 5-point likert scale , ranging from 1 ( not at all ) to 5 ( extremely ) . a positive screen for ptsd at baseline was defined as a report of a moderate or higher level of at least one intrusion symptom , three avoidance symptoms , and two hyperarousal symptoms ( criteria established by dsm - iv ) ( 21 ) . major depressive disorder was assessed using nine items from the phq ( sensitivity = 0.93 ; specificity = 0.89 ) corresponding to the depression diagnosis based on the dsm - iv ( 19 ) . a positive screen for depression at baseline was defined as 1 ) endorsement of depressed mood or anhedonia and 2 ) response of more than half the days or nearly every day to at least five items . other anxiety ( 6 items ) and panic ( 15 items ) symptoms problem drinking was defined using items from the phq on risky behaviors related to alcohol use , such as driving under the influence more than one time over the past year , where endorsement of at least one item indicated problem drinking . binge drinking was defined as consuming five or more drinks ( men ) or four or more drinks ( women ) in 1 day during the past year . smokers were identified as those ever smoking 100 cigarettes in their lifetime , with those persisting in this habit defined as current smokers and those having quit as past smokers . in longitudinal models , service branch , service component , pay grade , military occupation , birth year , race / ethnicity , and education were assessed at baseline . smoking status , alcohol use , bmi , combat deployment , ptsd , depression , anxiety , panic , and all sleep variables were assessed at all available time points . the outcome of interest was incident diagnosed diabetes self - reported on a follow - up survey among people without diabetes at the baseline assessment . at baseline , participants reported if they had ever been told by a doctor or health professional that they had diabetes or at follow - up assessments , participants responded to the same question in regard to the previous 3 years . univariate analyses assessed associations of incident diabetes with sleep , military , demographic , behavioral , and mental health characteristics while accounting for multiple periods of observation per subject . since repeated measurements were available with up to two follow - up assessments per person , generalized estimating equations ( gees ) were used to account for these multiple assessments while adjusting for fixed and time - varying covariates , to estimate adjusted odds of reporting incident diabetes ( 22 ) . incident diabetes was determined at each follow - up assessment , and all covariates were evaluated using data from the previous assessment ( 22 ) . in all multivariable models , adjustments were performed for known type 2 diabetes risk factors captured in the study : age , sex , education , bmi , and race / ethnicity . the final adjusted models were built using a stepwise backward reduction algorithm that retained all significant variables ( p < 0.10 ) ( table 1 ) while always keeping age , sex , race / ethnicity , education , and bmi in the model . additionally , mental health variables were added to the final models to determine if this altered the relationship between sleep and diabetes . first - order interaction terms between retained sleep variables and age , sex , and bmi were tested to determine whether the exposure - outcome associations varied by these characteristics . possible presence of multicollinearity in multivariable models was considered if the variance inflation factor exceeded 4 . best - fitting gee models were judged using the quasilikelihood information criterion , the measure developed for gee models analogous to the akaike information criterion . statistical analyses were performed using sas statistical software , version 9.3 ( sas institute , inc . , baseline demographic , military , and behavioral characteristics of millennium cohort participants by development of diabetes at follow - up of the 77,047 participants enrolled at baseline , follow - up surveys were completed by 55,021 participants at year 3 and 54,790 at year 6 . since incident diabetes was the outcome of interest , eligible participants included those who were followed until either the outcome was reported to have occurred at year 3 or 6 , or until their last completed follow - up if the outcome was not reported . exclusions included participants reporting provider - diagnosed diabetes at baseline , missing outcome or exposure data , or if dod outpatient or inpatient medical encounter data contained codes for type 1 diabetes ( n = 72 ) ( icd-9 codes 250.x1 and 250.x3 ) . the primary data sources were the self - administered millennium cohort surveys and dod electronic personnel files , managed by the defense manpower data center ( dmdc , seaside , ca ) . for these analyses , electronic records from dmdc provided information on age , sex , race / ethnicity , education , pay grade , service branch , service component , military occupation , dates of separation , and deployment dates . dmdc information was supplemented with self - reported data to reduce missing values when necessary . three sleep variables were included in the analyses : 1 ) trouble sleeping , 2 ) sleep duration , and 3 ) sleep apnea . trouble sleeping was assessed using the sleep items on the prime - md patient health questionnaire ( phq ) and the ptsd checklist civilian version ( pcl - c ) ( 1720 ) . the phq asks , over the last 4 weeks , how often have you experienced trouble falling asleep or staying asleep ? the pcl - c asks , in the past month , have you had trouble falling asleep or staying asleep ? participants with trouble sleeping were defined as those who responded moderately or above on the pcl - c sleep item or several days or longer on the phq sleep item . sleep duration was reported as whole number of hours slept in an average 24-h period over the past month . participants with sleep apnea were identified by self - report as being told by a doctor or other health professional they had sleep apnea . ptsd was assessed using the pcl - c ( 17 ) , a 17-item self - report screening tool for ptsd symptoms during the past 30 days on a 5-point likert scale , ranging from 1 ( not at all ) to 5 ( extremely ) . a positive screen for ptsd at baseline was defined as a report of a moderate or higher level of at least one intrusion symptom , three avoidance symptoms , and two hyperarousal symptoms ( criteria established by dsm - iv ) ( 21 ) . major depressive disorder was assessed using nine items from the phq ( sensitivity = 0.93 ; specificity = 0.89 ) corresponding to the depression diagnosis based on the dsm - iv ( 19 ) . a positive screen for depression at baseline was defined as 1 ) endorsement of depressed mood or anhedonia and 2 ) response of more than half the days or nearly every day to at least five items . other anxiety ( 6 items ) and panic ( 15 items ) symptoms problem drinking was defined using items from the phq on risky behaviors related to alcohol use , such as driving under the influence more than one time over the past year , where endorsement of at least one item indicated problem drinking . binge drinking was defined as consuming five or more drinks ( men ) or four or more drinks ( women ) in 1 day during the past year . smokers were identified as those ever smoking 100 cigarettes in their lifetime , with those persisting in this habit defined as current smokers and those having quit as past smokers . in longitudinal models , service branch , service component , pay grade , military occupation , birth year , race / ethnicity , and education were assessed at baseline . smoking status , alcohol use , bmi , combat deployment , ptsd , depression , anxiety , panic , and all sleep variables were assessed at all available time points . the outcome of interest was incident diagnosed diabetes self - reported on a follow - up survey among people without diabetes at the baseline assessment . at baseline , participants reported if they had ever been told by a doctor or health professional that they had diabetes or at follow - up assessments , participants responded to the same question in regard to the previous 3 years . univariate analyses assessed associations of incident diabetes with sleep , military , demographic , behavioral , and mental health characteristics while accounting for multiple periods of observation per subject . since repeated measurements were available with up to two follow - up assessments per person , generalized estimating equations ( gees ) were used to account for these multiple assessments while adjusting for fixed and time - varying covariates , to estimate adjusted odds of reporting incident diabetes ( 22 ) . incident diabetes was determined at each follow - up assessment , and all covariates were evaluated using data from the previous assessment ( 22 ) . in all multivariable models , adjustments were performed for known type 2 diabetes risk factors captured in the study : age , sex , education , bmi , and race / ethnicity . the final adjusted models were built using a stepwise backward reduction algorithm that retained all significant variables ( p < 0.10 ) ( table 1 ) while always keeping age , sex , race / ethnicity , education , and bmi in the model . additionally , mental health variables were added to the final models to determine if this altered the relationship between sleep and diabetes . first - order interaction terms between retained sleep variables and age , sex , and bmi were tested to determine whether the exposure - outcome associations varied by these characteristics . possible presence of multicollinearity in multivariable models was considered if the variance inflation factor exceeded 4 . best - fitting gee models were judged using the quasilikelihood information criterion , the measure developed for gee models analogous to the akaike information criterion . statistical analyses were performed using sas statistical software , version 9.3 ( sas institute , inc . , baseline demographic , military , and behavioral characteristics of millennium cohort participants by development of diabetes at follow - up a total of 47,093 participants were included in this analysis and followed for up to 6 years . incident diabetes was assessed at each 3-year period separately , with 13,621 participants followed for one 3-year period , and 33,472 participants followed for two 3-year periods . a total of 383 cases occurred during years 03 and 488 cases occurred during years 36 of follow - up . the 488 cases that did not occur until years 36 are also present in the first column of table 1 as not having diabetes from year 03 . at baseline , participants overall were on average young ( 34.9 years of age , sd 9.0 years ) , slightly overweight ( bmi 26.0 kg / m , sd 3.3 ) , 25.6% female , and more likely than not to be white , non - hispanic race , less than college educated , army service branch , and on active duty ( table 1 ) . overall , 871 participants newly self - reported diabetes , for a cumulative annual incidence of 3.6/1,000 person - years . participants who newly self - reported diabetes during follow - up were significantly more likely at baseline to have reported trouble sleeping , sleep duration 5 h , and sleep apnea ( table 1 ) . sleep duration > 8 h was reported by similar proportions of participants with and without newly self - reported diabetes . participants with new - onset diabetes were on average older , had a higher bmi , and were more likely of nonwhite race . characteristics associated with new - onset diabetes included no deployment , having separated from the military since baseline , army or navy / coast guard service branch , reserves / national guard service component , enlisted pay grade , and administration / supply occupation . binge or problem drinking was less likely to have been reported in people reporting new - onset diabetes , and current and former smoking frequency was similar in groups with and without new - onset diabetes . all mental health symptoms occurred significantly more frequently among those who newly reported diabetes during follow - up . factors associated with a higher odds of incident diabetes in models adjusted for sex and known type 2 diabetes risk factors ( age , bmi , education , and race / ethnicity ) included trouble sleeping , sleep duration 5 h compared with 7 h , and sleep apnea ( table 2 ) . several military service characteristics remained significantly related to the outcome , including no deployment , enlisted pay grade , administration / supply occupation , and having separated from the military since baseline ( table 2 ) . binge or problem drinking remained significantly associated , and current smoking emerged as a risk factor for new - onset diabetes after adjustment . screening positive for depression , panic , other anxiety disorder , and ptsd were all significantly associated with higher odds of new - onset diabetes in these adjusted models ( table 2 ) . relative odds of incident self - reported diabetes by each baseline characteristic of interest in the multivariable model ( table 3 ) , sleep characteristics remained significantly and independently associated with higher diabetes odds . the sleep variables that entered the final model included reported trouble sleeping and sleep apnea , which were associated with significant increases in the odds of diabetes of 1.21 to 1.78 , respectively . these associations were not only independent of each other but also the other covariates adjusted for in the model ( table 3 ) . military service characteristics significantly associated with diabetes odds included deployment without combat experience , administration or supply occupations , pay grade , and having separated from the military since baseline . a positive screen for ptsd was significantly associated with higher diabetes odds , independent of sleep characteristics and other covariates in the model , and panic disorder was of borderline significance . tests for interactions between the sleep variables included in the models in table 3 and age , sex , and bmi were all nonsignificant ( p > 0.10 ) . no variable had a variance inflation factor > 4 , indicating that collinearity was unlikely . the model in table 3 was rerun with sleep duration substituted for trouble sleeping but with the other included covariates unchanged . the quasilikelihood information criterion for the model containing trouble sleeping was slightly lower ( 8,640.9 ) than when sleep duration was included in the model ( 8,646.3 ) . the sleep duration variable was insignificant overall in this model ( p = 0.1585 ) , although two sleep categories were significantly related to diabetes odds ( < 5-h odds ratio [ or ] 1.52 [ 95% ci 1.092.14 ] ; 5-h or 1.28 [ 1.011.62 ] ) . several additional modifications of the multivariable model shown in table 3 were conducted . in the first modification , symptoms of depression and anxiety disorder were included in the model to assess whether adjustment for these additional factors altered the association between sleep and diabetes odds . in the second modification , all subjects reporting sleep apnea were removed from the model shown in table 3 . in each of these modifications , the statistical significance of the associations between the sleep variables and diabetes odds remained unchanged , and the associations were of similar magnitude to those seen in table 3 ( data not shown ) . longitudinal multivariable regression model of sleep characteristics and mental health symptoms as risk factors for incident self - reported diabetes in the millennium cohort our study findings confirm that sleep characteristics are associated with subsequent new - onset self - reported diabetes . these associations persisted after adjustment for multiple mental health conditions known to affect sleep duration and quality , including ptsd and depression ( 1113 ) . therefore , it is unlikely that sleep simply serves as a surrogate marker for associated mental health conditions previously shown to predict higher diabetes risk ( 9,10 ) . additionally , participants reporting provider - diagnosed sleep apnea were at higher risk of developing self - reported diabetes during follow - up . although sleep apnea is associated with higher bmi and both altered sleep quality and quantity , adjustment for these variables did not alter the significance of the higher diabetes risk associated with this condition ( 23 ) . overall , these results suggest that the associations between type 2 diabetes risk and mental health conditions and sleep characteristics , if causal , may involve different pathways . whereas trouble sleeping and shorter sleep duration were associated with higher risk of diabetes in a model adjusted for known diabetes risk factors , longer sleep duration was not , in contrast to findings that others have reported ( 4 ) . the reason for this discrepancy is unclear , but it may be due to the relatively younger age of our cohort compared with investigations reporting higher diabetes risk with longer sleep duration ( 24,25 ) . the independent role of mental health conditions associated with poor quality and quantity of sleep in predicting risk of diabetes has only been addressed in a limited manner in previous research . the nurses health study investigators examined sleep as a predictor of incident diabetes in this cohort from 19861996 and adjusted for an assessment of depression based on the short form 36 health survey that was obtained in 1992 , approximately midway through the follow - up period ( 24 ) . thus , it is unclear whether depression preceded or followed diabetes onset during the first 6 years of follow - up . an analysis using the national health and nutrition examination survey i epidemiologic follow - up study adjusted for depression while examining the association between sleep and diabetes incidence , although no association was seen between depression and diabetes incidence in this cohort ( 25 ) . lastly , a prospective study of swedish men assessed the presence of depression with a simple yes / no question , do you feel depressed ? and did not provide information on the accuracy of this question in capturing this condition ( 26 ) . to our knowledge , our study is the first to consider mental health conditions other than depression as potential confounding factors and to measure their occurrence prior to the onset of diabetes . previous prospective research on sleep characteristics and type 2 diabetes risk has not considered whether sleep duration or quality is related to this outcome independent from sleep apnea , a known cause of sleep disturbance . in our fully adjusted analysis , both sleep apnea and trouble sleeping had independent associations with diabetes risk , suggesting that the associations between sleep characteristics and diabetes risk do not merely serve as markers for the presence of sleep - disordered breathing . in addition to the known higher risk of obesity associated with sleep apnea , other factors associated with sleep - disordered breathing may be involved in higher diabetes risk and include abnormal sympathetic activity and release of proinflammatory mediators such as tumor necrosis factor- and interleukin-6 ( 27 ) . in support of the latter , we observed an independent association between sleep apnea and diabetes risk after adjustment for bmi . this suggests that manifestations of sleep apnea other than the impact of greater general adiposity may be associated with diabetes risk , although it is possible that incomplete adjustment due to residual confounding occurred due to the inaccuracy of bmi in characterizing body composition . the association between trouble sleeping or shorter sleep duration and diabetes risk independent of associated weight gain and sleep apnea is not well understood , but it may be related to both changes in insulin sensitivity , secretion , and glucose effectiveness . one investigation reported significant or borderline - significant reductions in insulin sensitivity , insulin secretion , glucose effectiveness , and the disposition index immediately after a 5-day period of sleep deprivation ( 4 h of sleep per night ) among 11 healthy lean males in a controlled setting ( 28 ) . all these changes have been associated with higher risk of the development of type 2 diabetes ( 29 ) . several characteristics of military service were significantly associated with lower diabetes risk in multivariable models , including deployment , officer pay grade , and remaining in military service . for most comparisons , military deployment with or without combat exposure was related to a lower risk for diabetes , possibly reflecting selection of people medically fit for deployment who are at lower risk for diabetes . the mental health conditions independently associated with diabetes risk included those characterized by excessive stress , including ptsd and panic disorder . a review of the literature on stress and diabetes risk concluded that both general emotional and job stress were repeatedly associated with a higher risk of developing diabetes in adults ( 30 ) . overactivation of the hypothalamic - pituitary axis and abnormal stimulation of the sympathetic nervous system are thought to accompany excessive levels of stress , with accumulation of visceral fat and promotion of an inflammatory state with greater insulin resistance ( 31 ) . a previous analysis of the members of this cohort recruited in 2001 and followed for 3 years similarly demonstrated a higher diabetes risk associated with ptsd symptoms . first , type 2 diabetes was determined using self - reported data , and it was not confirmed by medical record review . however , because self - reported diabetes compared with physician diagnosis or medical record data have been shown to have a high sensitivity ( 7099% ; median 81% ) and specificity ( 9299% ) , there is less likelihood for misclassification ( 32 ) . also , it is possible that individuals self - reporting type 1 diabetes were included in this analysis . however , individuals with a type 1 diabetes diagnosis in their dod medical records were excluded , although this elimination process would miss those participants no longer receiving care from military treatment facilities after discharge or as reservists . in addition , positive screens for mental disorders were determined from self - report . also , we were unable to adjust for diet or family history , which are known risk factors for diabetes . we expect these findings to generalize to younger populations who are sufficiently healthy to enter into the u.s . armed forces , but it is unclear whether the results would apply to dissimilar populations . self - report of clinician - diagnosed sleep apnea was obtained , and the sensitivity and specificity of this report compared with the medical record are not known . medical encounter data were not used to confirm diagnoses of diabetes or sleep apnea in this cohort due to the potential for some of its members , particularly those in the reserves or national guard , to have received care in non - dod facilities . inaccurate classification of sleep apnea and other covariates potentially may have limited our ability to adjust for confounding . finally , self - reported sleep data were assessed during the prior 4 weeks , and sleep duration was rounded to the nearest whole hour , potentially biasing the estimate of actual sleep time ( 3335 ) . this is the first study , to our knowledge , to examine baseline sleep characteristics in relation to incident diabetes while adjusting for several mental health conditions known to impair sleep and be associated with type 2 diabetes . furthermore , this research was conducted in a large population - based study of active duty and reserve / national guard u.s . military personnel from all service branches . additional strengths include the large sample size and up to 6-year duration of follow - up . moreover , no association was found between reporting type 2 diabetes at baseline and nonresponse to the first follow - up questionnaire among the first panel of enrollees , suggesting that the development of diabetes was unlikely to have influenced the probability of responding to the follow - up survey ( 36 ) . finally , previous evaluations of possible biases suggest the cohort is reasonably representative of military personnel in terms of baseline demographic and mental health characteristics , that participants report health and exposure data reliably , and that enrollment in the study is not influenced by health status ( 14,3740 ) . we conclude that trouble sleeping and sleep apnea predict higher diabetes risk , independent of multiple diabetes risk factors and several mental health conditions . of note , self - reported sleep apnea was also associated with a significantly higher risk for diabetes even after adjustment for other sleep characteristics , bmi , and mental health conditions . substitution of sleep duration for trouble sleeping in multivariable modeling yielded nearly identical results , with significantly higher risk seen with < 6 h of sleep . confirmation of these findings through further analyses may advance our understanding of diabetes pathophysiology and create new opportunities for prevention .

objectiveresearch has suggested that a higher risk of type 2 diabetes associated with sleep characteristics exists . however , studies have not thoroughly assessed the potential confounding effects of mental health conditions associated with alterations in sleep.research design and methodswe prospectively assessed the association between sleep characteristics and self - reported incident diabetes among millennium cohort study participants prospectively followed over a 6-year time period . surveys are administered approximately every 3 years and collect self - reported data on demographics , height , weight , lifestyle , features of military service , sleep , clinician - diagnosed diabetes , and mental health conditions assessed by the prime - md patient health questionnaire and the ptsd checklist civilian version . statistical methods for longitudinal data were used for data analysis.resultswe studied 47,093 participants ( mean 34.9 years of age ; mean bmi 26.0 kg / m2 ; 25.6% female ) . during 6 years of follow - up , 871 incident diabetes cases occurred ( annual incidence 3.6/1,000 person - years ) . in univariate analyses , incident diabetes was significantly more likely among participants with self - reported trouble sleeping , sleep duration < 6 h , and sleep apnea . participants reporting incident diabetes were also significantly older , of nonwhite race , of higher bmi , less likely to have been deployed , and more likely to have reported baseline symptoms of panic , anxiety , posttraumatic stress disorder , and depression . after adjusting for covariates , trouble sleeping ( odds ratio 1.21 [ 95% ci 1.031.42 ] ) and sleep apnea ( 1.78 [ 1.392.28 ] ) were significantly and independently related to incident diabetes.conclusionstrouble sleeping and sleep apnea predict diabetes risk independent of mental health conditions and other diabetes risk factors .

pubmed

we retrospectively reviewed the radiological and clinical findings of 13 patients with a pathologically confirmed mesenchymal hamartoma of the liver . the institutional review board approved the review of the radiological and clinical data for this study and waived the requirement for informed consent . a computed hospital information system was used to identify all patients with a pathologically proved mesenchymal hamartoma during a 15-year period . the pathological diagnosis for a mesenchymal hamartoma was based on overgrowth of the mesenchymal stroma and the proliferation of architecturally abnormal bile ducts with or without a cystic change , accompanied by periductal collaring of the stromal cells ( 1 , 9 ) . three pediatric radiologists retrospectively reviewed the radiological features of the hepatic mesenchymal hamartomas by consensus . ct scans were obtained with various third generation ct scanners at 80 - 120 ma , 100 - 120 kvp , and 5- to 10-mm thick sections . ct scans were obtained after an intravenous bolus injection of contrast media and portal venous phase scans of the liver were available for all patients . ct images of the tumor were evaluated for location , size , presence of septa within the cystic portion , ct attenuation of the tumor relative to the surrounding hepatic parenchyma ( low , iso , or high ) , pattern of contrast - enhancement ( homogeneous or heterogeneous enhancement of the solid portion , and septal enhancement of the cystic portion ) and presence of calcification on a pre - contrast ct scan . according to the extent of cystic component on the ct scans , we classified each tumor into one of three types : 1 ) multiseptated cystic - a purely cystic tumor with multiple thin septa ; 2 ) mixed cystic and solid - a partially cystic tumor with irregular thick septa ; 3 ) solid - a purely solid tumor without a cystic portion . the cystic portion was defined as an area of low attenuation that showed 0 - 40 hounsfield units ( hu ) of ct attenuation value . we classified the septal thickness as follows : thin ( < 2 mm in thickness ) or thick ( > 2 mm in thickness ) . ultrasonographic ( us ) scans of nine patients were available for review . among them , seven patients had a color or power doppler us examination . us images were evaluated for the presence of capsule , internal content of the cystic portion ( echogenic debris or fluid - fluid level ) , septal thickness within the cystic portion , and the echogenicity of the solid portion . the amount of mesenchymal stroma , the presence or absence of duct proliferation , the amount of hepatocytes , and the presence or absence of vascular proliferation was evaluated . the amount of mesenchymal stroma and hepatocytes were evaluated semi - quantitatively : little in < 10% of the mesenchymal hamartoma , moderate in 10 - 50% of the mesenchymal hamartoma and abundant in > 50% of the mesenchymal hamartoma . the age of the patients , size of the tumor and various histological findings were compared with the imaging features of the hepatic mesenchymal hamartomas among the three ct types using the kruskall - wallis test . chicago , il ) . a two - tailed p - value of < 0.05 was considered to be statistically significant . we retrospectively reviewed the radiological and clinical findings of 13 patients with a pathologically confirmed mesenchymal hamartoma of the liver . the institutional review board approved the review of the radiological and clinical data for this study and waived the requirement for informed consent . a computed hospital information system was used to identify all patients with a pathologically proved mesenchymal hamartoma during a 15-year period . the pathological diagnosis for a mesenchymal hamartoma was based on overgrowth of the mesenchymal stroma and the proliferation of architecturally abnormal bile ducts with or without a cystic change , accompanied by periductal collaring of the stromal cells ( 1 , 9 ) . three pediatric radiologists retrospectively reviewed the radiological features of the hepatic mesenchymal hamartomas by consensus . ct scans were obtained with various third generation ct scanners at 80 - 120 ma , 100 - 120 kvp , and 5- to 10-mm thick sections . ct scans were obtained after an intravenous bolus injection of contrast media and portal venous phase scans of the liver were available for all patients . ct images of the tumor were evaluated for location , size , presence of septa within the cystic portion , ct attenuation of the tumor relative to the surrounding hepatic parenchyma ( low , iso , or high ) , pattern of contrast - enhancement ( homogeneous or heterogeneous enhancement of the solid portion , and septal enhancement of the cystic portion ) and presence of calcification on a pre - contrast ct scan . according to the extent of cystic component on the ct scans , we classified each tumor into one of three types : 1 ) multiseptated cystic - a purely cystic tumor with multiple thin septa ; 2 ) mixed cystic and solid - a partially cystic tumor with irregular thick septa ; 3 ) solid - a purely solid tumor without a cystic portion . the cystic portion was defined as an area of low attenuation that showed 0 - 40 hounsfield units ( hu ) of ct attenuation value . we classified the septal thickness as follows : thin ( < 2 mm in thickness ) or thick ( > 2 mm in thickness ) . ultrasonographic ( us ) scans of nine patients were available for review . among them , seven patients had a color or power doppler us examination . us images were evaluated for the presence of capsule , internal content of the cystic portion ( echogenic debris or fluid - fluid level ) , septal thickness within the cystic portion , and the echogenicity of the solid portion . the amount of mesenchymal stroma , the presence or absence of duct proliferation , the amount of hepatocytes , and the presence or absence of vascular proliferation was evaluated . the amount of mesenchymal stroma and hepatocytes were evaluated semi - quantitatively : little in < 10% of the mesenchymal hamartoma , moderate in 10 - 50% of the mesenchymal hamartoma and abundant in > 50% of the mesenchymal hamartoma . the age of the patients , size of the tumor and various histological findings were compared with the imaging features of the hepatic mesenchymal hamartomas among the three ct types using the kruskall - wallis test . chicago , il ) . a two - tailed p - value of < 0.05 was considered to be statistically significant . the subjects were seven boys and six girls , aged from 6 months to 7 years 6 months ( mean age : 3 years 2 months ) . there was no significant difference of age ( p = 0.93 ) among the three ct types of tumors . the patients presented with abdominal distension ( n = 8) , a palpable mass ( n = 3 ) or an incidentally detected tumor during us ( n = 2 ) for other clinical indications : a patient ( case 8) with peutz - jeghers syndrome , and a patient ( case 13 ) that had a kasai operation for biliary atresia at one month of age , respectively . the serum level of alpha - fetoprotein , total and direct bilirubin level , alkaline phosphatase level , and level of transaminases ( aspartate transaminase and alanine transaminase ) were normal in all except two patients that had an elevated level of alpha - fetoprotein : the patient with biliary atresia ( case 13 ) and a 9-month - old girl ( case 5 ) . the tumor occurred in the right lobe of the liver in nine patients and in the left lobe in four patients . splenomegaly , ascites and retroperitoneal lymphadenopathy were noted in one patient that had biliary atresia . the longest diameter of the tumors ranged from 1.8 to 20 cm ( mean 13.0 cm ) and was larger than 10 cm in 11 patients ( 85% ) . there was no significant difference in the size of the tumors ( p = 0.79 ) among the three ct types of tumors . 1 ) , mixed solid and cystic in five patients ( 38% ) ( figs . 2 , 3 ) , and solid in four patients ( 31% ) ( figs . 4 , 5 ) . in four patients with a multiseptated cystic tumor , us ( n = 1 ) showed a well - defined tumor with variable cystic spaces and multiple thin septa in the liver ( fig . all of these tumors showed fine enhancing septa within the cystic tumor on post - contrast ct ( fig . neither calcification nor a solid portion was detected in these patients . in five patients with a mixed cystic and solid tumor , us ( n = 5 ) showed a well - defined tumor with variable - sized cystic spaces , irregularly thick septa , and a solid portion of variable extent with heterogeneous hyperechogenicity ( figs . 2a , 3a ) . in three patients , internal debris with fluid - fluid level was seen in the cystic portion of the tumor ( figs . 2a , 2c ) . on color and power doppler us ( n = 4 ) a tiny nodular intratumoral calcification was seen in one patient ( case 8) ( fig . = 3 ) showed a well - defined , isoechoic tumor ( n = 1 ) or slightly hyperechoic ( n = 2 ) ( fig . color or power doppler us ( n = 3 ) showed central ( fig . these tumors showed low attenuation on a pre - contrast ct scan in all patients , except for one with a high attenuating lesion . at post - contrast ct , 4b , 5c ) . in the patient that had biliary atresia ( case 13 ) , the tumor appeared as a 1.8-cm sized enhancing nodule with a peripheral rim of low attenuation at post - contrast ct ( fig . the mesenchymal hamartomas showed multiple variable sized cystic portions within the tumor in their gross morphology ( figs . each mesenchymal hamartoma with a multiseptated cystic appearance contained various amounts of myxoid or collagenous stroma and showed proliferation of the malformed bile ducts with periductal collaring of the stromal cells ( fig . all of the four patients with a multiseptated cystic tumor contained a small amount of hepatocytes ( < 10% ) on a semiquantitative evaluation . the mesenchymal hamartomas with a mixed solid and cystic appearance also contained various amounts of myxoid or collagenous stroma and showed proliferation of the malformed bile ducts with periductal collaring of the stromal cells ( fig . the amount of hepatocytes was small in three patients and moderate in two patients . in four patients with a solid mesenchymal hamartoma , a moderate ( n = 3 ) amount or abundant amount ( n = 1 ) of hepatocytes was found in the cords , islands or lobular pattern . the solid tumor also contained various amounts of mesenchymal stroma and proliferation of the small bile duct was seen ( fig . 4d ) . in one patient with an enhancing nodule with a peripheral rim of low attenuation ( case 13 ) , the enhancing portion revealed a large amount hepatocytes histologically with a rim of myxoid stroma . the amount of hepatocytes was significantly different among the multiseptated cystic , mixed solid and cystic , and solid tumors ( p = 0.042 ) . the other pathological findings such as the amount of mesenchymal stroma , the presence of a bile duct or vessel proliferation were not statistically significant among the three groups of the different imaging spectrum . the subjects were seven boys and six girls , aged from 6 months to 7 years 6 months ( mean age : 3 years 2 months ) . there was no significant difference of age ( p = 0.93 ) among the three ct types of tumors . the patients presented with abdominal distension ( n = 8) , a palpable mass ( n = 3 ) or an incidentally detected tumor during us ( n = 2 ) for other clinical indications : a patient ( case 8) with peutz - jeghers syndrome , and a patient ( case 13 ) that had a kasai operation for biliary atresia at one month of age , respectively . the serum level of alpha - fetoprotein , total and direct bilirubin level , alkaline phosphatase level , and level of transaminases ( aspartate transaminase and alanine transaminase ) were normal in all except two patients that had an elevated level of alpha - fetoprotein : the patient with biliary atresia ( case 13 ) and a 9-month - old girl ( case 5 ) . the tumor occurred in the right lobe of the liver in nine patients and in the left lobe in four patients . splenomegaly , ascites and retroperitoneal lymphadenopathy were noted in one patient that had biliary atresia . the longest diameter of the tumors ranged from 1.8 to 20 cm ( mean 13.0 cm ) and was larger than 10 cm in 11 patients ( 85% ) . there was no significant difference in the size of the tumors ( p = 0.79 ) among the three ct types of tumors . 1 ) , mixed solid and cystic in five patients ( 38% ) ( figs . 2 , 3 ) , and solid in four patients ( 31% ) ( figs . 4 , 5 ) . in four patients with a multiseptated cystic tumor , us ( n = 1 ) showed a well - defined tumor with variable cystic spaces and multiple thin septa in the liver ( fig . all of these tumors showed fine enhancing septa within the cystic tumor on post - contrast ct ( fig . neither calcification nor a solid portion was detected in these patients . in five patients with a mixed cystic and solid tumor , us ( n = 5 ) showed a well - defined tumor with variable - sized cystic spaces , irregularly thick septa , and a solid portion of variable extent with heterogeneous hyperechogenicity ( figs . 2a , 3a ) . in three patients , internal debris with fluid - fluid level was seen in the cystic portion of the tumor ( figs . 2a , 2c ) . on color and power doppler us ( n = 4 ) a tiny nodular intratumoral calcification was seen in one patient ( case 8) ( fig . = 3 ) showed a well - defined , isoechoic tumor ( n = 1 ) or slightly hyperechoic ( n = 2 ) ( fig . color or power doppler us ( n = 3 ) showed central ( fig . these tumors showed low attenuation on a pre - contrast ct scan in all patients , except for one with a high attenuating lesion . at post - contrast 4b , 5c ) . in the patient that had biliary atresia ( case 13 ) , the tumor appeared as a 1.8-cm sized enhancing nodule with a peripheral rim of low attenuation at post - contrast ct ( fig . the mesenchymal hamartomas showed multiple variable sized cystic portions within the tumor in their gross morphology ( figs . each mesenchymal hamartoma with a multiseptated cystic appearance contained various amounts of myxoid or collagenous stroma and showed proliferation of the malformed bile ducts with periductal collaring of the stromal cells ( fig . all of the four patients with a multiseptated cystic tumor contained a small amount of hepatocytes ( < 10% ) on a semiquantitative evaluation . the mesenchymal hamartomas with a mixed solid and cystic appearance also contained various amounts of myxoid or collagenous stroma and showed proliferation of the malformed bile ducts with periductal collaring of the stromal cells ( fig . the amount of hepatocytes was small in three patients and moderate in two patients . in four patients with a solid mesenchymal hamartoma , a moderate ( n = 3 ) amount or abundant amount ( n = 1 ) of hepatocytes was found in the cords , islands or lobular pattern . the solid tumor also contained various amounts of mesenchymal stroma and proliferation of the small bile duct was seen ( fig . 4d ) . in one patient with an enhancing nodule with a peripheral rim of low attenuation ( case 13 ) , the enhancing portion revealed a large amount hepatocytes histologically with a rim of myxoid stroma . the amount of hepatocytes was significantly different among the multiseptated cystic , mixed solid and cystic , and solid tumors ( p = 0.042 ) . the other pathological findings such as the amount of mesenchymal stroma , the presence of a bile duct or vessel proliferation were not statistically significant among the three groups of the different imaging spectrum . a mesenchymal hamartoma is the second most common benign hepatic tumor in children and accounts for 8% of all hepatic tumors in children ( 10 - 14 ) . histologically , a mesenchymal hamartoma appears as a disordered arrangement of mesenchyme , bile ducts and hepatocytes ( 1 - 3 , 9 , 15 ) . cords of normal - appearing hepatocytes are separated by zones of loose poorly cellular mesenchyme . cystic degeneration of the mesenchyme with resultant fluid accumulation from obstruction and dilatation of the lymphatics and/or by entrapped bile ducts may lead to enlargement of the tumor ( 12 ) . the margin between the liver and the tumor is distinct ; however , a true capsule is generally not present ( 2 ) . the level of serum alpha - fetoprotein , which is believed to be secreted by the proliferating hepatocytes within the tumor , can be elevated ( 13 , 14 ) . a multiseptated cystic appearance , which may be a typical finding of mesenchymal hamartoma , is rarely seen in other hepatic tumors in children . intratumoral calcification , which can be frequently detected in hepatoblastomas ( over 50% ) or infantile hemangioendotheliomas ( up to 40% ) ( 11 ) , has been reported very rarely for a mesenchymal hamartoma . ( 8) described the ct findings of a mesenchymal hamartoma containing central and peripheral calcifications in a 57-year - old woman . ( 15 ) also described the ct findings of the multiseptated cystic form of a mesenchymal hamartoma with a septal calcification in a 10-year - old boy . in this series although a mesenchymal hamartoma present most frequently as a multiseptated cystic or mixed solid and cystic tumor ( 1 - 5 ) , it can rarely occur as a solid tumor ( 6 - 9 ) . in this study , according to the extent of the cystic component on ct scans , we classified each tumor into one of three types for evaluating the imaging spectrum of this tumor . in this study , the tumor was totally solid in 31% of the patients , and one case of a solid mesenchymal hamartoma ( case 11 ) showed features of a so - called " mixed hamartoma . " the mixed hamartoma is a rare hamartomatous lesion composed of all cellular components of the normal liver , including a larger amount of hepatocytes , ductal proliferation , and hepatocellular - ductal transition . the mixed hamartoma and mesenchymal hamartoma may be in one disease spectrum , since there is some overlapping in the histological features of both hamartomas ( 16 , 17 ) . in children with a solid mesenchymal hamartoma , radiological differential diagnosis should include various hepatic tumors such as a hepatoblastoma , a hepatocellular carcinoma , an infantile hemangioendothelioma , a hepatic adenoma , or a focal nodular hyperplasia . heterogeneous enhancement and absence of a tumor capsule in a mesenchymal hamartoma can be helpful in the differential diagnosis from other tumors . as in one of our patients that had biliary atresia and a solid mesenchymal hamartoma ( case 13 ) , lack ( 18 ) described a mesenchymal hamartoma in a 5-month - old girl with biliary atresia , which was found incidentally during an exploratory laparotomy for obstructive jaundice . lack ( 18 ) showed an interest in this case because one of the theories for development of a mesenchymal hamartoma is biliary obstruction ( 19 ) . in conclusion , a mesenchymal hamartoma of the liver in children can show a wide spectrum of radiological features from a multiseptated cystic tumor to a mixed solid and cystic tumor , and even a solid tumor .

objectivea hepatic mesenchymal hamartoma is an uncommon benign tumor in children and little is known about the spectrum of its radiological features . the purpose of this study is to describe the spectrum of radiological features of a hepatic mesenchymal hamartoma in children.materials and methodsthirteen children with a pathologically confirmed hepatic mesenchymal hamartoma ( m : f = 7:6 ; mean age , 3 years 2 months ) were included in our study . ultrasonography ( us ) was performed in nine patients including color and power doppler us ( n = 7 ) . ct scans were performed in all patients . we evaluated the imaging findings of the hepatic mesenchymal hamartomas and the corresponding pathological features.resultseach patient had a single tumor ( mean diameter : 13 cm [ 1.8 - 20 cm ] ) . on ct and/or us , four patients ( 31% ) had a " multiseptated cystic tumor " , five patients ( 38% ) had a " mixed solid and cystic tumor " , and four patients ( 31% ) had a " solid tumor . " the septa of the cystic portion were thin in the multiseptated cystic tumors and irregularly thick in the mixed solid and cystic tumors as seen on us . on a post - contrast ct scan , solid portions or thick septa of the tumors showed heterogeneous enhancement . the amount of hepatocytes was significantly different among the three tumor groups according to the imaging spectrum ( p = 0.042).conclusiona hepatic mesenchymal hamartoma in children can show a wide spectrum of radiological features , from a multiseptated cystic tumor to a mixed solid and cystic tumor , and even a solid tumor .

pubmed

rheumatoid arthritis ( ra ) is a disabling and progressive chronic autoimmune disease that carries a significant burden.1 although ra affects people of all ages , its prevalence increases with increasing age and it is more common in women than in men.2,3 a prevalence of 0.5%1% and a mean annual incidence ( ie , rate of new cases ) of 0.02%0.05% have been estimated in studies carried out in northern europe and in north america.4 the prevalence of ra in southern europe has been estimated to be lower than that in northern europe , with rates of 0.45% and 0.66% reported in the two areas , respectively.5 the annual incidence of ra is also lower in southern than in northern european countries.6 estimates of prevalence are often very variable as a result of several factors , including the time period when the study was undertaken ; the age distribution of the country reporting the estimates ; differences in the health care organizations in the reporting countries ; and the source of information.6 similarly , changes in the epidemiology of ra are difficult to predict ; while some studies might suggest a decline in incidence among countries with high rates of ra,5 other investigators suggest that the incidence is expected to increase in europe because of the growing proportion of older people.7,8 ra is associated with a significant economic burden for patients , families , and health care systems.9 moreover , an increase in the costs associated with ra is anticipated as a result of the aging population.7 there is a great amount of variability in estimates of the cost of ra across countries ; these estimates are affected by methods of calculation , reimbursement policies , and availability ( and time of availability ) of drugs and social services . differences in country - specific treatment guidelines on when to introduce newer costly drug classes ( ie , biological agents ) for patients with severe active and erosive disease also contribute to the variation in costs across the european union ( eu).6 however , the macroeconomic condition and the treatment guidelines remain the most important determinants that drive choice of therapy.6 italy has the highest proportion of people aged 65 years or older in the eu and it has been estimated that , by 2029 , 25.7% of the population in italy will be 65 years.7 thus , the health care costs associated with age - related diseases like ra are likely to increase in italy . the introduction of newer costly therapies may further contribute to the expected increase in the economic burden of ra in italy . this review aims to evaluate the economic impact of ra in italy in the past two decades and to compare the costs of ra before and after the introduction of biological agents . in addition , the review will attempt to predict the future costs of ra in italy , and to compare these costs with those in other european countries . while this is not designed as a systematic review as such , it does follow a similar procedure for identification of publications of relevance . comprehensive electronic searches of pubmed / medline were performed using the search terms ( in any field ) : prevalence or cost and rheumatoid arthritis and italy , with no language restriction . the search identified 414 papers ; abstracts of these publications were assessed for their relevance . in total , six papers reporting data on the prevalence of ra in italy1015 and four papers reporting economic data1619 were initially considered as adequate for the objectives of this review . in addition , further data were extracted from the websites of italian or european public health agencies and from italian journals to add to the previously identified published literature on the health care costs of ra in italy . this led to the inclusion of a further two articles6,20 that add important information on the past and current costs of ra in italy , as well as on estimates of future costs . although the small population samples and limited coverage of the studies do not allow a firm estimate of the national prevalence of the disease , the studies do give a rough idea of the likely prevalence and also highlight potential geographical variations . a study10 undertaken in 19911992 in chiavari , a small town located on the ligurian coast , found that ra had a prevalence of 0.33% ( 95% confidence interval [ ci ] 0.130.53 ) in the general population , 0.13% ( 95% ci 00.31 ) in men , and 0.51% ( 95% ci 0.180.84 ) in women . this study included 3,294 individuals ( 73.9% of those contacted ) , aged 16 years , identified from four general practices . the mean age of subjects was 48.3 years , which may account for the relatively low prevalence reported in this study . a higher prevalence of 0.46% ( 95% ci 0.330.59 ) was reported in another study11 conducted from april 2004 to june 2004 in 2,155 subjects aged 18 years in the marche region . similar estimates were reported from a survey conducted in 20022003 in tempio pausania ( northern sardinia ) that involved 30,264 subjects aged 18 years;12 prevalence was estimated to be 0.46% in the general population , 0.73% in women , and 0.19% in men . another study carried out in the province of pisa in the years 2006 and 200714 evaluated the prevalence of ra , the reliability of the ra diagnosis and prevalence estimated by general practitioners ( gps ) , and the economic impact of the disease in a total of 26,709 subjects aged over 18 years . each gp completed a questionnaire on their patients ; patients considered by gps to have ra were then invited to attend an appointment at a specialist rheumatology center for confirmation or rejection of the diagnosis made . the estimated prevalence of correctly diagnosed ra was 0.51% ( 95% ci 0.440.57 ) and the reliability of gps when making a diagnosis of ra was 69% overall , although there was a high degree of heterogeneity among gps . benucci et al13 evaluated the incidence of early ra ( new cases ) in a suburban area of florence between september 2005 and august 2006 and suggested a rate of 0.98 ( 95% ci 0.641.32 ) in the overall population , and 1.42 ( 95% ci 0.851.99 ) for females and 0.51 ( 95% ci 0.160.87 ) for males . the mean age of newly diagnosed subjects was 47.7 years in females and 54.9 years in males . the incidence rate observed in this study was higher than that observed in other european countries , possibly due to tight adherence to diagnostic criteria for early ra and to the use of the criteria of the american college of rheumatology ( acr)21 as a method of assessment . the most recent treatment - driven estimation of the prevalence and incidence of ra in italy , performed in a cohort of 2,268,514 males and 2,446,769 females , and based on a diagnosis made according to prescription of at least three ra - specific drugs ( corticosteroids , disease - modifying anti - rheumatic drugs , and biological agents ) , was calculated for the year 2011.15 patients were classified as follows : those on specific drugs were classed as having active ra ; those who had never had more than four prescriptions in the past were classed as unlikely ra ; and those previously on chronic treatment but who had discontinued therapy for > 1 year were classed as having ra in remission . a diagnosis of ra was made by a qualified specialist in a total of 22,801 of these subjects ( 0.48% ) , with a prevalence of active ra , ra in remission , and confirmed ra ( active + remission ra ) of 0.32% , 0.09% , and 0.41% , respectively . the yearly incidence of active ra per 100,000 subjects was 48 ( 95% ci 4057 ) for females and 20 ( 95% ci 1030 ) for males . the female : male ratio for both prevalence and incidence was ~3.5:1 before the fifth decade of life and tended to decline to about 2:1 in subjects aged > 70 years . a modeling analysis of the prevalence of ra in 27 countries in the eu ( plus iceland , norway , and switzerland ) conducted in 20086 estimated that the overall prevalence of ra in italy was 0.49% and was exactly the same as the calculated average prevalence in europe . in this analysis , italy was ranked as eighth in terms of estimated prevalence of ra and was one of four european countries ( together with the uk , sweden , and germany ) in which subjects aged 65 years contributed more than 50% of the diagnosed cases of ra . the data also showed that italy and germany were the european countries with the highest proportion of people aged 65 years . taken together , these data6 suggest that the prevalence of ra will further increase in europe and in italy in line with projected increases in the elderly population . few studies have estimated the costs associated with ra in italy before the availability of biological agents . a retrospective , prevalence - based , multicenter , cost - of - illness study , undertaken in 1998 in ten rheumatology institutes located in northern , central , and southern italy,19 evaluated the direct ( hospitalizations , specialist visits , treatments , diagnostics , and nonmedical costs ) , indirect ( productivity loss and informal care ) , and intangible ( deterioration in health - related quality of life [ hrqol ] of patients , their families , and their friends ) costs associated with ra in 200 patients aged 1865 years . participants were categorized into four classes of severity according to the acr ra functional status index in place at the time of the study.22 the results showed that indirect costs accounted for the largest part of the disease management costs and that social costs ( direct plus indirect costs ) increased as ra worsened . the calculated average annual cost for each patient varied significantly depending on disease severity and correlated with the health assessment questionnaire disability index23 and acr criteria.22 total costs ranged from 3,718.3 for patients in functional class i to 22,946.0 for patients in class iv . both the direct costs ( respectively , 1,643.4 , 2,910.2 , 4,236.5 , and 5,696.8 per patient from class i to class iv ) and the indirect costs ( 2,074.9 , 9,566.4 , 12,183.1 , and 17,249.2 per patient , respectively ) over the 12 months of observation increased significantly ( p<0.0005 ) across the four functional classes . the modeling study conducted in 20086 used a specifically developed model to estimate the total cost of ra in europe . costs , extracted from the studies identified in a comprehensive literature review , were separated into different categories : medical costs , drugs , nonmedical costs , informal care costs , and indirect costs , and inflated to 2008 values . the estimated costs per patient were then combined with country- and age - specific prevalence data to obtain a figure for the total cost of ra in each country . the mean estimated annual cost per patient in europe with ra was 12,902 , with a clear difference between western europe ( mean 14,997 ) and central / eastern europe ( mean 3,752 ) . the total mean annual cost of the disease across all european countries was estimated at about 25.1 billion . the estimated mean annual cost per patient in the study countries ranged from about 2,000 to 21,000 , the estimated mean annual cost per patient in italy was 11,546 in line with the mean for europe as a whole . in italy , mean costs were estimated at 4,552 for direct costs ( excluding biological agents ) , 731 for biological agents , 3,290 for informal care , and 2,972 for indirect costs . this distribution indicates that , at the time of this modeling study ( 2008 ) , direct costs in europe overall represented 43% of the total expenditure . notably , at the time of the analysis , italy and germany had the lowest use of biologics in western europe due to tight budget control policies . for example , in italy , biologics could only be prescribed in the hospital setting and use of such drugs was limited to a small percentage of the total expenditure.6 despite individual differences in costs between countries , costs outside the health care sector , such as informal care and productivity loss , represented the greater part of the costs of ra in all countries.6 the structure of the costs of ra has changed over the years , with a substantial increase in the direct costs of the disease largely as a result of the availability of newer , more costly biological agents . for example , in france , the direct annual costs of ra were estimated to be 4,000 in 2000 ( before the introduction of biological agents)24 and about 12,000 in a 2005 study in which 20% of patients were receiving these agents.25 although the introduction of biological agents has contributed to increased direct medical costs,26,27 their use is associated with marked improvements in disease activity , joint damage , and loss of productivity.18 furthermore , data from the consortium of rheumatology researchers of north america registry have shown that the use of biological agents is associated with a reduction in the risk of cardiovascular events in patients with ra.28 in a health economic analysis carried out in the province of pisa in the years 2006 and 2007,14 the mean annual cost per patient with ra was estimated to be 5,878 ( median 6,434 ; interquartile range 6697,052 ) , with high variability , mainly depending on the degree of disability . in this analysis , more than 90% of the overall annual cost per patient was due to the medical and nonmedical components of direct costs . a more recently published literature review16 evaluated the mean annual social costs per patient and the total social cost of ra in the last decade in italy . data from each study ( table 2 ) , when further analyzed , showed that the mean annual social cost ( ie , overall direct plus indirect costs ) of ra was 13,595 per adult patient ( 2012 values ) . based on prevalence data , it was estimated that ra resulted in a total social cost of 3.5 billion in italy per year . direct medical costs accounted for 21% of the total costs , while the remaining 79% were nonmedical costs ( direct nonmedical costs and indirect costs ) . nonmedical and indirect costs were mainly sustained by individual households for informal care ( 40% of the total cost ) , and by the economy ( due to absence from work of patients and caregivers ; 31% of the total cost ) . based on these data , it is evident that assessment of the economic burden of ra based solely on the evaluation of direct medical costs gives a limited view of the total economic burden of the disease . italy has the second highest percentage of population 65 years of age in the world,29 therefore , an increase in the costs of ra in italy is expected due to the aging population . since mean costs per patient increase with increasing functional disability , reliable health - economic evaluations should estimate the long - term consequences of the changing course of the disease and the effects of preventing or delaying the development of severe disability.30 short - term analyses may be misleading as they tend to neglect the importance of modifications in the course of the disease as a result of treatment intervention and its positive impact on the deterioration of hrqol . a systematic literature review18 on the economic consequences of , and pharmacoeconomic issues relating to , ra , based on a total of 127 published articles , has shown that disease severity , functional disability , age , and socioeconomic status are the most relevant predictors of cost increase in ra . a published editorial20 has estimated the economic impact of ra in italy over the next 30 years using cost - of - illness modeling . this analysis assumed that the costs of ra varied as the disease evolved , and classified disease by stage according to the steinbrocker radiological classification.31 using inferential statistical methods , the probability of evolution toward a more severe stage of the disease in the absence of treatment was estimated . a total of 411,692 patients with ra were classified according to the four stages of the disease and their progression over the next 30 years was simulated . the results showed that , in the next 30 years , the annual cost of ra will increase from 3.3 billion ( for 2009 ) to a peak of 4 billion ( for 2029 ) , including both direct and indirect costs . cost estimates will vary between regions , with higher costs estimated in lombardy ( 430500 million ) , lazio ( 300330 million ) , sicily ( 238281 million ) , and emilia - romagna ( 131187 million ) . this suggests that costs will be only partially correlated with the number of inhabitants in different italian regions and will possibly depend on differing regional health care policies . expenditure will not greatly differ between workers ( from 320 million in 2009 to 420 million in 2029 ) and homemakers ( from 318 million in 2009 to 421 million in 2029 ) . the disease was also estimated to have a significant impact on costs due to loss of productivity that are supported by the italian national health care system ( nhs ) , which are predicted to increase from 0.67% of total social security spending in 2009 to 0.7% of this spending in 2029 . in addition , ra was predicted to account for a total of 1% of all nhs costs in 2029 . these data indicate that the costs associated with untreated ra would make a significant contribution to nhs and societal costs in the next decades , thus confirming that delaying early diagnosis and intervention will result in increased overall costs for the community . the development of biosimilar biologics may alter the landscape of biologics and their associated costs in the future . physicians attitudes toward the interchangeability of drugs , indications from regulatory authorities , and national recommendations will have a major impact on the use of biosimilars in the management of ra . while , a statement position from the italian society of rheumatology has raised concerns about the true equivalence of biosimilars in rheumatic diseases and hence on the potential for interchangeability of drugs , studies in ra currently support the similarity of biosimilars and their originators.32 furthermore , as biologics require a more sophisticated manufacturing process than that of existing conventional medications , tight regulation will be required to avoid any possible additional safety risks to make these agents truly cost - effective.33 although the small population samples and limited coverage of the studies do not allow a firm estimate of the national prevalence of the disease , the studies do give a rough idea of the likely prevalence and also highlight potential geographical variations . a study10 undertaken in 19911992 in chiavari , a small town located on the ligurian coast , found that ra had a prevalence of 0.33% ( 95% confidence interval [ ci ] 0.130.53 ) in the general population , 0.13% ( 95% ci 00.31 ) in men , and 0.51% ( 95% ci 0.180.84 ) in women . this study included 3,294 individuals ( 73.9% of those contacted ) , aged 16 years , identified from four general practices . the mean age of subjects was 48.3 years , which may account for the relatively low prevalence reported in this study . a higher prevalence of 0.46% ( 95% ci 0.330.59 ) was reported in another study11 conducted from april 2004 to june 2004 in 2,155 subjects aged 18 years in the marche region . similar estimates were reported from a survey conducted in 20022003 in tempio pausania ( northern sardinia ) that involved 30,264 subjects aged 18 years;12 prevalence was estimated to be 0.46% in the general population , 0.73% in women , and 0.19% in men . another study carried out in the province of pisa in the years 2006 and 200714 evaluated the prevalence of ra , the reliability of the ra diagnosis and prevalence estimated by general practitioners ( gps ) , and the economic impact of the disease in a total of 26,709 subjects aged over 18 years . each gp completed a questionnaire on their patients ; patients considered by gps to have ra were then invited to attend an appointment at a specialist rheumatology center for confirmation or rejection of the diagnosis made . the estimated prevalence of correctly diagnosed ra was 0.51% ( 95% ci 0.440.57 ) and the reliability of gps when making a diagnosis of ra was 69% overall , although there was a high degree of heterogeneity among gps . benucci et al13 evaluated the incidence of early ra ( new cases ) in a suburban area of florence between september 2005 and august 2006 and suggested a rate of 0.98 ( 95% ci 0.641.32 ) in the overall population , and 1.42 ( 95% ci 0.851.99 ) for females and 0.51 ( 95% ci 0.160.87 ) for males . the mean age of newly diagnosed subjects was 47.7 years in females and 54.9 years in males . the incidence rate observed in this study was higher than that observed in other european countries , possibly due to tight adherence to diagnostic criteria for early ra and to the use of the criteria of the american college of rheumatology ( acr)21 as a method of assessment . the most recent treatment - driven estimation of the prevalence and incidence of ra in italy , performed in a cohort of 2,268,514 males and 2,446,769 females , and based on a diagnosis made according to prescription of at least three ra - specific drugs ( corticosteroids , disease - modifying anti - rheumatic drugs , and biological agents ) , was calculated for the year 2011.15 patients were classified as follows : those on specific drugs were classed as having active ra ; those who had never had more than four prescriptions in the past were classed as unlikely ra ; and those previously on chronic treatment but who had discontinued therapy for > 1 year were classed as having ra in remission . a diagnosis of ra was made by a qualified specialist in a total of 22,801 of these subjects ( 0.48% ) , with a prevalence of active ra , ra in remission , and confirmed ra ( active + remission ra ) of 0.32% , 0.09% , and 0.41% , respectively . the yearly incidence of active ra per 100,000 subjects was 48 ( 95% ci 4057 ) for females and 20 ( 95% ci 1030 ) for males . the female : male ratio for both prevalence and incidence was ~3.5:1 before the fifth decade of life and tended to decline to about 2:1 in subjects aged > 70 years . a modeling analysis of the prevalence of ra in 27 countries in the eu ( plus iceland , norway , and switzerland ) conducted in 20086 estimated that the overall prevalence of ra in italy was 0.49% and was exactly the same as the calculated average prevalence in europe . in this analysis , italy was ranked as eighth in terms of estimated prevalence of ra and was one of four european countries ( together with the uk , sweden , and germany ) in which subjects aged 65 years contributed more than 50% of the diagnosed cases of ra . the data also showed that italy and germany were the european countries with the highest proportion of people aged 65 years . taken together , these data6 suggest that the prevalence of ra will further increase in europe and in italy in line with projected increases in the elderly population . few studies have estimated the costs associated with ra in italy before the availability of biological agents . a retrospective , prevalence - based , multicenter , cost - of - illness study , undertaken in 1998 in ten rheumatology institutes located in northern , central , and southern italy,19 evaluated the direct ( hospitalizations , specialist visits , treatments , diagnostics , and nonmedical costs ) , indirect ( productivity loss and informal care ) , and intangible ( deterioration in health - related quality of life [ hrqol ] of patients , their families , and their friends ) costs associated with ra in 200 patients aged 1865 years . participants were categorized into four classes of severity according to the acr ra functional status index in place at the time of the study.22 the results showed that indirect costs accounted for the largest part of the disease management costs and that social costs ( direct plus indirect costs ) increased as ra worsened . the calculated average annual cost for each patient varied significantly depending on disease severity and correlated with the health assessment questionnaire disability index23 and acr criteria.22 total costs ranged from 3,718.3 for patients in functional class i to 22,946.0 for patients in class iv . both the direct costs ( respectively , 1,643.4 , 2,910.2 , 4,236.5 , and 5,696.8 per patient from class i to class iv ) and the indirect costs ( 2,074.9 , 9,566.4 , 12,183.1 , and 17,249.2 per patient , respectively ) over the 12 months of observation increased significantly ( p<0.0005 ) across the four functional classes . the modeling study conducted in 20086 used a specifically developed model to estimate the total cost of ra in europe . costs , extracted from the studies identified in a comprehensive literature review , were separated into different categories : medical costs , drugs , nonmedical costs , informal care costs , and indirect costs , and inflated to 2008 values . the estimated costs per patient were then combined with country- and age - specific prevalence data to obtain a figure for the total cost of ra in each country . the mean estimated annual cost per patient in europe with ra was 12,902 , with a clear difference between western europe ( mean 14,997 ) and central / eastern europe ( mean 3,752 ) . the total mean annual cost of the disease across all european countries was estimated at about 25.1 billion . the estimated mean annual cost per patient in the study countries ranged from about 2,000 to 21,000 , the estimated mean annual cost per patient in italy was 11,546 in line with the mean for europe as a whole . in italy , ( excluding biological agents ) , 731 for biological agents , 3,290 for informal care , and 2,972 for indirect costs . this distribution indicates that , at the time of this modeling study ( 2008 ) , direct costs in europe overall represented 43% of the total expenditure . notably , at the time of the analysis , italy and germany had the lowest use of biologics in western europe due to tight budget control policies . for example , in italy , biologics could only be prescribed in the hospital setting and use of such drugs was limited to a small percentage of the total expenditure.6 despite individual differences in costs between countries , costs outside the health care sector , such as informal care and productivity loss , represented the greater part of the costs of ra in all countries.6 the structure of the costs of ra has changed over the years , with a substantial increase in the direct costs of the disease largely as a result of the availability of newer , more costly biological agents . for example , in france , the direct annual costs of ra were estimated to be 4,000 in 2000 ( before the introduction of biological agents)24 and about 12,000 in a 2005 study in which 20% of patients were receiving these agents.25 although the introduction of biological agents has contributed to increased direct medical costs,26,27 their use is associated with marked improvements in disease activity , joint damage , and loss of productivity.18 furthermore , data from the consortium of rheumatology researchers of north america registry have shown that the use of biological agents is associated with a reduction in the risk of cardiovascular events in patients with ra.28 in a health economic analysis carried out in the province of pisa in the years 2006 and 2007,14 the mean annual cost per patient with ra was estimated to be 5,878 ( median 6,434 ; interquartile range 6697,052 ) , with high variability , mainly depending on the degree of disability . in this analysis , more than 90% of the overall annual cost per patient was due to the medical and nonmedical components of direct costs . a more recently published literature review16 evaluated the mean annual social costs per patient and the total social cost of ra in the last decade in italy . data from each study ( table 2 ) , when further analyzed , showed that the mean annual social cost ( ie , overall direct plus indirect costs ) of ra was 13,595 per adult patient ( 2012 values ) . based on prevalence data , it was estimated that ra resulted in a total social cost of 3.5 billion in italy per year . direct medical costs accounted for 21% of the total costs , while the remaining 79% were nonmedical costs ( direct nonmedical costs and indirect costs ) . nonmedical and indirect costs were mainly sustained by individual households for informal care ( 40% of the total cost ) , and by the economy ( due to absence from work of patients and caregivers ; 31% of the total cost ) . based on these data , it is evident that assessment of the economic burden of ra based solely on the evaluation of direct medical costs gives a limited view of the total economic burden of the disease . italy has the second highest percentage of population 65 years of age in the world,29 therefore , an increase in the costs of ra in italy is expected due to the aging population . since mean costs per patient increase with increasing functional disability , reliable health - economic evaluations should estimate the long - term consequences of the changing course of the disease and the effects of preventing or delaying the development of severe disability.30 short - term analyses may be misleading as they tend to neglect the importance of modifications in the course of the disease as a result of treatment intervention and its positive impact on the deterioration of hrqol . a systematic literature review18 on the economic consequences of , and pharmacoeconomic issues relating to , ra , based on a total of 127 published articles , has shown that disease severity , functional disability , age , and socioeconomic status are the most relevant predictors of cost increase in ra . a published editorial20 has estimated the economic impact of ra in italy over the next 30 years using cost - of - illness modeling . this analysis assumed that the costs of ra varied as the disease evolved , and classified disease by stage according to the steinbrocker radiological classification.31 using inferential statistical methods , the probability of evolution toward a more severe stage of the disease in the absence of treatment was estimated . a total of 411,692 patients with ra were classified according to the four stages of the disease and their progression over the next 30 years was simulated . the results showed that , in the next 30 years , the annual cost of ra will increase from 3.3 billion ( for 2009 ) to a peak of 4 billion ( for 2029 ) , including both direct and indirect costs . cost estimates will vary between regions , with higher costs estimated in lombardy ( 430500 million ) , lazio ( 300330 million ) , sicily ( 238281 million ) , and emilia - romagna ( 131187 million ) . this suggests that costs will be only partially correlated with the number of inhabitants in different italian regions and will possibly depend on differing regional health care policies . expenditure will not greatly differ between workers ( from 320 million in 2009 to 420 million in 2029 ) and homemakers ( from 318 million in 2009 to 421 million in 2029 ) . the disease was also estimated to have a significant impact on costs due to loss of productivity that are supported by the italian national health care system ( nhs ) , which are predicted to increase from 0.67% of total social security spending in 2009 to 0.7% of this spending in 2029 . in addition , ra was predicted to account for a total of 1% of all nhs costs in 2029 . these data indicate that the costs associated with untreated ra would make a significant contribution to nhs and societal costs in the next decades , thus confirming that delaying early diagnosis and intervention will result in increased overall costs for the community . the development of biosimilar biologics may alter the landscape of biologics and their associated costs in the future . physicians attitudes toward the interchangeability of drugs , indications from regulatory authorities , and national recommendations will have a major impact on the use of biosimilars in the management of ra . while , a statement position from the italian society of rheumatology has raised concerns about the true equivalence of biosimilars in rheumatic diseases and hence on the potential for interchangeability of drugs , studies in ra currently support the similarity of biosimilars and their originators.32 furthermore , as biologics require a more sophisticated manufacturing process than that of existing conventional medications , tight regulation will be required to avoid any possible additional safety risks to make these agents truly cost - effective.33 few studies have estimated the costs associated with ra in italy before the availability of biological agents . a retrospective , prevalence - based , multicenter , cost - of - illness study , undertaken in 1998 in ten rheumatology institutes located in northern , central , and southern italy,19 evaluated the direct ( hospitalizations , specialist visits , treatments , diagnostics , and nonmedical costs ) , indirect ( productivity loss and informal care ) , and intangible ( deterioration in health - related quality of life [ hrqol ] of patients , their families , and their friends ) costs associated with ra in 200 patients aged 1865 years . participants were categorized into four classes of severity according to the acr ra functional status index in place at the time of the study.22 the results showed that indirect costs accounted for the largest part of the disease management costs and that social costs ( direct plus indirect costs ) increased as ra worsened . the calculated average annual cost for each patient varied significantly depending on disease severity and correlated with the health assessment questionnaire disability index23 and acr criteria.22 total costs ranged from 3,718.3 for patients in functional class i to 22,946.0 for patients in class iv . both the direct costs ( respectively , 1,643.4 , 2,910.2 , 4,236.5 , and 5,696.8 per patient from class i to class iv ) and the indirect costs ( 2,074.9 , 9,566.4 , 12,183.1 , and 17,249.2 per patient , respectively ) over the 12 months of observation increased significantly ( p<0.0005 ) across the four functional classes . the modeling study conducted in 20086 used a specifically developed model to estimate the total cost of ra in europe . costs , extracted from the studies identified in a comprehensive literature review , were separated into different categories : medical costs , drugs , nonmedical costs , informal care costs , and indirect costs , and inflated to 2008 values . the estimated costs per patient were then combined with country- and age - specific prevalence data to obtain a figure for the total cost of ra in each country . the mean estimated annual cost per patient in europe with ra was 12,902 , with a clear difference between western europe ( mean 14,997 ) and central / eastern europe ( mean 3,752 ) . the total mean annual cost of the disease across all european countries was estimated at about 25.1 billion . the estimated mean annual cost per patient in the study countries ranged from about 2,000 to 21,000 , the estimated mean annual cost per patient in italy was 11,546 in line with the mean for europe as a whole . in italy , ( excluding biological agents ) , 731 for biological agents , 3,290 for informal care , and 2,972 for indirect costs . this distribution indicates that , at the time of this modeling study ( 2008 ) , direct costs in europe overall represented 43% of the total expenditure . notably , at the time of the analysis , italy and germany had the lowest use of biologics in western europe due to tight budget control policies . for example , in italy , biologics could only be prescribed in the hospital setting and use of such drugs was limited to a small percentage of the total expenditure.6 despite individual differences in costs between countries , costs outside the health care sector , such as informal care and productivity loss , represented the greater part of the costs of ra in all countries.6 the structure of the costs of ra has changed over the years , with a substantial increase in the direct costs of the disease largely as a result of the availability of newer , more costly biological agents . for example , in france , the direct annual costs of ra were estimated to be 4,000 in 2000 ( before the introduction of biological agents)24 and about 12,000 in a 2005 study in which 20% of patients were receiving these agents.25 although the introduction of biological agents has contributed to increased direct medical costs,26,27 their use is associated with marked improvements in disease activity , joint damage , and loss of productivity.18 furthermore , data from the consortium of rheumatology researchers of north america registry have shown that the use of biological agents is associated with a reduction in the risk of cardiovascular events in patients with ra.28 in a health economic analysis carried out in the province of pisa in the years 2006 and 2007,14 the mean annual cost per patient with ra was estimated to be 5,878 ( median 6,434 ; interquartile range 6697,052 ) , with high variability , mainly depending on the degree of disability . in this analysis , more than 90% of the overall annual cost per patient was due to the medical and nonmedical components of direct costs . a more recently published literature review16 evaluated the mean annual social costs per patient and the total social cost of ra in the last decade in italy . data from each study ( table 2 ) , when further analyzed , showed that the mean annual social cost ( ie , overall direct plus indirect costs ) of ra was 13,595 per adult patient ( 2012 values ) . based on prevalence data , it was estimated that ra resulted in a total social cost of 3.5 billion in italy per year . direct medical costs accounted for 21% of the total costs , while the remaining 79% were nonmedical costs ( direct nonmedical costs and indirect costs ) . nonmedical and indirect costs were mainly sustained by individual households for informal care ( 40% of the total cost ) , and by the economy ( due to absence from work of patients and caregivers ; 31% of the total cost ) . based on these data , it is evident that assessment of the economic burden of ra based solely on the evaluation of direct medical costs gives a limited view of the total economic burden of the disease . italy has the second highest percentage of population 65 years of age in the world,29 therefore , an increase in the costs of ra in italy is expected due to the aging population . since mean costs per patient increase with increasing functional disability , reliable health - economic evaluations should estimate the long - term consequences of the changing course of the disease and the effects of preventing or delaying the development of severe disability.30 short - term analyses may be misleading as they tend to neglect the importance of modifications in the course of the disease as a result of treatment intervention and its positive impact on the deterioration of hrqol . a systematic literature review18 on the economic consequences of , and pharmacoeconomic issues relating to , ra , based on a total of 127 published articles , has shown that disease severity , functional disability , age , and socioeconomic status are the most relevant predictors of cost increase in ra . a published editorial20 has estimated the economic impact of ra in italy over the next 30 years using cost - of - illness modeling . this analysis assumed that the costs of ra varied as the disease evolved , and classified disease by stage according to the steinbrocker radiological classification.31 using inferential statistical methods , the probability of evolution toward a more severe stage of the disease in the absence of treatment was estimated . a total of 411,692 patients with ra were classified according to the four stages of the disease and their progression over the next 30 years was simulated . the results showed that , in the next 30 years , the annual cost of ra will increase from 3.3 billion ( for 2009 ) to a peak of 4 billion ( for 2029 ) , including both direct and indirect costs . cost estimates will vary between regions , with higher costs estimated in lombardy ( 430500 million ) , lazio ( 300330 million ) , sicily ( 238281 million ) , and emilia - romagna ( 131187 million ) . this suggests that costs will be only partially correlated with the number of inhabitants in different italian regions and will possibly depend on differing regional health care policies . expenditure will not greatly differ between workers ( from 320 million in 2009 to 420 million in 2029 ) and homemakers ( from 318 million in 2009 to 421 million in 2029 ) . the disease was also estimated to have a significant impact on costs due to loss of productivity that are supported by the italian national health care system ( nhs ) , which are predicted to increase from 0.67% of total social security spending in 2009 to 0.7% of this spending in 2029 . in addition , ra was predicted to account for a total of 1% of all nhs costs in 2029 . these data indicate that the costs associated with untreated ra would make a significant contribution to nhs and societal costs in the next decades , thus confirming that delaying early diagnosis and intervention will result in increased overall costs for the community . the development of biosimilar biologics may alter the landscape of biologics and their associated costs in the future . physicians attitudes toward the interchangeability of drugs , indications from regulatory authorities , and national recommendations will have a major impact on the use of biosimilars in the management of ra . while , a statement position from the italian society of rheumatology has raised concerns about the true equivalence of biosimilars in rheumatic diseases and hence on the potential for interchangeability of drugs , studies in ra currently support the similarity of biosimilars and their originators.32 furthermore , as biologics require a more sophisticated manufacturing process than that of existing conventional medications , tight regulation will be required to avoid any possible additional safety risks to make these agents truly cost - effective.33 the studies conducted to date have indicated that the prevalence of ra in italy is ~0.5% . although most of these studies have been undertaken in small communities or at a regional level , and are therefore poorly representative of the national situation , the calculated prevalence has not changed over the years . the largest of the studies on the prevalence and incidence of ra in italy,15 which involved more than 4.5 million people , has clearly indicated that both the prevalence and incidence of ra progressively increase in both men and women up to the age of 80 years . as italy is undergoing a process of demographic transition , in which higher life expectancy will lead to a dramatic increase in the number of elderly people in the near future,7 it is expected that ra will impose a heavy socioeconomic burden on the nhs in coming years . it is well established that costs associated with ra are likely to rise as the disease progresses , accompanied by a substantial reduction in hrqol.17 as in other disabling diseases , much of the cost of ra occurs outside the health care system , because it is associated with loss of work capability and the need for informal care in the later stages of the disease.25 the severity and the progression of ra have a great influence on the costs of the disease . disease progression is associated with increasing functional impairment and reduced hrqol , which may lead to work absenteeism and inability and hence increased indirect costs , which are estimated to be twice as high as direct costs when the disease progresses.34,35 the introduction of biological drugs for the treatment of ra highlights the role of economics in the considerations for patient access to innovative but expensive treatments . inclusion in the reimbursement system is a very important condition for access to innovative treatments . cost - effectiveness is a further criterion to fulfill , in addition to a favorable risk benefit profile , for marketing approval by the european medicines agency and national medicines agencies . health - economic analyses conducted on a cross - sectional basis or over a short - term period reflect differences in the costs of drugs and are less sensitive to the long - term evolution of the disease in terms of its impact on social costs and loss of productivity . therefore , a reliable estimate of the cost - effectiveness of a drug class should not be based solely on the net costs of the compounds , but should also take into consideration the effects of such drugs on the progression of the disease , as rapid / severe progression can lead to huge increases in indirect social costs and loss of productivity . in fact , the use of biologics in the early stage of ra may reduce other health care costs by slowing the evolution of the disease . the extent to which the high direct costs incurred from early use of biologics may be balanced in part by other health care cost offsets due to the reduction in the need for surgery or other interventions beyond the fourth year of the disease , remains to be elucidated.36 although cost benefit and cost - effectiveness analyses are recognized as standard techniques for pharmacoeconomic evaluations , other methods , such as the cost utility analysis , are better tailored to evaluate the health improvement attributable to a treatment , in which health improvement is generally measured in quality - adjusted life - years ( qalys ) gained.37 in particular , the qaly combines life expectancy and hrqol by weighing life - years with a quality index named utility . as hrqol is among the most important outcomes in ra , in which it is difficult to simply study morbidity and mortality , interest has been focused on how well the different treatment programs improve patients physical functioning , social functioning , and psychological well - being . accurate pharmacoeconomic estimates may drive the use of new and more expensive therapies in the treatment of ra and help health care providers and budget holders to choose the most effective strategies for patients . analyses should not be focused only on the effects of a therapy on the onset of disability and the progression of joint destruction , but also on the adverse event profile of the drug and on its efficacy and safety over the long term , which are generally not investigated in randomized clinical trials . pharmacoeconomic analysis of the existing therapies for ra should be based on the awareness that treatment will prevent progression to the next level of disease severity or that the disease will take longer to progress , thus avoiding or delaying the high costs associated with more severe stages of the disease.38 this will help attenuate the impact on indirect and social costs due to disease progression and , most importantly , on the intangible costs due to the progression of the disease and associated comorbidities over the years . indeed , there is a high prevalence of comorbidities and their associated risk factors among ra patients , as shown in a recent international observational study conducted in 17 countries worldwide.39 the study has shown a high intercountry variability in the prevalence of comorbidities in ra and suggests that the diagnosis and management of such comorbidities is far from optimal . depression ( 15% of patients on average across countries ) was the most commonly observed comorbidity , followed by asthma ( 6.6% ) , gastrointestinal ulcer ( 10.8% ) , ischemic cardiovascular disease ( 6.0% ) , and solid tumors ( 4.5% ) . italy had the highest prevalence of hepatitis b ( 9.0% ) and c ( 6.6% ) , rate of patients with > 40 skin nevi ( 12% ) and of framingham score > 20% ( 59% ) . although the high prevalence of some of these comorbidities may be related to the medications used ( eg , corticosteroids ) or to traditional risk factors ( eg , tobacco smoking ) , some others ( eg , depression ) may be a direct consequence of systemic inflammation and its associated symptoms and effect on hrqol.40 a task force of the acr and the european league against rheumatism has recently highlighted the importance of comorbidities in the management of ra and in the treatment decision - making strategies.41 while the use of biologic response modifiers is not associated with an increased risk of malignancy,42 improvements in disease activity following 1-year treatment with biologic agents is associated with important gains in terms of qalys and hrqol,43 suggesting that successful treatment of ra can result in lower levels of depression . the recent published editorial20 on the 30-year costs of untreated ra in italy , which has shown that actual costs may rise in the coming years if the disease is not managed , highlights that costly interventions aimed at sparing future expenses are of particular relevance in countries like italy , where ra and associated comorbidities will make a significant contribution to the economic burden for the nhs , given the increased proportion of elderly people expected in years to come . further research is needed to develop estimates of the economic impact of different therapeutic approaches in patients with ra in italy . in particular , accurate pharmacoeconomic analyses would be valuable to help guide treatment decisions , ensuring effective spending without compromising the quality of care delivered .

this literature review examines available evidence on the current and past costs associated with rheumatoid arthritis ( ra ) in italy , together with the future health - economic prospects for the disease . studies have been conducted to date on the prevalence , or the associated costs , of ra in italy . although future changes in the incidence of ra are a matter of debate , the impact of ra on health care costs is expected to grow in coming decades in line with projected increases in life expectancy and in the proportion of elderly people in italy . it has been estimated that the indirect ( productivity loss and informal care ) and intangible ( deterioration in health - related quality of life ) costs of the disease will contribute to an increase in national health service expenditure , which will correspond to 1% of the total health care costs of the nation in the near future . the introduction of biological agents for the treatment of rheumatic diseases has resulted in an increase in the direct costs of ra ; however , economic analyses that exclude indirect costs will underestimate the full economic impact of ra . the effectiveness of innovative therapies in preventing disease progression and functional impairment may , over time , attenuate the cost impact of ra in terms of hospitalizations and work absenteeism . further research is needed to develop estimates of the economic impact of different therapeutic approaches in patients with ra in italy , in order to provide tools that can drive the choice of the most cost - effective therapeutic option while maintaining high - quality care .

pubmed

in prosthodontic practice , limited mouth opening in patients is a common occurrence . prosthetic rehabilitation of patient with restricted mouth opening due to any causative factors , presents difficulties at all stages , right from the preliminary impressions to the insertion of prosthesis . a maximal oral opening smaller than the size of a complete denture can make prosthetic treatment challenging . limited oral opening itself is not a disease but manifest as a consequence of certain conditions namely surgical treatments of orofacial carcinoma , cleft lip , trauma , burns , plummer vinson syndrome , scleroderma , trismus , rheumatism , temporomandibular joint dysfunction syndrome , oral submucous fibrosis , and any damage to masticatory muscles . a 71-year - old male patient reported to the department of prosthodontics , rama dental college and hospital kanpur ( up ) , india , with the chief complaint of missing teeth and wanted replacement of the missing teeth by means of fabrication of complete denture . on examination , it was found that patient had oral submucous fibrosis with heavy vertical fibrous bands and homogenous leukoplakia on both sides of buccal mucosa which lead to reduced mouth opening of 2.5 cm . patient was also diagnosed with bilateral angular cheilitis which often bleed on opening mouth wide . treatment plan included sectional maxillary and mandibular final impression procedures , followed by fabricating maxillary hinged and mandibular sectional and hinged complete denture . primary impression for maxillary arch was taken using smallest size edentulous stock tray with impression compound ( y dents ; mdm corp . ) , and after proper disinfection ( 1:213 iodophore ) , primary cast was poured in type ii dental plaster ( dentico ; neelkanth mine chem.).two autopolymerizing acrylic resin special trays ( trevelon ; dentsply india pvt . ltd . ) both trays were trimmed one side short of maxillary tuberosity.border molding was done with each tray using low fusing compound ( dpi ; pinnacle , india ) and the final impression was made with light body polyvinyl siloxane addition silicon impression material ( examix nds ; gc company).one sectional impression tray was poured with dental stone ( denstone , grade iii ) . over that cast second sectional impression tray was assembled and the remaining part of the cast was poured.similarly , mandibular primary impression was taken using smallest size stock tray and after disinfection , primary cast was poured . a conventional special tray was fabricated over the primary cast and that was later sectioned from the midline into two halves . one hole was drilled in the center of the tray handle and a brass dowel pin was passed through it , from one end to the other end of the hole . similarly , two holes were drilled over the premolar region on both sides of the tray and the two brass dowel pins were placed vertically in those holes and were connected by acrylic plate which was detachable.border molding and final impression were made individually with the each half of the tray and the tray was joined extra - orally with the help of dowel pin and acrylic plate . the master cast was poured with dental stone.after obtaining maxillary and mandibular master casts , denture bases were fabricated and then were sectioned from the midline . after sectioning , the two ends of the denture bases were rejoined from the midline using press buttons ( snap fasteners ; needles ind ) . wax rims ( link , modeling wax , mdm corp . ) were prepared over the denture bases . the wax rim was not prepared in the mandibular anterior region because of the presence of the press buttons over the anterior segment of the ridge . jaw relation was recorded.then the teeth arrangement was done [ figure 1 ] ( lactodent ; pyrax enterprises ) . patient was called for try-in.after try - in , the press buttons were removed from the maxillary denture base and the gap between the two parts of the denture base was closed by incorporating wax between them and the processing of denture was done in a conventional manner . similarly , the press buttons were also removed from the anterior segment of the mandibular denture base and the anterior teeth were arranged . an index was made for the anterior teeth with the help of putty addition polyvinyl siloxane impression material ( exaflex , gc company ) . the anterior teeth were then removed and placed back in putty index , rest of the denture was processed with heat cure acrylic resin ( trevlon , dentsply ) , without anterior teeth in a conventional manner so that the hinge assembly can be placed later in the anterior teeth region.after processing , the dentures were retrieved , finished , and polished and then again sectioned from the midline with the help of thin disk bur.an orthodontic mandibular molar tube with 1-mm stainless steel wire was used to form a hinge assembly for the maxillary and mandibular sectional dentures so that the dentures can collapse at the midline.for maxillary denture , two mandibular molar tubes ( orthotechno wire and co. ) were used with 1-mm stainless steel wire which was passing through the slot of the tube . the maxillary denture was placed back over the cast and these assemblies were incorporated between the two sections of the denture in the center of the palatal surface using self - cure acrylic resin and the rest of the gap created during sectioning of the maxillary denture was also filled with self - cure acrylic resin . proper care was taken to avoid the entrapment of acrylic material inside the hinge , which can block the hinge movement of the dentures.for mandibular denture , one orthodontic mandibular molar tube with 1-mm stainless steel wire was used to form a hinge assembly so that the denture can collapse at the midline . this assembly was incorporated between the two sections of the denture using self - cure acrylic resin.the mandibular hinge denture was placed back over the master cast and the index of the mandibular denture was made with irreversible hydrocollo i d ( alginate , zelgan 2002 , dentsply ) and the cast was poured with dental stone.over that cast , mandibular anterior teeth were arranged back in their correct position with the help of previously made putty index . two clasps were adapted over the adjacent teeth to retain the acrylic removable partial denture [ figure 2 ] . wax - up was done and the acrylic removable partial denture was processed in a conventional acrylization manner.maxillary hinged denture and the mandibular sectional ( separate anterior removable segment ) and hinged denture [ figure 3 ] were placed in patient 's mouth.patient was educated about the insertion and removal of dentures . instructions were given to maintain the denture.patient was recalled for the follow - up visits to check for the maintenance and patient 's adaptability with the dentures . first follow - up was done after 24 h of denture insertion followed by 1 week to check for the comfort and any tissue reaction . initially , patient felt difficulty while placing and removal of the dentures from the oral cavity but with time patient got used to it . figure 1(a ) maxillary and ( b ) mandibular sectional denture bases with occlusion rims and teeth arrangement figure 2(a ) maxillary denture sectioned from the midline incorporating two hinge assemblies in the palatal region of the maxillary denture ; ( b ) mandibular acrylized denture with hinge assembly in anterior region . the index of the denture made and the cast obtained , anterior teeth arranged back in position with putty index to make anterior removable partial denture figure 3(a ) collapsed maxillary hinged and ( b ) mandibular sectional and hinged complete denture with anterior removable partial denture primary impression for maxillary arch was taken using smallest size edentulous stock tray with impression compound ( y dents ; mdm corp . ) , and after proper disinfection ( 1:213 iodophore ) , primary cast was poured in type ii dental plaster ( dentico ; neelkanth mine chem . ) . two autopolymerizing acrylic resin special trays ( trevelon ; dentsply india pvt . ltd . ) border molding was done with each tray using low fusing compound ( dpi ; pinnacle , india ) and the final impression was made with light body polyvinyl siloxane addition silicon impression material ( examix nds ; gc company ) . one sectional impression tray was poured with dental stone ( denstone , grade iii ) . over that cast second sectional impression tray similarly , mandibular primary impression was taken using smallest size stock tray and after disinfection , primary cast was poured . a conventional special tray was fabricated over the primary cast and that was later sectioned from the midline into two halves . one hole was drilled in the center of the tray handle and a brass dowel pin was passed through it , from one end to the other end of the hole . similarly , two holes were drilled over the premolar region on both sides of the tray and the two brass dowel pins were placed vertically in those holes and were connected by acrylic plate which was detachable . border molding and final impression were made individually with the each half of the tray and the tray was joined extra - orally with the help of dowel pin and acrylic plate . after sectioning , the two ends of the denture bases were rejoined from the midline using press buttons ( snap fasteners ; needles ind ) . wax rims ( link , modeling wax , mdm corp . ) were prepared over the denture bases . the wax rim was not prepared in the mandibular anterior region because of the presence of the press buttons over the anterior segment of the ridge . then the teeth arrangement was done [ figure 1 ] ( lactodent ; pyrax enterprises ) . after try - in , the press buttons were removed from the maxillary denture base and the gap between the two parts of the denture base was closed by incorporating wax between them and the processing of denture was done in a conventional manner . similarly , the press buttons were also removed from the anterior segment of the mandibular denture base and the anterior teeth were arranged . an index was made for the anterior teeth with the help of putty addition polyvinyl siloxane impression material ( exaflex , gc company ) . the anterior teeth were then removed and placed back in putty index , rest of the denture was processed with heat cure acrylic resin ( trevlon , dentsply ) , without anterior teeth in a conventional manner so that the hinge assembly can be placed later in the anterior teeth region . after processing , the dentures were retrieved , finished , and polished and then again sectioned from the midline with the help of thin disk bur . an orthodontic mandibular molar tube with 1-mm stainless steel wire was used to form a hinge assembly for the maxillary and mandibular sectional dentures so that the dentures can collapse at the midline . for maxillary denture , two mandibular molar tubes ( orthotechno wire and co. ) were used with 1-mm stainless steel wire which was passing through the slot of the tube . the maxillary denture was placed back over the cast and these assemblies were incorporated between the two sections of the denture in the center of the palatal surface using self - cure acrylic resin and the rest of the gap created during sectioning of the maxillary denture was also filled with self - cure acrylic resin . proper care was taken to avoid the entrapment of acrylic material inside the hinge , which can block the hinge movement of the dentures . for mandibular denture , one orthodontic mandibular molar tube with 1-mm stainless steel wire was used to form a hinge assembly so that the denture can collapse at the midline . this assembly was incorporated between the two sections of the denture using self - cure acrylic resin . the mandibular hinge denture was placed back over the master cast and the index of the mandibular denture was made with irreversible hydrocollo i d ( alginate , zelgan 2002 , dentsply ) and the cast was poured with dental stone . over that cast , mandibular anterior teeth were arranged back in their correct position with the help of previously made putty index . two clasps were adapted over the adjacent teeth to retain the acrylic removable partial denture [ figure 2 ] . wax - up was done and the acrylic removable partial denture was processed in a conventional acrylization manner . maxillary hinged denture and the mandibular sectional ( separate anterior removable segment ) and hinged denture [ figure 3 ] were placed in patient 's mouth . patient was recalled for the follow - up visits to check for the maintenance and patient 's adaptability with the dentures . first follow - up was done after 24 h of denture insertion followed by 1 week to check for the comfort and any tissue reaction . initially , patient felt difficulty while placing and removal of the dentures from the oral cavity but with time patient got used to it . ( a ) maxillary and ( b ) mandibular sectional denture bases with occlusion rims and teeth arrangement ( a ) maxillary denture sectioned from the midline incorporating two hinge assemblies in the palatal region of the maxillary denture ; ( b ) mandibular acrylized denture with hinge assembly in anterior region . the index of the denture made and the cast obtained , anterior teeth arranged back in position with putty index to make anterior removable partial denture ( a ) collapsed maxillary hinged and ( b ) mandibular sectional and hinged complete denture with anterior removable partial denture several techniques have been described in the literature for the fabrication of sectional and hinged tray / complete dentures , utilizing various mechanisms for connecting each of the components . pubmed database for the last two decades was reviewed using key words : microstomia , hinged and sectional dentures and different techniques for patients with microstomia are found to be documented [ table 1 ] . different techniques and denture modifications for microstomia other management techniques described in the literature for patients with restricted mouth opening are surgery , use of dynamic opening devices , and modification of denture design . in cases where microstomia is not manageable by surgeries or dynamic opening devices , modified impression procedure and prosthesis design facilitate prosthetic rehabilitation . the denture aids for a better function , health , esthetics , and overall well - being of the patient and thus a rewarding service can be provided by the prosthodontist . the reduction in maximal oral opening ( microstomia ) less than the normal ( 35 - 40 mm ) hinders conventional prosthetic treatment of edentulous patients . it is often difficult to apply conventional clinical procedures to construct dentures for patients who demonstrate limited mouth opening . however , with careful treatment planning and prudent designing , the use of either sectional impression techniques and/or sectional dentures , many of the apparent clinical difficulties can be overcome . here , the sectional impression procedures and the prosthesis designs are described which aid in fabrication of prosthesis for a patient with the limited mouth opening which aids for a better function , health , esthetics , and overall well - being of the patient . simplified sectional tray design and ease of fabrication are the major advantages of this technique . this technique can be accomplished in any dental laboratory , without using complicated machinery or attachment devices for sectioning or assembling the trays / prosthesis together . the press buttons and mandibular molar bands are easily available and are easy to maintain . this technique shares disadvantages common to all sectional tray / prosthesis designs , such as additional time , labor , and materials . however , to determine the long - term success of this technique , periodic recall , maintenance , and further improvements in design are needed .

restricted mouth opening is a definite prosthodontic hindrance to carry out treatment successfully . restricted mouth opening can be due to many reasons such as microstomia , oral submucous fibrosis , some genetic disorder , and as a result of some surgical treatment . in the past , various techniques for prosthetic rehabilitation of limited oral opening have been tried such as surgeries , use of dynamic opening devices , magnetic devices , and modification of denture design . here we present ; a simplified technique and simple design for fabrication of maxillary hinged and mandibular hinged and sectional complete denture for a patient with restricted mouth opening due to oral submucous fibrosis .

pubmed

hepatocellular carcinoma ( hcc ) is the sixth most common malignancy worldwide and the third most common cause of global cancer related mortality [ 1 , 2 ] . hcc burden disproportionately impacts developing countries and males ; as of 2008 , 85% of cases occurred in africa and asia , with worldwide male : female sex ratio of 2.4 . risk factors for the development of hcc include chronic liver inflammation from hepatitis b and c infection , autoimmune hepatitis , excessive alcohol use , nonalcoholic steatohepatitis , primary biliary cirrhosis , environmental carcinogens such as aflatoxin b , and genetic metabolic disease ( such as hemochromatosis and alpha-1 antitrypsin deficiency ) . prognostic and therapeutic options are dependent upon the severity of underlying liver disease , and median overall survival ( os ) for metastatic or locally advanced disease is estimated at 58 months . hcc is relatively refractory to cytotoxic chemotherapy , likely due to overexpression of multidrug - resistant genes , protein products such as heat shock 70 and p - glycoprotein , and p53 mutations . presently , systemic therapeutic options in the locally advanced or metastatic setting are limited to sorafenib , an oral multikinase inhibitor targeting raf kinase , vascular endothelial growth factor ( vegf ) , and platelet - derived growth factor ( pdgf ) receptor tyrosine kinase signaling . although the transition from normal hepatocyte to hcc is not fully understood , hepatocarcinogenesis is a complex multistep process driven by accumulation of heterogeneous molecular alterations from initial hepatocyte injury to metastatic invasion . dysplastic nodules subsequently progress to early hcc through cumulative genetic alterations , while advanced hcc often involves intrahepatic metastasis and portal vein invasion . molecular alterations implicated in hcc development include mutations in oncogenes and tumor suppressor genes ( p53 and p16 ) , epigenetic alterations , chromosomal changes , and aberrant activation of signaling cascades necessary for proliferation , angiogenesis , invasion and metastasis , and survival . pathogenesis of early and advanced hcc may be modulated through different mechanisms ; for example , p53 mutations , p16 gene silencing , and aberrant akt signaling are more frequently observed in advanced hcc [ 46 ] . the molecular pathogenesis of hcc is multifactorial and is reliant upon dysregulation of multiple pathways including wnt / b - catenin , mitogen - activated protein kinase ( mapk ) , phosphatidylinositol-3 ( pi3k)/akt / mammalian target of rapamycin ( mtor ) , vegf , pdgf , insulin - like growth factor ( igf ) , epidermal growth factor ( egf ) , tgf - beta , and hepatocyte growth factor [ 6 , 7 ] . the hepatocyte growth factor ( hgf ) and its transmembrane tyrosine kinase receptor , cellular met ( cmet ) promote cell survival , proliferation , migration , and invasion via modulation of epithelial - mesenchymal interactions . hgf - cmet signaling is critical for normal processes such as embryogenesis , organogenesis , and postnatal tissue repair after acute injury . hgf - cmet axis activation is also implicated in cellular invasion and metastases through induction of increased proliferation ( mitogenesis ) , migration and mobility ( motogenesis ) , three - dimensional epithelial cell organization ( morphogenesis ) , and angiogenesis . hgf was first discovered in 1984 as a mitogenic protein for rat hepatocytes in vitro . hgf was subsequently found to be indistinguishable from scatter factor , a fibroblast - derived motility factor promoting epithelial cell dispersal and three - dimensional branching tubulogenesis . hgf is secreted primarily by mesenchymal cells ( or by stellate and endothelial cells in the liver ) as an inactive single - chain precursor ( pro - hgf ) which is bound to heparin proteoglycans within the extracellular matrix . hgf transcription is upregulated by inflammatory modulators such as tumor necrosis factor alpha , il-1 , il-6 , tgf - beta , and vegf [ 11 , 12 ] . circulating pro - hgf undergoes proteolytic conversion via extracellular proteases including hgf activator ( hgfa ) , urokinase - type plasminogen activator , factors xii and xi , matriptase , and hepsin into an active two - polypeptide chain heterodimeric linked by a disulfide bond . hgfa is a serine protease which is secreted primarily by the liver and circulates as pro - hgfa ; pro - hgfa is activated by thrombin in response to tissue injury and malignant transformation [ 13 , 14 ] . the active form of hgf includes an -chain containing four kringle domains ( k1 to k4 ) and an amino - terminal loop domain ( n ) , and a -chain ( c - terminal ) containing a serine protease homology ( sph ) domain . hgf is a ligand for the met receptor , also known as cellular met or cmet . the met protooncogene was first isolated in 1984 from a human osteosarcoma - derived cell line driven by a chromosomal rearrangement tpr - met , resulting from fusion of translocated promoter region located on chromosome 1q25 and met sequence located on chromosome 7q31 . the tpr - met rearrangement encodes for a prototype of the cmet receptor tyrosine kinase family . the cmet receptor is expressed predominantly on the surface of endothelial and epithelial cells of many organs , including the liver , kidney , prostate , pancreas , kidney , muscle , and bone marrow . like hgf , cmet is synthesized as an inactive single - chain precursor and undergoes proteolytic cleavage into a disulfide linked heterodimer consisting of an extracellular -chain and transmembrane -chain . the extracellular domain includes an amino - terminal semaphorin domain , terminal cysteine - rich psi domain , and four ipt repeat immunoglobulin domains . the cytoplasmic portion includes a juxtamembrane region including two phosphorylation sites , a tyrosine kinase ( tk ) domain and a carboxyl terminal region for substrate docking . under normal conditions , hgf interacts with cmet via a paracrine signaling loop and triggers cmet kinase activation within the cytoplasmic portion ( see figure 1 ) . hgf contains two cmet binding sites including a high affinity constitutively active site on the -chain ( n - terminal and first kringle domain , or n - k1 ) and a low affinity site on the -chain ( c - terminal ) [ 8 , 19 ] . the exact mechanism of cmet receptor activation and the contribution of both binding sites to receptor activation by full length hgf are not yet established , although it is evident that both - and -chain sites have distinct functions . while -chain n - k1 portion activates cmet and induces met dimerization , -chain residues bind cmet once the receptor is already occupied by hgf n - k4 and subsequently induce phosphorylation and downstream signaling . hgf binding induces autophosphorylation of tyrosine residues y1234 and y1235 within the cmet tk domain activation loop . phosphorylation also occurs at two sites within the carboxyl terminal region ( y1349 and y1356 ) , forming a multifunctional high affinity binding docking site that recruits a range of intracellular adaptors containing src homology-2 domains [ 2124 ] . recruited adaptor proteins and kinase substrates include growth factor receptor - bound protein 2 ( grb2 ) , grb2-associated binder ( gab1 ) , phospholipase c- , stat3 , shc , src , shp2 , pi3k , and ship1 [ 23 , 24 ] . these and other adaptor proteins provide scaffolding for a larger apparatus of network proteins , ultimately promoting activation of multiple signaling pathways . of these , grb2 and gab1 are critical effectors that interact directly with the receptor ; grb2 binding to the cmet docking site through y1356 results in downstream signaling via the ras / mapk pathway , while gab1 phosphorylation by met kinase activates the pi3k pathway . other signaling proteins that are activated by cmet include p38 , jnk , and nuclear factor kb . alterations in transcription induce cell cycle progression , antiapoptosis , increased cell motility , angiogenesis , and survival . the hgf - cmet pathway serves as a hub for multiple heterogeneous signaling networks and is also modulated by the activation of other receptor tk families such as the epidermal growth factor receptor ( egfr ) , human epidermal growth factor receptor 2 , insulin - like growth factor 1 receptor , raf kinase , and vegf . the hgf - cmet pathway plays an essential role in mammalian development in terms of morphogenesis , mitogenesis , and motogenesis , and angiogenesis hgf - cmet signaling is likely to be critical in many aspects of adult homeostasis including cardiac and hepatic tissue injury repair [ 26 , 27 ] , skin wound repair , and skin immunity regulation . targeted disruption of the hgf or met genes results in embryonically lethal knockouts with impaired organogenesis of the liver and placenta . preclinical models demonstrate that hgf functions as a hepatotrophic factor enhancing hepatic regeneration and suppressing hepatocyte apoptosis [ 31 , 32 ] ; expression of hgf is increased in response to liver injury , while neutralization of endogenous hgf or met knockout facilitates liver damage and fibrotic changes with delayed repair . under normal physiologic conditions , hgf - induced cmet activation is strictly controlled by paracrine ligand delivery followed by ligand activation at target cell surfaces and ligand - activated receptor internationalization / degradation . despite multiple checkpoints , hgf - cmet axis dysregulation occurs in a variety of solid tumors and hematopoietic derived malignancies and plays a key role in malignant transformation by promoting tumor cell migration , epithelial to mesenchymal transition , invasion , proliferation , and angiogenesis . dysregulated cmet signaling upregulates protease production ( plasminogen dependent and independent ) and increased cell dissociation via extracellular matrix degradation , facilitating tumor invasiveness and metastasis . mechanisms of pathogenic activation include aberrant paracrine or autocrine ligand production , overexpression of hgf and cmet , upregulation of genes encoding proteases for hgf / cmet processing , constitutive kinase activation independent of cmet gene amplification , and cmet gene mutations leading to ligand - independent kinase activity [ 3436 ] . the hgf - cmet axis is implicated in hepatocarcinogenesis through multiple mechanisms , many of which are still being elucidated . overexpression of hgf and cmet [ 3741 ] is observed in 33% and 2048% of human hcc samples , respectively . the prognostic utility of cmet and hgf overexpression is uncertain ; while some studies show no correlation between cmet overexpression and tumor size or invasive behavior [ 38 , 42 ] or hgf levels and survival , others demonstrate an inverse relationship with survival . specifically , cmet overexpression was found to correlate with poorly differentiated hcc and increased intrahepatic metastases along with decreased five - year survival . after hepatectomy , cmet overexpression in hcc tissue has been correlated with early tumor recurrence , metastasis , and shorter 5-year survival [ 41 , 45 , 46 ] . a cmet - regulated gene expression signature was found to define a subset of human hcc with poor prognosis and aggressive phenotype and correlated with increased vascular invasion , increased microvessel density , and decreased mean survival time . higher hgf levels negatively correlate with survival per biomarker analysis of the sharp trial and prior data and positively correlate with tumor size ; however given that hgf is secreted as an inactive precursor , overexpression alone is unlikely to guarantee pathway dysregulation . hgf has also been investigated as a potential biomarker for hcc development , but may also be a specific marker for liver cirrhosis . while gains in 7q have been reported in hcc , gene amplification has not been reported as a significant source of increased cmet . crosstalk between cmet and egfr [ 55 , 56 ] and cmet and vegf signaling pathways is also implicated in promoting tumor survival . cmet - csrc has been shown to mediate egfr phosphorylation and cell survival in the presence of egfr inhibitors . cmet is both an independent angiogenic factor and interacts with angiogenic survival signals promoted through vegf . by upregulating hypoxia - inducible factor , hypoxia results in increased hgf expression in tumor and surrounding normal interstitial cells and hgf - cmet signaling induces upregulation of tumoral vegf expression and endothelial vegfr2 expression and downregulation of endogenous inhibitors of angiogenesis [ 58 , 59 ] . dual vegf and cmet axis activity demonstrates increased capillary formation in vivo , tubulogenesis in vitro , and tumoral microvessel density . given the predominant role of dysregulated hgf - cmet signaling in hepatocarcinogenesis , pharmacologic cmet inhibition is a promising therapeutic strategy . targets for inhibition of the cmet signal transduction pathway include ligand - receptor interaction , cmet kinase activity , and cmet and adaptor protein interaction . hgf - cmet axis inhibitors can be broadly classified into biologic antagonists , c - met adaptor protein inhibitors , small - molecule downstream pathway inhibitors , and small synthetic met tyrosine kinase inhibitors ( tki ) . of these , tkis are the most common and the only ones to have completed phase 2 testing in hcc as of march 2013 . table 1 shows the selected hgf - cmet inhibitors in active clinical trials for either hcc or advanced solid malignancies ( including hcc ) as of march 2013 . a comprehensive listing of hgf - cmet inhibitors in active clinical trials for all malignancies is maintained by the bottaro nci lab and is available at https://ccrod.cancer.gov/confluence/display/ccrhgf/home . biologic antagonists inhibit cell surface interactions such as ligand - receptor binding or receptor clustering , preventing activation of downstream signaling . these include hgf competitive analogs , met decoy receptor , and anti - hgf - cmet monoclonal antibodies . hgf competitive analogs compete with ligand for receptor binding without causing cmet dimerization and activation and include nk2 [ 60 , 61 ] , nk4 [ 6264 ] , and uncleavable hgf ; preliminary safety and drug development data in humans are pending . nk2 may be the least promising hgf competitive analog due to being a potent mitogen and promoting metastases . met decoy receptors are soluble forms of the cmet extracellular domain which compete with hgf and inhibit cmet dimerization ; in vitro and in vivo mice models demonstrate suppression of hgf - induced tumor cell migration and metastasis [ 68 , 69 ] . monoclonal antibodies targeting hgf and the extracellular domain of cmet are currently being explored in clinical trials ( see table 1 ) , but data are not yet available for hcc . given the importance of adaptor proteins in propagating downstream cmet signaling , cmet adaptor inhibitors offer unique potential for cmet specific inhibition . as described above , cmet signaling is initiated through autophosphorylation of cytoplasmic tyrosines that form docking sites for adaptor proteins . grb2 and gab1 are critical effectors that interact directly with the cmet receptor , ultimately recruiting a larger apparatus of network proteins necessary for cmet signaling . gab1 coupling with cmet requires gab1 binding to the sh3 domain of grb2 , and cmet binding to the sh2 domain of grb2 [ 72 , 73 ] . c90 is a selective grb2 antagonist with demonstrable inhibition of gastric cancer cell motility and matrix invasion in vitro and impaired metastatic spread of human prostate cancer cells in vivo ; data in human studies have not been reported to date [ 74 , 75 ] . small - molecule downstream pathway inhibitors directed towards inhibition of stat3 phosphorylation showed preliminary tolerability in a phase i trial of advanced solid tumors , but data are not yet available for hcc . synthetic small met tkis inhibit downstream signal transduction by preventing phosphorylation , either via competitive binding of the intracellular adenosine triphosphate ( atp ) site in cmet 's tk domain , or noncompetitive binding of a cmet region outside of the atp binding site . while some of the tkis in development are cmet specific , others target multiple pathways including vegf , pdgfr , fms - related tyrosine kinase 3 ( flt3 ) , v - kit feline sarcoma viral oncogene homolog protein ( kit ) , and anaplastic lymphoma kinase ( alk ) . preclinical studies and clinical trials show tolerability and efficacy of cmet tkis across a variety of solid malignancies . as of march 2013 , promising results in the phase 2 randomized setting for hcc are available for two cmet inhibitors : tivantinib and cabozantinib . tivantinib ( arq 197 ) is an oral low - molecular - weight tki which is non - atp competitive . safety and tolerability without drug - related worsening of hepatic function were previously reported in a phase ib trial of 20 cirrhotic patients ( child - pugh a and b ) with hcc , with 2 or less prior systemic chemotherapy regimens . rimassa and colleagues reported results at asco 2012 of a phase ii trial assigning 107 patients with unresectable hcc with ecog ps 0 - 1 and child - pugh a in a 2 : 1 randomization to either second - line tivantinib or placebo with crossover allowed . although no difference in median os occurred , the primary endpoint of time to progression ( ttp ) was met and favored tivantinib versus placebo ( 6.6 versus 6.2 months , hr = 0.90 , p = 0.63 ; 6.9 versus 6 weeks , hr = 0.64 , p = 0.04 , resp . ) . patients with high met expression ( defined as 50% or more cells in the tumor specimen with 2 + or 3 + staining intensity ) versus low met expression receiving tivantinib demonstrated a significant improvement in both os and ttp ( 7.2 versus 3.8 months , hr 0.38 p = 0.01 ; 11.7 versus 6.8 weeks , hr 0.43 , p = 0.03 , resp . ) . no detrimental effect was reported in patients with low met expression , and common adverse events were neutropenia , asthenia , poor appetite , and anemia . as of november 2012 , a phase iii trial for tivantinib and hcc patients is in development . cabozantinib ( xl 184 ) is an unselective oral multikinase tki targeting cmet , kit , rearranged during transfection ( ret ) , vegfr1 and 2 and 3 , flt3 , axl receptor tyrosine kinase ( axl ) , and tie family angiopoietin 1 receptor . cabozantinib has shown efficacy in the phase 3 setting for medullary thyroid carcinoma with progression - free survival ( pfs ) improvement and phase 2 setting for advanced solid tumors ( including hcc ) . verslype and colleagues reported preliminary results of a phase 2 randomized discontinuation study of cabozantinib in 41 patients with hcc , child - pugh score a , and one prior line of systemic treatment . all patients initiated cabozantinib for a 12-week lead in time frame ; patients with partial response continued on study drug while patients with progression of disease discontinued . 32 patients with stable disease were blindly randomized 1 : 1 to continue cabozantinib or receive placebo . the primary endpoint was overall response rate ( rr ) during the lead in time phase and pfs for patients entering the randomization phase . independent of prior sorafenib use , median os was 15.1 months , median pfs was 4.4 months , and median time on study was 6 months . common grade 3 adverse effects were hand - foot syndrome , diarrhea , and thrombocytopenia . met expression was not evaluated prospectively , and given the broad activity of cabozantinib , it is unclear how much activity is attributable to met inhibition alone . in fact , the inhibition of both vegf and met concurrently may be particularly effective . vegf inhibition leads to increased met signaling , either from resultant hypoxia or direct interactions between vegfr2 and met [ 81 , 82 ] . preliminary evidence of efficacy against hcc was seen in a phase i combination study of tivantinib with sorafenib including a complete response and another prolonged partial response lasting greater than one year . inc280 is a selective met tki in phase i testing for early hcc , currently accruing . foretinib ( gsk 1363089 ) is a small - molecule tki targeting both met and vegf in phase i / ii testing for advanced hcc . based on preclinical trials and phase 2 data for tivantinib and cabozantinib , inhibition of cmet signaling is a promising therapeutic strategy in hcc . although it is unclear which genetic and molecular abnormalities implicated in hgf - cmet dysregulation are predictive of sensitivity to cmet targeted therapy , ongoing trials must address the challenge of identifying patients most likely to achieve maximal benefit and minimal toxicity . two current strategies for patient selection based on tumor biomarkers are quantification of tumor cmet content via immunohistochemical ( ihc ) and immunoassay tissue analysis and assessment of met sequence status . pharmacodynamic serum markers are under evaluation in ongoing trials as a strategy to assess clinical response . tumoral cmet overexpression as measured by commercially available ihc kits appears to correlate with efficacy of cmet inhibitors in hcc and other solid tumors , although more prospective data are necessary for validation . identification of cmet phosphorylation status is a potentially powerful target for targeted antibodies ; two site immunoassays of flash - frozen tissue samples yielding precise measurements of met content and phosphorylation activation are currently under development . another promising stratification strategy is assessment of met sequence status including met mutations , met amplification , and chromosome 7 polysomy . preclinical studies of cmet targeted agents demonstrate variable efficacy based on met mutation location ; for example , pf-2341066/4217903 is a selective inhibitor with increased activity against certain cmet atp binding site mutations in comparison to met kinase domain activation loop mutations . in vitro studies of su11274 , a small - molecule tk competitive atp binding site inhibitor , show selective inhibition of two out of four identified met mutations . amplification of cmet is associated with increased clinical response to foretinib ( xl 880 ) in phase 2 gastric cancer data , and cmet copy number correlates with increased clinical response to tivantinib in addition to erlotinib in advanced nsclc . ancillary pharmacodynamic and pharmacokinetic marker studies in a variety of solid tumors show variable correlation with clinical response . preclinical data with crizotinib in gastric cancer cell lines demonstrate variable biomarker modulation of cmet inhibition with cmet dependent gastric cancer cell lines versus cmet - independent lines [ 88 , 89 ] . clinical data shows changes in plasma concentrations of hgf , vegf , soluble met , soluble vegfr2 , pigf , and epo during treatment with various tkis [ 90 , 91 ] . the predictive utility of these biomarkers in patients with cmet - dependent hcc is unclear ; further analyses and prospective validation are necessary . hgf - cmet is an intriguing target for the development of companion diagnostic tools as an adjunct tool for patient selection and stratification for cmet therapy . in vitro and in vivo animal studies suggest that radiolabelled dyes containing cmet binding peptide successfully targets cmet receptors with higher imaged based tumor uptake . a variant of su11274 with radiomethylation modification is being utilized as a pet visualization agent to quantify cmet receptor in xenograft models . inhibition of cmet is a promising therapeutic strategy in hcc . given the heterogeneous mechanisms underlying cmet dysregulation , there is an urgent and unmet need for the development of predictive biomarkers to identify which subsets of cmet - dependent hcc tumors are most likely to benefit from specific classes of inhibitors . as more agents move into phase 2 and 3 trials for hcc , one important consideration is the emergence of acquired and primary resistance mechanisms from de novo or preexisting mutations . these may be overcome by rational combination therapy directed against multiple pathways , different levels of ligand - receptor - tki interaction , and the presence of cmet addiction . innovative clinical trial designs ( such as the discontinuation cabozantinib design ) with incorporation of enriched patient cohorts , biomarker analyses , pharmacodynamic markers , and companion diagnostics are essential in moving forward .

under normal physiological conditions , the hepatocyte growth factor ( hgf ) and its receptor , the met transmembrane tyrosine kinase ( cmet ) , are involved in embryogenesis , morphogenesis , and wound healing . the hgf - cmet axis promotes cell survival , proliferation , migration , and invasion via modulation of epithelial - mesenchymal interactions . hepatocellular cancer ( hcc ) is the third most common cause of worldwide cancer - related mortality ; advanced disease is associated with a paucity of therapeutic options and a five - year survival rate of only 10% . dysregulation of the hgf - cmet pathway is implicated in hcc carcinogenesis and progression through activation of multiple signaling pathways ; therefore , cmet inhibition is a promising therapeutic strategy for hcc treatment . the authors review hgf - cmet structure and function in normal tissue and in hcc , cmet inhibition in hcc , and future strategies for biomarker identification .

pubmed

the associations between diabetes and cancer have been the subject of debate for over a century . the length of this debate suggests , perhaps , that any such associations are relatively weak , and it is safe to say that most practicing diabetologists do not view cancer as a specific , diabetes - associated risk in day - to - day practice . that diabetes is not more clearly associated with cancer is surprising , since diabetes may be viewed as a disease of increased intracellular oxidative stress , as well as accelerated biochemical aging caused by the accumulation of advanced glycation end - products ( ages ) on proteins and other macromolecules ( reviewed in ) . an additional concern is the role of insulin , which , in various forms , is increasingly used in the management of type 2 diabetes , a condition that is now epidemic worldwide . specifically , the ability of insulin to bind and activate the insulin - like growth factor-1 ( igf-1 ) receptor has raised concerns that it may promote the growth of nascent malignancies and even the establishment of new ones . , there has been a recent surge in interest in the role of diabetes , insulin therapy , and specifically the effects of newer forms of insulin in promoting cancer . recent rigorous epidemiologic studies have confirmed that type 2 diabetes does indeed confer increased risk for certain types of malignancies , in particular breast , colon , and pancreatic tumors . many patients with type 2 diabetes eventually require insulin , and the long - acting insulin analogue , glargine , is one of the most commonly used agents . several recent studies have demonstrated the potential mitogenic and anti - apoptotic effects of glargine on cultured cancer cells , and have implicated activation of the igf-1 pathway . however , the clinical implications remain controversial , particularly when studies pertaining to untransformed cells did not support any mitogenic potential . in addition , according to some authors , glargine compared with human insulin does not seem to display increased mitogenic effects , despite its higher affinity for the igf-1 receptor ; rather , any type of insulin has the potential to promote growth in cancer cells , especially if administered at sufficiently high , supra - physiologic doses . the question now is whether glargine is associated with increased cancer risk compared with other commonly used forms of insulin . this question has come to the fore with the publication of several studies in diabetologia [ 12 - 15 ] , which will be reviewed briefly here . the epidemiologic study that initiated the recent debate was performed in germany and suggested a specific cancer risk associated with glargine . based upon the mixed opinions of six reviewers , the editors of diabetologia , the journal to which it was submitted , commissioned three additional studies [ 13 - 15 ] , aiming to confirm or refute its findings . in the initial study , hemkens et al . followed 127,031 german patients who were being treated with human insulin and human insulin analogues ( aspart , lispro , or glargine ) over a mean of 1.6 years between january 1998 and june 2005 . those on two insulin analogues or a combination of human insulin and an analogue were excluded from the analysis . patients with known or suspected malignant disease within 3 years prior to study start were also excluded . the primary outcome was occurrence of a malignant neoplasm , and the secondary outcome was all - cause mortality . the gross malignancy incidence rates for human insulin , aspart , lispro , and glargine were 2.50 , 2.16 , 2.13 , and 2.14 per 100 patient - years , respectively , in the unadjusted analysis . however , when the data were adjusted for daily insulin dose , glargine was found to be associated with a greater risk of malignancy , and the risk was incremental with higher glargine doses when compared with human insulin { adjusted hazard ratio [ hr ] of 1.09 for a daily dose of 10 iu [ 95% confidence interval ( ci ) 1.00 - 1.19 ] , 1.19 for a daily dose of 30 iu [ 95% ci 1.10 - 1.30 ] , and 1.31 for a daily dose of 50 iu [ 95% ci 1.20 - 1.42]}. aspart and lispro were not associated with an increased risk for cancer compared with human insulin . in addition , regardless of the type of insulin , higher doses and longer duration of exposure increased the risk of malignancy . finally , the mean insulin dose until occurrence of a neoplasm ( cumulative dose divided by time until an event ) was significantly lower for glargine ( 25.9 iu / day ) than for human insulin , aspart , and lispro ( 43.8 , 38.9 , and 36.2 iu / day , respectively ) . the major weaknesses of the study included the fact that patients were not allocated to treatment groups in a prospective manner and that insulin dose was calculated during follow - up from mean values , and not determined at baseline . in addition , the exclusion of patients on combination insulin therapy may not reflect common practice , in which patients are typically on basal / bolus regimens requiring the use of different types of insulin . finally , the duration of diabetes , as well as smoking status , body mass index ( bmi ) , and other potential confounding factors were not taken into account . the three additional , commissioned studies were published in the same issue of diabetologia . in sweden , jonasson and colleagues evaluated 114,842 patients with diabetes on insulin and noted that women on glargine alone , but not glargine combined with other types of insulin , had a higher risk of breast cancer than women on other types of insulin ( hr adjusted for age , smoking , bmi , and other confounding factors was 1.97 , 95% ci 1.29 - 3.00 ) . however , this increased risk was not associated with an increased mortality rate ; instead , all - cause mortality was actually decreased in women on glargine alone versus women on other types of insulin ( relative risk [ rr ] 0.83 , 95% ci 0.71 - 0.96 ) . the associations with other malignancies such as prostate and gastrointestinal tumors did not reach statistical significance . no analysis taking into account insulin dose was possible in this study , and therefore the incremental risk conferred by higher doses as observed by hemkens et al . the short duration of exposure and observation as well as the lack of data on how much insulin the patients were taking limit the strength of the conclusions . in a study in scotland , no increased risk of total or site - specific cancer associated with overall glargine use was found in a cohort of 12,852 patients followed for 4 years ; in particular , no increase in breast cancer risk ( hr 1.49 , 95% ci 0.79 - 2.83 ) was found . however , exclusive use of glargine did seem to confer higher risk ( adjusted hr 3.39 , 95% ci 1.46 - 7.85 ) , but the number of cases was small ( six events ) . in addition , glargine - only users tended to be older , were more likely to be women , and to have a higher bmi , reflecting a baseline imbalance . finally , the restricted use of glargine in scotland may have contributed to a skewing of the results , since there were a limited number of patients taking this form of insulin . in a uk studied 62,809 patients with diabetes on metformin , sulfonylurea monotherapy , a combination of both , or insulin therapy . the latter were further divided into glargine - treated versus other human insulin - treated ( long - acting , biphasic , and biphasic analogue ) . the results indicated that any type of insulin therapy increased the risk of occurrence of solid tumors and that concomitant metformin therapy attenuated this effect . indeed , the adjusted hrs relative to metformin monotherapy were 1.08 ( 95% ci 0.96 - 1.21 ) for the metformin / sulfonylurea combination , 1.36 ( 95% ci 1.19 - 1.54 ) for sulfonylurea alone , and 1.42 ( 95% ci 1.27 - 1.60 ) for insulin - based therapies . concomitant metformin therapy decreased the risk of malignancy associated with insulin by 46% ( hr 0.54 , 95% ci 0.43 - 0.66 ) . when compared with glargine , other human insulins displayed comparable rates , suggesting no added risk attributable to glargine ( adjusted hr 1.24 , 95% ci 0.90 - 1.17 for basal human insulin ; hr 0.88 , 95% ci 0.66 - 1.19 for biphasic human insulin ; and hr 1.02 , 95% ci 0.76 - 1.37 for analogue biphasic insulin ) . malignancies that had the strongest association with insulin therapy compared with metformin were colorectal ( hr 1.69 , 95% ci 1.23 - 2.33 ) and pancreatic ( hr 4.63 , 95% ci 2.64 - 8.10 ) tumors . breast and prostate cancer risks did not seem to be affected by treatment with insulin or insulin secretagogues , and a prior history of solid tumor strongly increased the overall risk ( hr 3.86 , 95% ci 3.46 - 4.31 ) . in a letter published in a subsequent issue of diabetologia , rosenstock et al . , reported a study of glargine versus neutral protamine hagedorn ( nph ) insulin in type 2 diabetes patients randomly assigned to one insulin or the other for over 4 years . although malignancy risk was not a primary endpoint for the study , no increased cancer risk with glargine compared to nph was found ( rr 0.90 , 95% ci 0.64 - 1.26 ) . home and lagarenne , using the sanofi - aventis pharmacovigilance database for all randomized company - sponsored clinical trials , published a systematic review of 31 trials comparing glargine to other agents ( nph for most studies ) . no evidence for an increased risk of malignancy attributable to glargine was found ( rr 0.90 , 95% ci 0.60 - 1.36 ) . all but one of these studies lasted less than 12 months ( on average , 6 months ) , so their major limitation was the short duration of exposure and follow - up . indeed , the majority of reported cases of malignancy were derived from the study of rosenstock and colleagues , discussed above , in which follow - up exceeded 4 years . finally , a meta - analysis comparing cancer risk associated with another new , long - acting insulin , insulin detemir , versus nph and glargine was performed . it used data from randomized controlled trials sponsored by novo nordisk ( bagsvaerd , denmark ) and found comparable malignancy risks associated with detemir and glargine in the five trials that compared both drugs . however , the number of events was very small ( n = 8 ; event rate per 100 exposure - years = 0.87 for detemir , and n = 8 ; event rate per 100 exposure - years = 1.27 for glargine ) . the issues are highly emotive : cancer and diabetes eventually affect a large proportion of the world 's population . in addition , there are enormous financial implications surrounding the controversy , and unfortunately , complex issues tend to be oversimplified and are often sensationalized . either a diagnosis of cancer or the institution of insulin therapy for diabetes provokes great anxiety in many people . criticism of the publication of the diabetologia papers came in the correspondence section of the lancet , where pocock and smeeth raised concerns regarding the quality of the statistical analyses and contended that these analytical flaws , especially in the german paper , had raised unsubstantiated anxieties . they criticized the swedish study for its finding of an association of insulin glargine with breast cancer , arguing that the latter was not a pre - defined site for analysis but rather a secondary finding ultimately not confirmed by the scottish study ( the latter did find an increased risk of breast cancer associated with the exclusive use of glargine , but only affecting very few , older patients ) . further criticism came in an editorial by garg , hirsch , and skyler in diabetes technology and therapeutics , questioning the motivations behind the german study and raising concerns similar to those brought up by pocock and smeeth . this paper also emphasized the lack of evidence of an increased overall cancer risk associated with glargine based on the swedish , scottish , and uk studies , concluding that glargine use should not be discontinued based on unsubstantiated allegations. the letter of pocock and smeeth elicited a prompt response from the editor of diabetologia . the editor , while acknowledging the imperfections of the studies ( which he had already discussed in detail in an accompanying editorial ) , argued that the epidemiologic data can not and should not be dismissed , especially when considered in the context of animal and cell culture work , but rather should generate further research to define risk . indeed , in his initial editorial on the subject , he had earlier stated that the evidence presented in this set of papers is sufficient to establish that there is a case to answer , but is entirely insufficient to bring a verdict. at least on the latter conclusion , all parties agreed . these thought - provoking studies , although inconclusive , do raise concerns about the long - term effects of diabetes itself , insulin treatment , and specifically , glargine , on cancer development . it must be remembered that the greatly increased mortality among patients with diabetes has little to do with cancer , but instead is related to vascular complications . the overall benefit - to - risk ratio of good glycemic control is likely to support extensive use of insulin . all are agreed that the current evidence does not warrant any change in clinical practice , specifically with regard to the use of glargine . further research will need to address the relationship between diabetes , insulin , insulin analogues and secretagogues , and cancer . it will also explore potentially protective effects of metformin and perhaps the most tantalizing question : why is diabetes not more strongly associated with cancer ?

diabetes , in particular type 2 , is associated with an increased incidence of cancer . although the mortality attributable to cancer in type 2 diabetes is overshadowed by that due to cardiovascular disease , emerging data from epidemiologic studies suggest that insulin therapy may confer added risk for cancer , perhaps mediated by signaling through the igf-1 ( insulin - like growth factor-1 ) receptor . co - administered metformin seems to mitigate the risk associated with insulin . a recent series of publications in diabetologia addresses the possibility that glargine , the most widely used long - acting insulin analogue , may confer a greater risk than other insulin preparations , particularly for breast cancer . this has led to a heated controversy . despite this , there is a consensus that the currently available data are not conclusive and should not be the basis for any change in practice . further studies and more thorough surveillance of cancer in diabetes are needed to address this important issue .

pubmed

adamantinoma of the tibia is a rare slow - growing primary tumor of the bone . there has been much controversy on the origins of this tumor with one major focus being the relationship between osteofibrous / fibrous dysplasia and adamantinoma . there have been approximately 300 cases of tibial adamantinoma reported in the published english language literature . of these cases , 1520% the most common site of metastasis is to the lungs . as metastases to the regional lymph nodes are rare , there is limited information regarding the best practice plan for their management in the literature . we report 2 cases of tibial adamantinoma , which were problematic from a diagnostic point of view . case 2 is of a tibial adamantinoma that presented with groin metastases which was initially diagnosed as a cutaneous adnexal carcinoma . a review of inguinal nodal metastatic adamantinoma is outlined in addition to the clinic - pathological data on these cases . in 2007 , a 29-year - old female presented with a swelling in her left inguinal area . investigations including a computed tomography ( ct ) scan showed an extremely large heterogeneous , hyperdense , lobular lymph node centered in the left inguinal region measuring 6.54.6 cm . histopathological examination revealed the presence of a malignant neoplasm that was composed of sheets of basaloid cells with round vesicular nuclei and minimal cytoplasm . this lesion was interpreted as a cutaneous eccrine carcinoma ( figure 1a c ) . due to the unusual diagnosis , an external consultation and a request for additional clinical history were obtained . on detailed review , the pathological report was reinterpreted as metastatic adamantinoma in conjunction with a confirmed past clinical history of surgical excision of a left tibial adamantinoma three years previously in 2004 . the specimen consisted of a firm rubbery mass of fibro - fatty tissue that measured 18.511.37.2 cm with an ellipse of skin measuring 820.3 cm . the cut surface revealed a multi - lobulated light brown well - circumscribed mass that measured 10.475.5 cm . on microscopic examination , the neoplastic cells were composed of predominantly basaloid and tubular nests of malignant cells with some peripheral palisading ( figure 1d f ) . the histomorphological appearance of these cells was identical to the previous histology in this case ( figure 1a c ) confirming the presence of inguinal lymph nodal metastases from adamantinoma of the tibia . biopsy of left inguinal mass ( a , b , c ) and left inguinal lymphadenopathy ( d , e , f ) . photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of a malignant neoplasm in the subcutaneous fat ( a ) . lesional cells are composed predominantly of basaloid cells with round vesicular nuclei and minimal cytoplasm as seen at medium power ( b ) ( lens objective 4 ) and at high power ( c ) ( lens objective 10 ) . photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of a metastatic malignant neoplasm in the lymph node ( ) ( d ) . the neoplastics cells were composed of basaloid and tubular nests of malignant cells with some peripheral palisading as seen at medium power ( e ) ( lens objective 4 ) and at high power ( f ) ( lens objective 10 ) . biopsy of left inguinal mass ( a , b , c ) and left inguinal lymphadenopathy ( d , e , f ) . photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of a malignant neoplasm in the subcutaneous fat ( a ) . lesional cells are composed predominantly of basaloid cells with round vesicular nuclei and minimal cytoplasm as seen at medium power ( b ) ( lens objective 4 ) and at high power ( c ) ( lens objective 10 ) . photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of a metastatic malignant neoplasm in the lymph node ( ) ( d ) . the neoplastics cells were composed of basaloid and tubular nests of malignant cells with some peripheral palisading as seen at medium power ( e ) ( lens objective 4 ) and at high power ( f ) ( lens objective 10 ) . in april 2009 , a new node along the left external iliac in the retroperitoneum was detected and biopsied under radiological guidance . histopathological examination showed similar findings confirming the presence of metastatic adamantinoma ( figure 2a and b ) . ct scans of the chest showed the presence of two nodules in the right lung , which remained stable on follow up over the following year . follow up in early 2011 shows stable residual disease with no additional findings of note . photomicrographs of hematoxylin and eosin stained slide at low power ( a ) ( lens objective 2 ) shows the presence of a malignant neoplasm . the neoplastic cells ( b ) at high power ( lens objective 10 ) shows a similar morphology of basaloid cells as seen in figure 1 . photomicrographs of hematoxylin and eosin stained slide at low power ( a ) ( lens objective 2 ) shows the presence of a malignant neoplasm . the neoplastic cells ( b ) at high power ( lens objective 10 ) shows a similar morphology of basaloid cells as seen in figure 1 . in 1999 , a 58-year - old female presented with a painful swelling on the mid shaft of her left tibia . histopathological examination showed the presence of islands of woven bone in a background of bland spindled fibrous tissue , which was interpreted as fibrous dysplasia ( figure 3a and b ) . nine years later , in 2008 , she was seen for a painful swelling in the same region , which had been increasing in size since her previous operation . a wide excision of the lesion with a tibial allograft and intramedullary nail fixation was performed in october 2009 . there was a predominant fibrous dysplasia like pattern consisting of cellular bland , spindled fibroblastic proliferation with scattered islands of woven bone . embedded within the spindle cells were occasional clusters of plump epitheliod appearing cells that had a corded arrangement in focal regions ( figure 3c ) . immunohistochemistry confirmed the epithelial nature of these cells with strong positive staining to pankeratin antibodies ( figure 3d ) . such areas were not identified even on retrospective detailed examination of the previous biopsy from 1999 . photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of islands of woven bone amidst a background of bland fibrous spindle cells ( a ) . medium power examination ( b ) ( lens objective 4 ) shows the presence of a rich fibro - collagenous stroma . ( c and d ) 2009 histopathological examination of the tibial lesion photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of occasional clusters of plump epitheliod appearing cells with a corded arrangement in focal regions amidst a fibrous spindle background as seen ( c ) which are strongly positive to pankeratin antibodies on immunohistochemical examination ( d ) . photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of islands of woven bone amidst a background of bland fibrous spindle cells ( a ) . medium power examination ( b ) ( lens objective 4 ) shows the presence of a rich fibro - collagenous stroma . ( c and d ) 2009 histopathological examination of the tibial lesion photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of occasional clusters of plump epitheliod appearing cells with a corded arrangement in focal regions amidst a fibrous spindle background as seen ( c ) which are strongly positive to pankeratin antibodies on immunohistochemical examination ( d ) . the patient underwent revision surgery for non - union of the left tibia in 2010 . in 2007 , a 29-year - old female presented with a swelling in her left inguinal area . investigations including a computed tomography ( ct ) scan showed an extremely large heterogeneous , hyperdense , lobular lymph node centered in the left inguinal region measuring 6.54.6 cm . histopathological examination revealed the presence of a malignant neoplasm that was composed of sheets of basaloid cells with round vesicular nuclei and minimal cytoplasm . this lesion was interpreted as a cutaneous eccrine carcinoma ( figure 1a c ) . due to the unusual diagnosis , an external consultation and a request for additional clinical history were obtained . on detailed review , the pathological report was reinterpreted as metastatic adamantinoma in conjunction with a confirmed past clinical history of surgical excision of a left tibial adamantinoma three years previously in 2004 . the specimen consisted of a firm rubbery mass of fibro - fatty tissue that measured 18.511.37.2 cm with an ellipse of skin measuring 820.3 cm . the cut surface revealed a multi - lobulated light brown well - circumscribed mass that measured 10.475.5 cm . on microscopic examination , the neoplastic cells were composed of predominantly basaloid and tubular nests of malignant cells with some peripheral palisading ( figure 1d f ) . the histomorphological appearance of these cells was identical to the previous histology in this case ( figure 1a c ) confirming the presence of inguinal lymph nodal metastases from adamantinoma of the tibia . biopsy of left inguinal mass ( a , b , c ) and left inguinal lymphadenopathy ( d , e , f ) . photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of a malignant neoplasm in the subcutaneous fat ( a ) . lesional cells are composed predominantly of basaloid cells with round vesicular nuclei and minimal cytoplasm as seen at medium power ( b ) ( lens objective 4 ) and at high power ( c ) ( lens objective 10 ) . photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of a metastatic malignant neoplasm in the lymph node ( ) ( d ) . the neoplastics cells were composed of basaloid and tubular nests of malignant cells with some peripheral palisading as seen at medium power ( e ) ( lens objective 4 ) and at high power ( f ) ( lens objective 10 ) . biopsy of left inguinal mass ( a , b , c ) and left inguinal lymphadenopathy ( d , e , f ) . photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of a malignant neoplasm in the subcutaneous fat ( a ) . lesional cells are composed predominantly of basaloid cells with round vesicular nuclei and minimal cytoplasm as seen at medium power ( b ) ( lens objective 4 ) and at high power ( c ) ( lens objective 10 ) . photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of a metastatic malignant neoplasm in the lymph node ( ) ( d ) . the neoplastics cells were composed of basaloid and tubular nests of malignant cells with some peripheral palisading as seen at medium power ( e ) ( lens objective 4 ) and at high power ( f ) ( lens objective 10 ) . in april 2009 , a new node along the left external iliac in the retroperitoneum was detected and biopsied under radiological guidance . histopathological examination showed similar findings confirming the presence of metastatic adamantinoma ( figure 2a and b ) . ct scans of the chest showed the presence of two nodules in the right lung , which remained stable on follow up over the following year . follow up in early 2011 shows stable residual disease with no additional findings of note . photomicrographs of hematoxylin and eosin stained slide at low power ( a ) ( lens objective 2 ) shows the presence of a malignant neoplasm . the neoplastic cells ( b ) at high power ( lens objective 10 ) shows a similar morphology of basaloid cells as seen in figure 1 . photomicrographs of hematoxylin and eosin stained slide at low power ( a ) ( lens objective 2 ) shows the presence of a malignant neoplasm . the neoplastic cells ( b ) at high power ( lens objective 10 ) shows a similar morphology of basaloid cells as seen in figure 1 . in 1999 , a 58-year - old female presented with a painful swelling on the mid shaft of her left tibia . histopathological examination showed the presence of islands of woven bone in a background of bland spindled fibrous tissue , which was interpreted as fibrous dysplasia ( figure 3a and b ) . nine years later , in 2008 , she was seen for a painful swelling in the same region , which had been increasing in size since her previous operation . a wide excision of the lesion with a tibial allograft and intramedullary nail fixation was performed in october 2009 . there was a predominant fibrous dysplasia like pattern consisting of cellular bland , spindled fibroblastic proliferation with scattered islands of woven bone . embedded within the spindle cells were occasional clusters of plump epitheliod appearing cells that had a corded arrangement in focal regions ( figure 3c ) . immunohistochemistry confirmed the epithelial nature of these cells with strong positive staining to pankeratin antibodies ( figure 3d ) . such areas were not identified even on retrospective detailed examination of the previous biopsy from 1999 . photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of islands of woven bone amidst a background of bland fibrous spindle cells ( a ) . medium power examination ( b ) ( lens objective 4 ) shows the presence of a rich fibro - collagenous stroma . ( c and d ) 2009 histopathological examination of the tibial lesion photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of occasional clusters of plump epitheliod appearing cells with a corded arrangement in focal regions amidst a fibrous spindle background as seen ( c ) which are strongly positive to pankeratin antibodies on immunohistochemical examination ( d ) . photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of islands of woven bone amidst a background of bland fibrous spindle cells ( a ) . medium power examination ( b ) ( lens objective 4 ) shows the presence of a rich fibro - collagenous stroma . ( c and d ) 2009 histopathological examination of the tibial lesion photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of occasional clusters of plump epitheliod appearing cells with a corded arrangement in focal regions amidst a fibrous spindle background as seen ( c ) which are strongly positive to pankeratin antibodies on immunohistochemical examination ( d ) . the patient underwent revision surgery for non - union of the left tibia in 2010 . a literature search limited to the english language using the search terms adamantinoma of tibia and tibial adamantinoma and metastases and inguinal lymph node metastases was conducted . research engines used included pubmed , medline , scopus , embase , and google / google scholar . table 1 provides a comprehensive chronological review of 10 cases with inguinal lymph node metastases with details of reference number , year reported , author , sex , age at diagnosis , bone involved , location in bone , character and duration of symptoms + / history of trauma , latent period , treatment , and outcome . , six cases with lymph node metastases as described in keeney 's study are not included in this table due to the lack of supporting clinical information . similarly , cases with inguinal lymph node metastases as described in moon 's review have been excluded from this table as the original reports are not in english . table 1inguinal lymph node metastases from adamantinoma of the tibia as reported in the english literature ( pub med , medline , scopus , embase , google / google scholar ) since 1930-search terms included adamantinoma of the tibia , and tibial adamantinoma and metastases , and inguinal lymph node metastases.reference numberyear reportedauthorsexage at diagnosisbone involvedlocation in bonecharacter and duration of symptoms + /history of traumalatent periodtreatmentoutcome#101938dunnem32left tibiaupper and middle thirdsswelling , 4yr ; pain later months severe contusionnine monthshigh voltage roentgen , ak amputation 9 months laterdied , 8 years inguinal lymph nodes*#111942dockerty , meyerdingf24left tibiamiddlerecurrent pain , 8 yr . , swelling , 4yr . no history of traumanonelocal excision , ak amputation 15 months laterdied , 1 year , inguinal lymph node metastases#121952mangalik , lal mehrotram40femur , tibia , fibuladistal proximal middleduration of symptoms for 12 months . previous injury to leg1 yearak amputation , irradiation of metastatic nodes#131962knappf14left tibiamiddlemass for 4 years no history of traumaak amputationtotal survival 16 years . pulmonary and inguinal lymph node metastasis#141976winter wgm30left tibiadistalnoneresection , disarticulation of lk , inguinal node dissection , pleurectomydied with inguinal nodes , pulmonary , brain - right occipital metsm37tibiamiddlenoneresection and bone grafting very late knee disarticulationdied , 8 years after initial surgery inguinal lymph nodes and pulmonary metastasis#151990de keyserf13tibiapathological fractureintramedullary nailing several curettages , en bloc resection bk amputation , ak amputationdied 4 years after ak amputation had pulmonary and lymph node metastasis#61994hazelbagm26left tibiadistalnoneamputation metastasis to groin lymph node , pelvisdied of diseasem15left tibiadistalnonechemotherapy , amputationdied of disease . lung / lymph metastases#162008freym20tibianadnadtotal tibia allograft knee disarticulation polychemotherapypulmonary and inguinal lymph node metastases amputation2011tharmabala et al.f24left tibiaswelling left tibia swelling in left groin4 yearssurgical excision in left tibia 2004 . left retroperitoneal mass excision 2009left inguinal nodal metastasis , stable pulmonary nodules with routine follow up in 2011*this is reported in reference # 17 by moon in 1965 . it was first described by maier in 1900 as a primary bone tumor with epithelial characteristics . later in 1913 , fischer named the tumor primary adamantinoma of the tibia because of its resemblance to adamantinoma ( also known as ameloblastoma ) of the jaw and the pituitary gland . although , adamantinoma of the tibia histologically resembles adamantinomas of the jaw and pituitary , there has been no evidence to suggest that these tumors are of similar origin . adamantinoma has also been associated with osteofibrous / fibrous dysplasia and in this regard is considered by some authors to represent a spectrum of disease with evidence of progression from osteofibrous / fibrous dysplasia to adamantinoma in some cases . due to the rarity of this lesion and the varied histological phenotypes , adamantinoma of the tibia can be a diagnostic dilemma leading to misdiagnosis , delay , and suboptimal treatment , as seen in our cases . it can present at any age but mostly occurs in the second to fourth decade . they are most commonly ( 8590% ) localized in the tibial diaphysis rarely , it has been described in other bones of the appendicular skeleton including the fibula , ulna , femur , humerus , radius , and short bones of the feet . , , adamantinoma of the tibia can be divided into two main groups based on their histology and biological behavior . classical adamantinoma refers to the prototype cases with a predominance of epithelial tumor cells and are associated with a destructive growth pattern on radiology . patients with classical adamantinoma are usually of an older age group with a reported range of 1565 years . this variant of classical adamantinoma has a stronger predisposition to local recurrences and a higher potential for distant metastases . in contrast , differentiated adamantinoma of the tibia has a predominance of a osteofibrous / fibrous dysplasia like pattern with scant epithelial tumor elements and is commonly seen in an intracortical location . many authors suggest that osteofibrous/ fibrous dysplasia , a benign lesion occurring commonly in the tibia of children under 10 years of age , is related to adamantinomas and may even be a precursor lesion for adamantinomas . , in such scenarios , tibial adamantinomas have often been misdiagnosed as fibrous dysplasia , as seen in our case . the association of fibrous dysplasia with adamantinoma was first described by cohen et al . in 1962 who proposed that the fibrous pattern seen in such cases was different from that seen in classical fibrous dysplasia . this variant of differentiated adamantinoma is considered by some authors as a secondary reparative process that is associated with a more benign behavior and better prognosis . distant metastases have been reported in adamantinoma of the long bones in about 1520% of cases this may be falsely low due to the slow growing nature of the tumor as long term follow up is required to evaluate its true metastatic potential . regional or other distant metastases as seen in the lung are found more frequently in patients who have a history of repeated local recurrences linked to inadequate primary excision of this lesion . however , in our case ( case 1 ) inguinal nodal metastases occurred with no evidence of local recurrence . risk factors for metastases include male sex , persistent pain , symptoms of less than five years duration , and inadequate initial treatment by biopsy curettage / excision , or resection . the only histological feature associated with an increased recurrence rate was lack of squamous differentiation . in view of the relationship between adamantinoma and osteofibrous dysplasia , czerniak et al . proposed two biological courses that a tibial adamantinoma may follow . one is by the differentiated adamantinomas , which are predominantly intracortical and behave as a reparative process with a low potential to metastasize . the second , such as that seen in our case ( case # 2 ) of classical adamantinoma , is one of progressive tumor growth with a higher potential to metastasize . early metastases within the first two years of initial diagnosis is commonly seen to occur to the regional lymph nodes , whereas delayed metastases is almost always pulmonary or osseous . given that lymph node metastasis usually occurs within the first two years , this is suggestive that these were present at the time of initial diagnosis . in this context , we recommend ultrasound imaging of regional lymph nodes as an essential component in the follow - up examinations of these cases . the current practice recommends surgical extirpation as the primary mode of treatment for localized regional lymph node metastases with radiotherapy and chemotherapy as suggested alternatives . the exact role for sentinel node biopsy and its cost / benefit ratio is unknown due to limited clinical experience in these rare lesions . in conclusion , tibial adamantinoma is a rare primary bone tumor that can cause diagnostic dilemmas especially with clinical presentations of the uncommon pattern of regional nodal metastases . increased clinical awareness of such rare neoplasms with recognition of uncommon patterns of metastases is essential for accurate diagnosis and optimal patient management . additionally , due to the slow - growing nature of tibial adamantinomas we recommend continuous long - term follow up for these patients .

adamantinoma is a rare primary bone tumor that commonly arises in the jaw and has also been described in the appendicular skeleton such as the tibia . we report 2 cases of tibial adamantinomas that were originally misdiagnosed ; one as fibrous dysplasia of the tibia and the other as a cutaneous eccrine carcinoma in a groin mass , which was metastatic adamantinoma to the inguinal lymph nodes . such metastatic adamantinoma to the groin lymph nodes is extremely rare . the clinical and pathological data with a review of the available literature on inguinal lymph node metastases from primary tibial adamantinoma are reported . increased clinical awareness and accurate recognition of such uncommon patterns of inguinal nodal metastases are imperative for appropriate planning of therapeutic strategies and risk management in these patients .

pubmed

antireflection ( ar ) technology plays an important role in the fabrication of high - efficiency solar cells by increasing light coupling into the active region of devices . generally , a relatively low - light reflectivity can be obtained for crystalline silicon solar cells by combining surface texturization with sixny - thin - film deposition . however , in order to further reduce the light reflectivity for the improvement of solar cell efficiency , a variety of nanostructures with size comparable to the wavelength of m : most of the utilized solar spectrum have been proposed , such as photonic crystals , porous structures , plasm : monics [ 5 - 8 ] , etc . the subwavelength structures can be nanowires , nanocones , frustum nanorods , biomimetic nanostructures [ 13 - 16 ] , dielectric nanoparticles etc . all these nanostructures should be based on the wide bandgap materials which , themselves , can not absorb sunlight . the zno material has a wide bandgap , which has been popularly used in the fields of photocatalysis and optoelectronic nanodevices . for photovoltaic application , the zno nanostructures as antireflective layers could be fabricated by various approaches like pulsed laser deposition , chemical vapor deposition , vapor liquid - solid growth , hydrothermal method , and electrochemical deposition technique . am : mong them , a seeding and growth two - step process in zinc salt and amide mixed solutions seems quite acceptable for photovoltaics due to its low cost and good potential for scale - up . experiments have shown that zno nanorod arrays can exhibit better antireflective properties than other oxide compounds . a weighted global reflectance of 6.6% has been achieved in the broadband range from 400 to 1,200 nm . however , m : most work on zno nanorod arrays as antireflective layers is performed on the naked silicon wafer by now . the application of zno nanostructures in the practical silicon solar cells is seldom reported . in this work , we have grown zno nanowhiskers ( nws ) with different lengths by the seeding and growth two - step process , and meanwhile have integrated the zno nw technique into the fabrication of czochralski ( cz ) silicon solar cells based on the current standard celling technology . the density and the length of zno nws have been optimized for the improvement of solar cell performances by controlling the time of growth . the result shows that the efficiency of cz silicon solar cell can be improved by a value of 3% relatively using the zno nws as the ar layer in practice . the starting samples are p - type cz silicon solar cells fabricated by a standard process including surface texturization , phosphorus diffusion , sixny film deposition , screen printing , and metallization . the seed layer of zno was first deposited on the front surface of solar cells by a magnetron sputtering system . the deposition temperature is 150c , and the seed layer thickness is about 10 nm . after protecting the parts without the zno seed film with waterproof tapes , the samples were immersed in the solution of zinc nitrate hexahydrate ( zn(no3)26h2o ) and ( ch2)6 n4 mixture for 30 to 60 min for the growth of zno nw . the zno nw growth temperature is 90c . for characterization , a scanning electron microscope ( sem ; hitachi s-4800 , hitachi , ltd . , chiyoda , tokyo , japan ) was used to observe the zno nws on the solar cells . the reflection spectra of the samples were measured by a uv vis spectrophotometer ( hitachi u-4100 ) in the wavelength range of 300 to 1,100 nm . the external quantum efficiencies ( eqes ) of the final solar cells were obtained from the spectral responses . the current voltage characteristics of the cells under the dark and illumination circumstances were measured . for illumination , one sun ( 100 mw / cm ) with am 1.5 global spectrum was used . figure 1 shows the sem micrographs of a silicon solar cell with the zno nws after a two - step growth process . it can be seen that the zno nws are uniformly fabricated on the surface of silicon solar cell . generally , the fabrication of zno nws based on aqueous solution consists of nucleation and growth . the main chemical reactions through the whole process can be described as follows : ( 1)znno326h2ozn2++ 2no3+6h2o ( 2)ch26n4 + 6h2o6hcho+4nh3 sem images of top ( a ) and cross - sectional ( b ) views of zno nw - coated solar cell surface . the decomposition of hexamethylenetetramine produces amm : monia that can supply the oh needed for the zno nw formation reaction and acts as a ph buffer to regulate the ph value of the solution . the ph value of the solution should be 6 or 7 for the whole reaction process . during the process , the zno nanocrystals existing in the seed layer serve as the nucleation centers where the growth of zno nws takes place . due to its growth habit , c - axis is the m : most preferred growth orientation , which leads to the formation of zno nws . figure 2 shows the sem micrographs of zno nws on the solar cells after growing at different times . figure 2a , d shows the plan view and its cross - sections of zno nws after growing 30 min , respectively . one can see that the tapered zno nws with the length of approximately 100 nm are obtained on the pyramid - like textured surface of solar cell via this method . with the growth time increasing , the density of zno nws increases ( see figure 2b , c , e , f ) . meanwhile , the length of zno nws becomes larger , e.g. , approximately 200 nm after 45 min and approximately 300 nm after 60 min . sem images of zno nws grown under different times and their cross - section view . ( a ) and ( d ) 30 min , ( b ) and ( e ) 45 min , and ( c ) and ( f ) 60 min . figure 3 shows the reflectance spectra of the solar cells without or with the zno nw coating . it can be seen that , compared with the solar cell without zno nw coating ( reference ) , all the solar cells with zno nw coating exhibit lower reflectance than in the whole wavelength range . the reflectivity of zno nw - coated solar cells decreases with an increase of zno nw growth time . the solar cell with zno nws grown for 45 min has the lowest reflectivity . however , when the zno nw growth time reaches to 60 min , the reflectivity of coated solar cell becomes higher in the wavelength range of 500 to 1,100 nm . since the density and length of zno nws increase with an increase of growing time , it is suspected that the zno nws grown for 60 min could have formed something like this finally results in a higher reflection of zno nw grown for 60 min . nevertheless , these results clarify that the zno nw - coated solar cells have better antireflection effect than the reference without zno nw coating . the decrease of reflectivity in the long wavelength range mainly results from the effect of zno nw trapping light by the multiple scattering of the incident light . meanwhile , their gradually and continuously changing refractive index profile from the air to the substrate is also beneficial for its antireflection property . the drastic decrease of reflectance in the low - wavelength range should be mainly attributed to the absorption of light by zno nws , themselves , which hardly makes any contribution to the improvement of the solar cell performance . after a prolonged growing time , e.g. , 60 min , the light absorption of long zno nws becomes even stronger , and therefore , their contribution to the solar cell efficiency is further smaller . the reflectance ( r ) spectra for the solar cell with zno nw grown under different times . figure 4 shows the eqes of the solar cells with zno nws grown for different times . it can be seen that the solar cells with zno nws grown for 30 and 45 min generally have higher eqes than the referenced one in the whole spectral region . it signifies that zno nws on cz silicon solar cells can effectively improve the response of light spectrum . for the solar cell with zno nws grown for 60 min , the eqes in the short - wavelength range are smaller than that of the referenced due to the stronger light absorption of long zno nws . table 1 lists the detailed parameters of the solar cells coated with zno nws grown for different times . it can be seen that even though the open - circuit voltages for all the solar cells keep the same , the short - circuit current density of the solar cells coated by zno nws are higher . this finally results in that the efficiency of the solar cells coated by zno nws grown for 30 and 45 min are improved by a value of 0.1% and 3.0% , respectively , in comparison with the referenced cell . figure 5 examples the current density as a function of bias voltage for the solar cells with zno nws grown for 45 min . it reveals that zno nw - coated solar cells have higher short - circuit current density than the referenced one , while the open - circuit voltages keep constant . the improvement of short - circuit current density is obviously a result of zno nws suppressing the light reflectivity . in the case of zno nws grown for 60 min , the fill factor of the cell has a low fill factor and , therefore , a low efficiency . this deterioration of the cell performance should be related to the long - time immersion of the solar cell in the 90c aqueous solution during the zno nw growth process . the external quantum efficiencies ( eqes ) of the solar cells with zno nws grown for different times . performance parameters of c - si solar cells with and without zno nws ff fill factor , jsc short - circuit current density , voc , open - circuit voltage . the current density - voltage curves of the solar cells with 45-min grown zno nws . under a standard am 1.5 illumination . in summary , around 100 to 300 nm zno nws are grown on the surface of czochralski si solar cells based on a low cost aqueous solution method . zno nws grown for different times , such as 30 , 45 , and 60 min , can reduce the reflectivity of the solar cells effectively ( r < 3% ) in a large spectral region and improve the external quantum efficiency in the corresponding range . furtherm : more , zno nws with 45-min growth time can improve the efficiency of the solar cells by 3% , corresponding to their increment of short - circuit current density and fill factor . finally , long - time immersion of solar cells in aqueous solution during the zno nw growth process can deteriorate the solar cell performance drastically . deren yang and xuegong yu are both professors and doctorate degree holders in the state key laboratory of silicon materials , department of materials science & engineering , zhejiang university . dong wang , dong lei , and genhu li are masters degree holder candidates of the same university . this work is supported by the national natural science foundation of china ( grant nos . 60906002 and 50832006 ) , national key technology r&d program ( 2011bae03b13 ) , key laboratory of optoelectronic materials chemistry and physics , cas ( 2008dp173016 ) , and innovation team project of zhejiang province ( 2009r50005 ) .

an efficient antireflection coating is critical for the improvement of silicon solar cell performance via increased light coupling . here , we have grown well - aligned zno nanowhisker ( nw ) arrays on czochralski silicon solar cells by a seeding - growth two - step process . it is found that the zno nws have a great effect on the macroscopic antireflection effect and , therefore , improves the solar cell performance . the zno nw array - coated solar cells display a broadband reflection suppression from 500 to 1,100 nm , and the minimum reflectance smaller than 3% can easily be achieved . by optimizing the time of zno nw growth , it has been confirmed that an increase of 3% relatively in the solar cell efficiency can be obtained . these results are quite interesting for the application of zno nanostructure in the fabrication of high - efficiency silicon solar cells .

pubmed

little is understood about this condition 's mechanism , exacerbating factor , and definite treatment . hyperhidrosis generally occurs in the axillae , the palms , plantar surface of the feet , and the face . it is estimated to affect 0.6 to 1.0% of the population , generally young adults . the principal characteristics of this condition include intense discomfort experienced by the patients , affecting their social , affective , and professional lives . primary hyperhidrosis generally manifests in childhood and early adolescence , does not occur during sleep , and can be exacerbated by emotional stress . excessive sweating of the palms and axillae can cause utmost embarrassment in professional and social interaction . the fact that such sweating is not socially accepted , and the discomfort , odor , and stained clothes caused by constant wetness significantly decrease quality of life ( qol ) . although various treatment options are available , including topical and systemic therapies , iontophoresis , regional nerve block , and botulinum toxin injection , have their limitations . video - assisted thoracoscopic sympathetic surgery has been accepted globally as a form of treatment for primary palmar hyperhidrosis . in the literature , different methods , including resection ( sympathectomy ) , transection ( sympathicotomy ) , and clipping much effort to reduce compensatory hyperhidrosis and determine its contributory factors has been attempted during surgical procedures . it has been used to reduce severe compensatory hyperhidrosis related to the procedural level of the sympathetic chain . procedures that involve r3 or / and r4 sympathetic ganglions are widely accepted in many treatment centers , and the results have been favorable . however , some patients still present with postoperative compensatory sweating or overly dry hands . the aim of this study was to compare two simplified methods for the treatment of palmar and axillary hyperhidrosis , in which the sympathetic chain was transected in merely one segment at the level of either the third or fourth rib , respectively referred to as r3 or r4 sympathicotomy . emphasis was placed on comparing the impact of sympathicotomy at r3 and r4 , and to evaluate the efficacy and adverse effects of palmar and axillary hyperhidrosis by surgery . between january of 1999 and april of 2009 , 133 patients with primary hyperhidrosis underwent video - assisted thoracic sympathicotomy . there were 62 males and 71 females , with a mean age of 22.56.5 years . the most common clinical manifestation of hyperhidrosis was palmer and axillary sweating ( in 39.9% ) , followed by palmer sweating alone ( in 33.8% ) , and axillary sweating alone ( in 26.3% ) . the severity of palmar sweating , sweating in other parts of the body , other concomitant symptoms and past medical history were recorded . chest radiography , electrocardiogram ( ecg ) and routine blood examination were performed before surgery . severe primary palmar hyperhidrosis that significantly interfered with daily life , including work , was the common primary indication for surgery . secondary hyperhidrosis caused by hyperthyroidism or neurotic anxiety , and patients with bradycardia ( heart rate < 60/min ) were excluded from surgery . patients were randomized into two therapeutic groups ( table 1 ) . patients were placed under single lumen intubation general anesthesia in the semi - fowler 's position ( dorsal decubitus position with back lifted 45 degrees from horizontal , and abduction of both arms ) . a 2 mm trocar was introduced into the pleural cavity through the fourth intercostals space of the mid - axillary line . after thoracoscope insertion , an initial panoramic view of the upper pleural cavity and mediastinal structures was obtained . another operating channel ( 2 mm ) was made through the fourth intercostal space of the anterior axillary line . after identification of the thoracic sympathetic ganglion , the t3 or t4 sympathetic chains were then destroyed by electric cautery . the anesthesiologist re - inflated the lung . following lung re - expansion by positive inflation with a 6 fr feeding catheter , the trocar was then removed . the patients were asked simple questions related to symptom resolution , surgical outcome satisfaction , and occurrence / intensity of reflex sweating . compensatory sweating in the other regions of the body was graded as " none " when compensatory sweating was not noticeable and did not interfere with daily activities , " mild " when it was tolerable but sometimes interfered with daily activities , " moderate " when it was barely tolerable and frequently interfered with daily activities , and " severe " when it was intolerable and always interfered with daily activities . the degree of satisfaction in each patient was recorded as " satisfied " or " dissatisfied " . self - reported satisfaction , recurrence , and improvement of sweating after intervention constituted the three recorded aspects of the patients ' subjective experience with sweat - triggering situations . recurrence was defined as severe discomfort experienced by patients similar to that experienced before surgery , despite improvement in sweating . statistical analysis of comparisons between the 2 groups was conducted using the student 's unpaired t test and chi - square test with spss 13.0 ( spss , inc , chicago , il ) . all operations were successfully performed using video - assisted thoracoscopy without associated severe morbidity and mortality . no life - threatening event or death occurred and other complications , such as horner syndrome , hemothorax , intercostal neuralgia , pulmonary edema and atelectasis were not observed . mild subcutaneous emphysema and residual pneumothorax on follow - up chest radiograph resolved spontaneously , and no further therapy was required . there was no significant difference between the 2 groups in terms of age , sex , and positive family history ( table 1 ) . in addition , there was no significant difference between the 2 groups in the rate of satisfaction and recurrence . the incidence of " moderate " to " severe " compensatory sweating was higher in the r3 ( 71.8% ) group than r4 ( 33% , p=0.02)(table 2 ) . no patient requested reoperation in either group , and no patient expressed regret after surgery . the dominating indication for sympathetic surgery is hyperhidrosis of the upper limb . when conservative and medical treatment fails , endoscopic thoracic surgery is performed . the first sympathectomy for the treatment of hyperhidrosis was performed in 1920 , in which anterior cervical access was used . in 1942 , great advance in surgical technique was made , which rendered it unnecessary to sacrifice the stellate ganglion in order to denervate the upper limb . as such , one of the most feared complications in thoracic sympathectomy - claude bernard - horner syndrome - was eliminated . in this study , patients were informed about the possible side effects of sympathicotomy , including compensatory sweating , prior to the procedure . chou et al . asserted that reflex response in the sweating center of the hypothalamus could cause the underlying mechanism of compensatory sweating . according to previous studies , various factors , such as the location of hyperhidrosis , sympathicotomy level , and surgical procedures including clamp , cut and resect can induce compensatory sweating . also , the severity of compensatory sweating is influenced by patient condition , such as obesity , primary complaint severity and t2 sympathetic ganglion resection . therefore , more selective sympathicotomy was proposed to minimize side effects , by lowering the level to preserve sympathetic tone in place of upper t2 sympathicotomy or multilevel sympathicotomy . selective sympathicotomy is an effective surgical method to eliminate compensatory sweating in the treatment of hyperhidrosis . although compensatory sweating remains the main secondary effect , numerous studies have reported that sympathicotomy is a safe and effective procedure for the management of hyperhidrosis and reduction of compensatory sweating . the frequency and severity of compensatory sweating were reduced , with less interruption of sympathetic trunk in group t4 compared to group t3 ( table 2 ) . this explains the differences at the other levels in sympathicotomy at palmar sweating and compensatory sweating are still controversial topic . lin et al . suggested that postoperative results including compensatory sweating is influenced by changes in the sympathetic tone and disturbance of the reflex arc in the hypothalamus secondary to the procedures of the upper thoracic sympathetic system . furthermore , anatomical variation , a mistake at the level of interruption , and incomplete resection of the sympathetic trunk may also cause compensatory sweating . the high personal expectations of patients could be the reason of dissatisfaction despite the development of minor complications . difference in surgical technique , such as sympathicotomy level and surgical procedure ( clamp , cut and resect ) , and classification of the extent of compensatory sweating could impact on the complication rate . many authors felt that the more sympathetic segments excised , particularly including t2 , the greater the incidence of severe compensatory symptoms . dewey et al . limited the extent of resection for hyperhidrosis to a single level , and found reduction in the incidence of severe compensatory symptoms . however , until now , no prospective study has been performed to compare r3 and r4 single level sympathicotomy for the treatment of palmar and axillary hyperhidrosis . in this study , patients did not experience discomfort in their daily activities . these results suggest that , compared to r3 sympathicotomy , r4 sympathicotomy decreases the extent of side effects but does not compromise efficacy . as such , it should be the method of choice for the treatment of palmar and axillary hyperhidrosis . r3 and r4 have similar success rates in the treatment of palmar and axillary hyperhidrosis .

backgroundvideo - assisted thoracic sympathicotomy plays an important for the treatment of essential hyperhidrosis . patients are usually satisfied with the surgical outcome at the early post - operative period , but suffer recurrence and compensatory sweating in the late post - operative period . there are many sympathicotomy methods to minimize recurrence and compensatory sweating . we compared the outcome of sympathicotomy methods above the third rib ( r3 ) and the fourth rib ( r4 ) with regards to symptoms , satisfaction , recurrence , and compensatory palmar and axillary hyperhydrosis.materials and methodsfrom january 1999 to april 2009 , 39 cases of thoracoscopic sympathicotomy at the third rib ( r3 ) , and 94 cases of thoracoscopic sympathicotomy at the fourth rib ( r4 ) for palmar and axillary hyperhidrosis were compared for early and late post - operative satisfaction , compensatory sweating and recurrence.resultsthere was no sex or age difference between groups . early satisfaction was 94.9% and 98.9% in the r3 group and r4 group , respectively . there was no difference in early satisfaction ( 94.9% in r3 and 98.9% in r4 ) , late satisfaction ( 84.6% in r3 and 89.4% in r4 ) , or recurrence ( 17.9% in r3 and 17.0% in r4 ) between groups . there was significant difference in compensatory sweating ( 71.8% in r3 and 33% in r4 , p=0.002).conclusionr4 sympathicotomy demonstrated superior efficacy in the treatment of compensatory sweating compared to r3 in palmar and/or axillary hyperhidrosis .

pubmed

parkinson disease ( pd ) , which is the most common neurodegenerative parkinsonian syndrome , is characterized by the degeneration of both dopaminergic and nondopaminergic neurons with neuronal intracytoplasmic inclusions known as lewy bodies 1 . although the clinical diagnosis of pd is often straightforward and robust in the cases with the typical cardinal symptoms and characteristic signs , the correct diagnosis can be challenging , especially in clinically mild or uncertain cases . furthermore , it remains difficult to accurately differentiate pd from other neurodegenerative parkinsonian syndromes ( non - pd ) , such as multiple system atrophy ( msa ) , progressive supranuclear palsy ( psp ) , and corticobasal degeneration . an accurate and early diagnosis is the key to correct management and prognostication . in recent years , molecular imaging techniques using pet or single - photon emission computed tomography ( spect ) have offered a variety of tests that can improve the diagnostic accuracy of pd and non - pd . dopamine transporter ( dat ) ligands 24 and the sympathetic innervation of the heart 1,5 have become very popular and are used widely in clinical practice . for example , imaging of dat binding with [ i]-2-carbomethoxy-3-(4-iodophenyl)-n-(3-fluoropropyl)-nortropane ( fp - cit ) successfully visualizes the presynaptic dopaminergic degeneration of the nigrostriatal tract 4,6 . dat spect abnormalities can help to support a diagnosis of degenerative parkinsonian syndromes by excluding patients with essential tremor 7 , and psychogenic 8 and vascular parkinsonism 9 who have a normal nigrostriatal function . however , dat imaging as a stand - alone tool can not differentiate between the various types of degenerative parkinsonian syndromes with sufficient accuracy 10,11 . similarly , several studies have shown the utility of meta-[i]-iodobenzylguanidine ( mibg ) myocardial scintigraphy in assessing the sympathetic cardiac nerve terminals in pd patients with a decreased cardiac mibg uptake in comparison with other parkinsonian syndrome patients in whom the mibg uptake is normal 12 . because these molecular imaging tools are limited in terms of their test accuracy ( ta ) , the use of either dat spect or mibg scintigraphy alone is insufficient for distinguishing pd from non - pd 13 . in this retrospective study , we compared the results of spect studies of striatal dat receptors and myocardial mibg scintigraphy with the clinical diagnoses in 34 patients with suspected parkinsonian syndromes to evaluate the diagnostic accuracy and usefulness of combined spect imaging in the same patient population . thirty - four patients ( male / female : 19/15 , mean age : 62.113.7 years , age range : 3181 years ) , who underwent both [ i]-mibg scintigraphy and dat spect within a 4-month period for a differential diagnosis between pd and other parkinsonian syndromes were consecutively enrolled in this retrospective study , which was carried out from april 2014 until june 2016 . because many patients underwent two tests during a period ranging from 3 to 4 months at our hospital , we established a 4-month interval between the tests . the study was approved by the institutional ethics committee and all procedures were in accordance with the ethical standards on human investigation and with the principles of the declaration of helsinki . all of the patients underwent molecular in - vivo brain and cardiac diagnostic examinations , which consisted of presynaptic striatal dat scintigraphy with fp - cit and the assessment of myocardial sympathetic innervation with mibg . the final diagnosis was made by the neurologists of tokushima university hospital , department of neurology , who carefully assessed the medical history and symptoms , the possible response to dopaminergic treatment , and the diagnostic mri findings . furthermore , five patients were diagnosed with probable msa - p according to the established criteria 15,16 and two patients fulfilled the national institute of neurologic disorders and stroke criteria for probable psp 17 . the brain spect scans were acquired according to the european association of nuclear medicine procedure guidelines for brain neurotransmission spect using [ i]-labeled dat ligands ( fp - cit , datscan ; nihon medi - physics co. ltd , tokyo , japan ) 18 . after an injection of 167 mbq of datscan , the projection data were obtained in a 128128 matrix on a ecam , toshiba ( toshiba medical systems corp . , tochigi , japan ) ( 15 april 201414 april 2015 ) , or symbia t16 , siemens ( siemens healthcare , erlangen , germany ) ( 15 april 201530 july 2016 ) mounted with low - energy to medium - energy general - purpose collimators . the data were reconstructed using the filtered back projection method without attenuation and scatter correction from 15 april 2014 to 14 april 2015 . the data were reconstructed by the ordered subset expectation maximization method ( iteration 12 , subset 6 ) using a syngo miva10c with the symbia net software program ( siemens ag , munich , germany ) and corrected for attenuation by computed tomography and the triple energy window scatter correction method from 15 april 2015 to 30 july 2016 . the specific binding ratio ( sbr ) was calculated semiquantitatively using the dat view software program ( nihon medi - physics , tokyo , japan ) on the basis of bolt s method as described in detail elsewhere 19 . for the quantitative analysis , irregular regions of interest ( rois ) were drawn on single - slice views in areas corresponding to the left and right striata on either side . although dat spect in pd typically shows an asymmetric loss of dat binding ( most prominent in the putamen ) , the tracer uptake is more symmetric in msa 20 and psp 21 . in one report of pathology - confirmed pd and msa cases with a similar disease duration 22,23 , visual inspection and a semiquantitative analysis with dat spect detected a bilateral reduction in striatal dat binding in all patients , but the trend toward greater asymmetric binding was greater in msa than in pd . for this study , we adopted the average value of the right and left sbrs as the sbr . after the patient had rested for 15 min in the supine position , 111 mbq of [ i]-mibg ( myomibg ; fuji ri pharma , tokyo , japan ) was injected intravenously . early and delayed spect were performed at 15 min and 3 h after the injection , respectively . planar imaging for 5 min in the anterior projection was performed automatically during spect . planar scanning and spect were performed using a dual - head gamma camera equipped with a low - energy to medium - energy general purpose parallel - hole collimator ( toshiba ecam ) . the heart to mediastinum ( h / m ) ratio was calculated by dividing the count density of the left ventricular roi by that of the mediastinal roi , according to the standard method , which has been described previously 23 , 24 . the h / m ratios calculated from the roi counts obtained by delayed spect were used for the comparison study because delayed scans show the neuronal uptake of mibg more explicitly 24 . the values were expressed as meansd and were analyzed using student s t - test and -test . p values of less than 0.05 were considered to indicate a statistically significant difference between pd and non - pd groups . the sensitivity and specificity of the respective diagnostic index ( h / m ratios of the mibg uptake in the delayed phase , sbr on dat spect , and combined dat spect and mibg myocardial scintigraphy ) for differentiation between pd and non - pd were assessed using a receiver - operating characteristic ( roc ) analysis . for the combined use of dat spect and mibg myocardial scintigraphy , the cutoff delayed - phase h / m and sbr values were 2.745 and 3.24 , respectively . the statistical analyses were carried out using the spss software program ( ibm spss statistics , version 23 ; ibm corp . , armonk , new york , usa ) . the normality of the data distribution was assessed using the shapiro wilk test . the mann whitney u - test was used for the comparison of non - normally distributed data . differences with a p value of less than 0.05 were considered to be biologically significant . the analysis of the descriptive statistics and basic comparisons were carried out using the spss software program . the sensitivity and specificity of the respective diagnostic index ( the h / m ratios of the mibg uptake in the delayed phase , sbr on dat spect , and combined dat spect and mibg myocardial scintigraphy ) for the differentiation between pd and non - pd were assessed in a roc analysis . we also calculated the specificity , sensitivity , positive predictive value , negative predictive value , and ta of the combination of the two methods using the cutoff value for their combination . the brain spect scans were acquired according to the european association of nuclear medicine procedure guidelines for brain neurotransmission spect using [ i]-labeled dat ligands ( fp - cit , datscan ; nihon medi - physics co. ltd , tokyo , japan ) 18 . after an injection of 167 mbq of datscan , the projection data were obtained in a 128128 matrix on a ecam , toshiba ( toshiba medical systems corp . , tochigi , japan ) ( 15 april 201414 april 2015 ) , or symbia t16 , siemens ( siemens healthcare , erlangen , germany ) ( 15 april 201530 july 2016 ) mounted with low - energy to medium - energy general - purpose collimators . the data were reconstructed using the filtered back projection method without attenuation and scatter correction from 15 april 2014 to 14 april 2015 . the data were reconstructed by the ordered subset expectation maximization method ( iteration 12 , subset 6 ) using a syngo miva10c with the symbia net software program ( siemens ag , munich , germany ) and corrected for attenuation by computed tomography and the triple energy window scatter correction method from 15 april 2015 to 30 july 2016 . the specific binding ratio ( sbr ) was calculated semiquantitatively using the dat view software program ( nihon medi - physics , tokyo , japan ) on the basis of bolt s method as described in detail elsewhere 19 . for the quantitative analysis , irregular regions of interest ( rois ) were drawn on single - slice views in areas corresponding to the left and right striata on either side . although dat spect in pd typically shows an asymmetric loss of dat binding ( most prominent in the putamen ) , the tracer uptake is more symmetric in msa 20 and psp 21 . in one report of pathology - confirmed pd and msa cases with a similar disease duration 22,23 , visual inspection and a semiquantitative analysis with dat spect detected a bilateral reduction in striatal dat binding in all patients , but the trend toward greater asymmetric binding was greater in msa than in pd . for this study , we adopted the average value of the right and left sbrs as the sbr . after the patient had rested for 15 min in the supine position , 111 mbq of [ i]-mibg ( myomibg ; fuji ri pharma , tokyo , japan ) was injected intravenously . early and delayed spect were performed at 15 min and 3 h after the injection , respectively . planar imaging for 5 min in the anterior projection was performed automatically during spect . planar scanning and spect were performed using a dual - head gamma camera equipped with a low - energy to medium - energy general purpose parallel - hole collimator ( toshiba ecam ) . the heart to mediastinum ( h / m ) ratio was calculated by dividing the count density of the left ventricular roi by that of the mediastinal roi , according to the standard method , which has been described previously 23 , 24 . the h / m ratios calculated from the roi counts obtained by delayed spect were used for the comparison study because delayed scans show the neuronal uptake of mibg more explicitly 24 . the values were expressed as meansd and were analyzed using student s t - test and -test . p values of less than 0.05 were considered to indicate a statistically significant difference between pd and non - pd groups . the sensitivity and specificity of the respective diagnostic index ( h / m ratios of the mibg uptake in the delayed phase , sbr on dat spect , and combined dat spect and mibg myocardial scintigraphy ) for differentiation between pd and non - pd were assessed using a receiver - operating characteristic ( roc ) analysis . for the combined use of dat spect and mibg myocardial scintigraphy , the cutoff delayed - phase h / m and sbr values were 2.745 and 3.24 , respectively . the statistical analyses were carried out using the spss software program ( ibm spss statistics , version 23 ; ibm corp . , armonk , new york , usa ) . the mann whitney u - test was used for the comparison of non - normally distributed data . differences with a p value of less than 0.05 were considered to be biologically significant . the analysis of the descriptive statistics and basic comparisons were carried out using the spss software program . the sensitivity and specificity of the respective diagnostic index ( the h / m ratios of the mibg uptake in the delayed phase , sbr on dat spect , and combined dat spect and mibg myocardial scintigraphy ) for the differentiation between pd and non - pd were assessed in a roc analysis . we also calculated the specificity , sensitivity , positive predictive value , negative predictive value , and ta of the combination of the two methods using the cutoff value for their combination . although there was no significant difference in the age of the patients in the two groups , a statistically significant difference was observed in the male - to - female ratio . the demographic and clinical features of the pd and non - pd patients the mean h / m ratio of the mibg uptake in the delayed phase ( 2.190.55 vs. 2.440.80 , cutoff=2.745 , p=0.025 ) was significantly lower in the pd patients than in the non - pd patients ; however , the mean sbr ( 2.460.87 vs. 2.941.40 , cutoff=3.24 , p=0.08 ) did not differ to a statistically significant extent ( table 1 and fig . roc analyses were used to set the cutoff sbr value ( in dat spect ) and the h / m ratio ( in delayed scanning ) in mibg scintigraphy for differentiating pd from non - pd ( fig . 2 ) . in the comparison with the separate use of the sbr and the delayed h / m ratio , the combined use of the two semiquantitative analyses using cutoff values of less than 3.24 and less than 2.745 , respectively , allowed for more accurate differentiation between pd and non - pd . the sensitivity and specificity of sbr were 86.7 and 52.6% , respectively ( table 2 ) . the sensitivity and specificity of the delayed h / m ratio on mibg scintigraphy were 93 and 47% , respectively . the accuracy of these analyses in differentiating between pd and non - pd was the same using each of the molecular methods ( dat spect , 67.6% ; mibg scintigraphy , 67.6% ) . in comparison with the sbr and delayed h / m ratio , the combined use of two semiquantitative analyses enabled pd and non - pd to be differentiated more accurately ( cutoff values : sbr<3.24 ; delayed h / m ratio<2.745 ) . these indices distinguished pd from non - pd with a sensitivity of 86.7% , a specificity of 73.7% , an accuracy of 79.4% , a positive predictive value of 72.2% , and a negative predictive value of 87.5% ( table 2 ) . box plots of the striatal fp - cit and cardiac delayed mibg accumulation in the pd and non - pd patients . the box plots show the striatal fp - cit and cardiac delayed mibg accumulation in the pd and non - pd patients . whiskers represent the minimum and maximum values . there was a statistically significant difference in the delayed h / m ratios of the pd and non - pd groups . the sbr values of the two groups did not differ to a statistically significant extent . - cit , n--fluoropropyl-2-carbomethoxy-3-(4-iodophenyl)-nortropane ; h / m , heart to mediastinum ratio ; mibg , meta-[i]-iodobenzylguanidine ; non - pd , degenerative parkinsonian syndrome other than pd ; pd , parkinson s disease ; sbr , specific binding ratio . the receiver - operating characteristic curves of the delayed h / m ratio and sbr . delayed h / m ratio : auc=0.698 ; cutoff value=2.745 ; sensitivity=0.93 ; specificity=0.47 . auc , area under the curve ; h / m , heart to mediastinum ratio ; sbr , specific binding ratio . the statistical parameters that were evaluated when investigating the combined use of fp - cit spect and mibg scintigraphy in differentiating pd from non - pd this retrospective study investigated the extent to which the combined use of mibg scintigraphy and dat spect may improve diagnostic accuracy in differentiation between pd and non - pd . the analysis showed that the individual molecular approaches had a mild ta in differentiating between pd and non - pd ( mibg scintigraphy , 62% ; fp - cit spect , 69% ) . in agreement with previous studies 25,26 , we confirmed that the combined use of dat spect and mibg scintigraphy was more useful for differentiating between pd from non - pd in comparison with the use of either method alone . dat spect is therefore very popular in the clinical setting and enables highly accurate differentiation between degenerative parkinsonian syndromes and movement disorders that are not associated with a dopamine deficit 79 . according to plotkin , the resulting accuracy , sensitivity , and specificity of fp - cit spect in the detection of pd and non - pd were 94 , 93 , and 100% , respectively 28 . the loss of dopaminergic neurons in the substantia nigra and the reduction of striatal dopamine projections are histopathological characteristics that are common to the degenerative parkinsonian syndromes and the low accumulation of striatal dat shows a moderate ta in differentiating pd from non - pd . the sensitivity ( 86.7% ) and specificity ( 52.6% ) that were observed in the present study were consistent with previous studies 10,20,29,30 . in our study , the sensitivity and specificity in differentiating pd from non - pd using the h / m ratio of the mibg uptake were 93 and 47% , respectively . the usefulness of mibg myocardial scintigraphy in the diagnosis of dlb was suggested recently in a meta - analysis . the sensitivity and specificity of mibg scintigraphy in the differential diagnosis between pd and other parkinsonian syndromes ranged from 71 to 100% and from 50 to 100% , respectively , with pooled estimates of 88% ( 95% confidence interval : 8690% ) and 85% ( 95% confidence interval : 8188% ) 5 . the specificity observed in the present study was lower than expected . in terms of the diagnostic performance , mibg scintigraphy is usually a sensitive , but not specific , test for pd 5 . there might be several reasons for this . as the myocardial [ i]-mibg uptake in patients with non - pd ( especially in patients with msa and psp ) is often found to be slightly reduced in comparison with healthy controls 31 , [ i]-mibg scintigraphy may yield some false - positive results in patients with msa and psp . the myocardial sympathetic nervous system of msa patients typically shows mild degeneration 32 ; thus , false - positive results may account for this scintigraphic finding . in addition to lbd - related postganglionic sympathetic degeneration , false - positive results on [ i]-mibg scintigraphy may also occur because of age - related degeneration because the myocardial [ i]-mibg uptake has been reported to decrease significantly with age 30,33 . the combination of dopaminergic and sympathetic scintigraphic imaging tests for differentiating between pd and non - pd has been explored by other authors 25,26,28,30,34 . some studies showed the complementary role of [ i]-mibg scintigraphy and dat spect in the differential diagnosis between pd and non - pd 25,34 . a recent study showed that [ i]-fp - cit spect presents high sensitivity in the diagnosis of lewy body disease ; thus , [ i]-mibg scintigraphy may play a complementary role in the differential diagnosis between pd and other parkinsonism 30 . in the present study , we therefore explored the extent to which the combination of these two tracers would improve the ta . our data show that by combining fp - cit spect and mibg scintigraphy , the ta can be increased to 79.4% . first , this study was carried out in a single university hospital ; thus , the study population was small . second , the clinical diagnosis was used as the gold standard in our study because of the absence of histopathological confirmation ; thus , the clinical diagnosis may have been incorrect in some cases . third , before a molecular examination is performed , it is necessary to establish an appropriate withdrawal period in which to cease the administration of interfering drugs by considering their biological half - lives . dat spect is therefore very popular in the clinical setting and enables highly accurate differentiation between degenerative parkinsonian syndromes and movement disorders that are not associated with a dopamine deficit 79 . according to plotkin , the resulting accuracy , sensitivity , and specificity of fp - cit spect in the detection of pd and non - pd were 94 , 93 , and 100% , respectively 28 . however , the loss of dopaminergic neurons in the substantia nigra and the reduction of striatal dopamine projections are histopathological characteristics that are common to the degenerative parkinsonian syndromes and the low accumulation of striatal dat shows a moderate ta in differentiating pd from non - pd . the sensitivity ( 86.7% ) and specificity ( 52.6% ) that were observed in the present study were consistent with previous studies 10,20,29,30 . in our study , the sensitivity and specificity in differentiating pd from non - pd using the h / m ratio of the mibg uptake were 93 and 47% , respectively . the usefulness of mibg myocardial scintigraphy in the diagnosis of dlb was suggested recently in a meta - analysis . the sensitivity and specificity of mibg scintigraphy in the differential diagnosis between pd and other parkinsonian syndromes ranged from 71 to 100% and from 50 to 100% , respectively , with pooled estimates of 88% ( 95% confidence interval : 8690% ) and 85% ( 95% confidence interval : 8188% ) 5 . the specificity observed in the present study was lower than expected . in terms of the diagnostic performance , mibg scintigraphy is usually a sensitive , but not specific , test for pd 5 . there might be several reasons for this . as the myocardial [ i]-mibg uptake in patients with non - pd ( especially in patients with msa and psp ) is often found to be slightly reduced in comparison with healthy controls 31 , [ i]-mibg scintigraphy may yield some false - positive results in patients with msa and psp . the myocardial sympathetic nervous system of msa patients typically shows mild degeneration 32 ; thus , false - positive results may account for this scintigraphic finding . in addition to lbd - related postganglionic sympathetic degeneration , false - positive results on [ i]-mibg scintigraphy may also occur because of age - related degeneration because the myocardial [ i]-mibg uptake has been reported to decrease significantly with age 30,33 . the combination of dopaminergic and sympathetic scintigraphic imaging tests for differentiating between pd and non - pd has been explored by other authors 25,26,28,30,34 . some studies showed the complementary role of [ i]-mibg scintigraphy and dat spect in the differential diagnosis between pd and non - pd 25,34 . a recent study showed that [ i]-fp - cit spect presents high sensitivity in the diagnosis of lewy body disease ; thus , [ i]-mibg scintigraphy may play a complementary role in the differential diagnosis between pd and other parkinsonism 30 . in the present study , we therefore explored the extent to which the combination of these two tracers would improve the ta . our data show that by combining fp - cit spect and mibg scintigraphy , the ta can be increased to 79.4% . first , this study was carried out in a single university hospital ; thus , the study population was small . second , the clinical diagnosis was used as the gold standard in our study because of the absence of histopathological confirmation ; thus , the clinical diagnosis may have been incorrect in some cases . third , before a molecular examination is performed , it is necessary to establish an appropriate withdrawal period in which to cease the administration of interfering drugs by considering their biological half - lives . the combination of dat spect and mibg myocardial scintigraphy may improve the sensitivity of the diagnosis of pd . in particular , this approach is based on commercially available radiotracers and can therefore be used in clinical practice without short - lived pet tracers . the sample size of the present study was small and the study was carried out in a single center . the results should therefore be confirmed in a multicenter study with a large population of pathologically diagnosed patients .

objectivemolecular imaging of nigrostriatal dopamine transporters ( dat ) and sympathetic cardiac innervation with single - photon emission computed tomography ( spect ) are useful tools for differentiating idiopathic parkinson disease ( pd ) from other degenerative parkinsonian syndromes ( non - pd ) . nevertheless , these modalities are often insufficient for achieving a definite diagnosis . the aims of this study were to evaluate the diagnostic accuracy of the combination of these tools.materials and methodsthe spect radiotracers [ 123i]-n--fluoropropyl-2-carbomethoxy-3-(4-iodophenyl)-nortropane ( fp - cit ) and meta-[123i]-iodobenzylguanidine ( mibg ) were used to research presynaptic dopaminergic projections ( dat spect ) and myocardial adrenergic innervation ( mibg scintigraphy ) , respectively . pd patients ( n=15 ; age : 61.513.6 years ) and non - pd patients ( n=19 ; age : 62.614.2 years ) who underwent both tests were enrolled in this study . receiver - operating characteristic analyses were used to set the cutoff values of the specific binding ratio in dat spect and the heart to mediastinum ratio in delayed scan in mibg scintigraphy for differentiating pd from non - pd . we calculated the sensitivity , specificity , and test accuracy of the individual methods and also the combination of these two modalities.resultswhen dat spect and mibg scintigraphy were used individually , they showed mild accuracy in differentiating pd from non - pd ( dat , 67.6% ; mibg , 67.6% ) . the combination of the two approaches using cutoff values of less than 3.24 for the specific binding ratio and less than 2.745 for the delayed heart to mediastinum ratio enabled more accurate differentiation between pd and non - pd . the accuracy of these indices in distinguishing pd from non - pd was 79.4%.conclusionthese results suggested that the combination of dat spect and mibg scintigraphy may improve the diagnostic accuracy in differentiating pd from non - pd .

pubmed

the ticks that belong to the genus amblyomma are large , ornate , longirostrate blood - feeding ectoparasites of a wide variety of vertebrates . amblyomma testudinarium is one of the species that has been frequently reported to bite humans in asian countries . usually , only a few ticks attached to and fed on a human , but 3 reported cases were infested with more than 100 larval ticks . in malaysia , a patient repeatedly infested with a. testudinarium was reported . it is well known that a. testudinarium is distributed in korea . however , human infestation of a. testudinarium tick is rare , with 2 previously reported cases in elderly korean persons . we report here an additional case of perianal infestation with a. testudinarium and provide histopathologic observations of its attachment to the skin . a 73-year - old korean man with a round nodule in the left perianal area presented to our clinic in september 2010 . the solitary nodule had been found about 2 weeks before admission and was not accompanied by hemorrhage and pain . he recalled a trip to a water reservoir located at yeonggwang - gun ( between 35 to 36 north latitude ) , a rural county in jeollanam - do province , south korea 1 month before the presentation . at that time , he defecated in the sitting posture on the grass near the water reservoir and felt the pain of sting around anus . gross appearance of the tick bite site was characterized by a mild swelling and erythema . the patient agreed to surgical treatment of the skin lesion to remove the perianal tick . 1a ) , placed in 10% buffered formalin and submitted for evaluation . during histologic processing , the tick was accidentally separated from the biopsied skin tissue . it was 25 mm in length and 18 mm in width and engorged with blood meal ( fig . the pedipalps had a long article ii with approximately 2.5 times as long as the article iii . the denticles of the inner file were much smaller than those of the other files . the ornamented scutum was relatively small , and its punctuations were dark brown or brownish - black spots on a pale yellow background ( fig . the eyes were located on the lateral edges of the scutum but not projecting ( fig . 2c ) and located on the ventrolateral surface posterolaterally to the coxa iv ( fig . the genital aperture was located at the level between the coxa i and coxa ii ( fig . the anus was located at the level posterior to the spiracular plates and embraced by y - shaped anal groove posteriorly ( fig . the coxa i had 2 subequal spurs , and the external spur was slightly longer than the internal spur ( fig . the coxae ii and iii had a single salient ridge - like spur ( fig . the coxa iv had a single spur which was longer than those of the coxae ii and iii ( fig . microscopical examinations revealed an intense skin reaction at the site where the tick had attached . due to deep penetration , the pathological changes were observed through all skin layers from the epidermis to the subcutaneous fat layer . in a low power field , the lesion comprised of central amorphous eosinophilic material and surrounding inflammatory cell infiltrations ( fig . 4 ) . at the site of the tick bite , a small epidermal defect and invaginated squamous epithelium were seen . the epidermal defect was about 1.8 mm in width and adjacent epidermis revealed parakeratosis , hyperkeratosis , and mild spongiosis . scattered inflammatory cells , mainly neutrophils , were seen in the epidermis . in the dermis , a characteristic feeding lesion was formed immediately beneath the attachment site . it was composed of 2 different portions ; superficial perirostral portion and deep cavitary portion ( fig . the perirostral portion was 2.1 mm in depth and much wider at the distal part than at its entrance . the cement , however , was found slightly at the dermal entrance of the tick mouthparts but not on the surface of the epidermis . it was lamellate and had projections into the adjacent tissue in some areas ( fig . it was relatively small and filled with purulent exudates containing inflammatory cells and erythrocytes ( fig . a dense and mixed cellular infiltrate was also noted around the feeding lesion and extended into neighboring dermis and subcutaneous tissues . however , there were very few inflammatory cells immediately adjacent to the site which abutted the eosinophilic cement ( fig . 5 ) . there was no evidence of infection or residual disease up to 1 month after surgical removal of the tick . among the hard ticks , only 3 genera , ixodes , haemaphysalis , and amblyomma have been recorded from humans in the republic of korea . this means that it does not belong to the genus ixodes which is the most common cause of human tick - bites in korea . the pedipalps of the present specimen are slender and longer than the basis capituli , and their second segment is not salient laterally . the eyes and fastoons are present , the small scutum is ornate , and the spiracular plate is comma - shaped the denticles of the inner file are much smaller than those of the other files . the spur of the coxa iv is longer than those of coxae ii and iii . the tarsi iii and iv have 2 distinct subapical ventral spurs . from the above observations , this tick is easily distinguished from amblyomma nitidum and amblyomma geoemydae which are distributed in the far east asia . therefore , our tick has been identified as a female adult of a. testudinarium based on morphological characteristics . the warm temperature and subtropical vegetation fauna are known to be a proper environmental condition for a. testudinarium . thus , this tick is more common in the tropic areas , including south east asia and indian peninsula . in japan , the closest geographical neighbor to korea , a. testudinarium is one of the most common ticks infesting humans and especially important in southwestern areas . in korea , it is also distributed in jeju island and the southern coastal area of the mainland . the first human case was reported in suncheon city ( 35 north latitude ) , a southern coastal area of jeollanam - do . the second case occurred in tongyeong city ( below 35 north latitude ) , southern coastal area of gyeongsangnam - do . in the present case , the patient was infested in yeonggwang - gun , a western coastal area of jeollanam - do . therefore , a. testudinarium extends the geographic range of distribution from the southern coastal area more northwards to the southwestern costal area ( below 36 north latitude ) of the korean peninsula . after a site for feeding has been chosen , ticks embed their anterior part calling capitulum into the skin . the capitulum is composed of 4 different components ; the palps , chelicerae , hypostome , and basis capituli . ticks attached on the skin lacerate the epidermal layers using the chelicerae which are paired appendages for cutting , ripping , and tearing skin . after epidermal laceration , ticks insert midline mouthparts ( the chelicerae and hypostome ) into the opening at a depth that varies by species . in the present case , the depth of penetration is similar to the length of the hypostome which has been deeply inserted into the dermis . however , the width of epidermal laceration is not similar to that of mouthparts but similar to that of the basis capituli . this means that the basis capituli is embedded shallowly in the host epidermis during the feeding period . the histopathology of skin responses to infection with the present female a. testudinarium shows that the dermis is infiltrated with various inflammatory cells associated with both acute and chronic reactions , i.e. , lymphocytes , neutrophils , histiocytes , and eosinophils . these inflammatory findings are similar to those reported by other researchers on amblyomma species experimentally and naturally infested in various vertebrate hosts . around the lesion abutting the cement , we observed an interesting area having relatively few inflammatory cells . this cell - free area is called the paucicellular zone . the biologic factors causing a zone of minimal cellular inflammation are not exactly known but are likely due to the anti - inflammatory effects of the tick saliva . according to the study on the effects of tick saliva and salivary gland extracts , this paucicellular zone might reflect tick - induced suppression of cellular migration into the injury site as a result of saliva constituents suppressing the movement of cells into the feeding lesion . the salivary glands of hard ticks secret eosinophilic cement material that help attachment of the tick to the host for the feeding period . it is well known that there is considerable difference in the distribution of the cement within the host tissue . generally , ixodids with long mouthparts ( longirostrate ticks ) penetrate the skin deeply to the dermis and have a perirostal secretion of the cement . ixodids with short mouthparts ( brevirostrate ticks ) have a copious superficial secretion of the cement which spreads over the skin . amblyomma and hyalomma are longirostrate ticks with relative long mouthparts . in the present amblyomma case , eosinophlic cement was found mostly in the perirostral portion but little on the skin surface .

a perianal tick and the surrounding skin were surgically excised from a 73-year - old man residing in a southwestern costal area of the korean peninsula . microscopically a deep penetrating lesion was formed beneath the attachment site . dense and mixed inflammatory cell infiltrations occurred in the dermis and subcutaneous tissues around the feeding lesion . amorphous eosinophilic cement was abundant in the center of the lesion . the tick had y - shaped anal groove , long mouthparts , ornate scutum , comma - shaped spiracular plate , distinct eyes , and fastoons . it was morphologically identified as a fully engorged female amblyomma testudinarium . this is the third human case of amblyomma tick infection in korea .

pubmed

hypertrophic cardiomyopathy ( hcm ) is characterized by left and/or right ventricular hypertrophy , with predominant involvement of the interventricular septum in the absence of other causes of hypertrophy , such as hypertension , valvular heart disease , or metabolic disease [ 17 ] . familial hcm is an autosomal dominant disorder caused by mutations in genes that encode sarcomere proteins . it has been reported that at least 15 genes are implicated in 5570 % of hcm , and the major genes causing hcm include cardiac -myosin heavy chain ( myh7 ) , -tropomyosin , cardiac troponin t ( tnnt2 ) , cardiac myosin - binding protein c ( mybpc3 ) , cardiac troponin i ( tnni3 ) , cardiac myosin regulatory light chains ( myl2 ) , and cardiac myosin essential light chains ( myl3 ) [ 1 , 4 , 5 , 811 ] . cardiac troponin t is a thin - filament protein that takes part in muscle contraction . the troponin complex on the actin filament regulates the force and velocity of muscle contraction . troponin c functions as a calcium receptor while troponin i inhibits adenosine triphosphatase ( atpase ) activity when bound to actin . troponin t fixes the troponin group to tropomyosin . during relaxation , the troponin group is bound to actin and tropomyosin , blocking the interaction of myosin and actin . mutations of tnnt2 were thought to account for approximately 15 % of familial hcm , with most missense mutations located in exons 816 , and were associated with a particularly severe form of the disease characterized by a poor overall prognosis with a high incidence of sudden death despite only mild left ventricular hypertrophy ( lvh ) [ 14 ] . most tnnt2 mutations of familial hcm alter the contractile properties of cardiac muscle , especially the ca sensitivity of force development and atpase activity in vitro and in vivo [ 1317 ] . previous reports have suggested that there is a more consistent relationship between certain genetic mutations and clinical outcome , allowing for the classification of benign and malignant mutations [ 1830 ] . for example , a favorable prognosis has been reported in patients with a phe110ile mutation of the tnnt2 mutation ( in 16 individuals of 6 japanese families ) [ 19 , 20 ] . from these data , genetic analysis and determination of genotype however , these findings were based on limited experience ; to date , only 30 different mutations have been identified in tnnt2 [ 911 , 18 ] . in addition to this genetic diversity , the phenotypic expression of these mutations varies considerably , ranging from asymptomatic individuals with a normal life expectancy to those with sudden cardiac death or the need for an early heart transplant . clinical parameters such as the degree of lvh , the presence or absence of a left ventricular outflow tract gradient , and electrophysiology testing have not been predictive markers of poor prognosis [ 15 ] . more recently , it was reported that late gadolinium enhancement with cardiac magnetic resonance can be a predictive marker of the ventricular arrhythmia and poor prognosis in hcm . in recent years , mutation - specific risk stratification was considered to be not possible , but genetic test - based risk stratification seemed to be clinically informative . the tnnt2 mutation has the third - ranked incidence of familial hcm , and the genotype phenotype correlation of this gene still remains insufficient in japanese familial hcm . we genetically evaluated 173 patients ( 101 men and 72 women ; 079 years old , median age 20 years ) who were clinically diagnosed with familial hcm . written informed consent was obtained from all study subjects in accordance with the declaration of helsinki . if patients were younger than 16 years , informed consent was given by their guardians . we assessed each patient by taking their history and performing a physical examination , and reviewed their medical records . all assessments were done with the approval of the ethics committees of tokyo women s medical university . the diagnosis of hcm was determined through clinical evaluation , chest radiography , electrocardiography , echocardiography , and cardiac catheterization based on current international consensus criteria . diseases of the heart , hypertension , valvular heart disease , or metabolic disease attributable to hcm were excluded from this study by pediatric cardiologists . genomic dna was prepared from peripheral blood lymphocytes or lymphoblast cell lines transformed by the epstein tnnt2 coding regions and exon intron boundaries , including regions approximately 30100 bp upstream and downstream from the exons , were amplified from genomic dna using primers , as described in previous reports [ 34 , 35 ] . genomic dna ( 50 ng ) was amplified through the use of primers designed from flanking intron sequences ( table 1).table 1primers used for amplification of fragments from tnnt2 exonforward ( 53)reverse ( 53)2gagctcttctgaggaaggcactacccagaatccgagggac3 and 4cagggcagcgtggactccccccagggctcccaggattt5ccattctctgctctgggttcgtgcacatgggaaagctgttct6tagggcttatctgtggggaaggccttccctggaaagagcactg7gaaatggaaatccacagggatactgcaccccgttccatca8 and 9ctctaggaaggatcagggcccctcacaaaagggatggagga10gcgatgtcaccttctccctacaccgcacccggccaata11ggtttccaatcctttcccctaagctgcagtggacacctcattc12gcctttgtcttcctgccttctccagccagcccaatctcttcact13acagggagggggcaatctggcccccagagcagatgcgggcagtg14actgggtgctgccgtctggtcaagggggctgttggggaatagg15cactcagcccccttctccaagcttctccgccccacatttc16ggcaccccagtcctacccgtccccctcaacagcactttt primers used for amplification of fragments from tnnt2 amplified products were purified using a multiscreen polymerase chain reaction ( pcr ) plate ( millipore , billerica , ma , usa ) and directly sequenced using the abi - prism bigdye - terminator cycle sequencing reaction kit and abi 3130xl genetic analyzer ( applied biosystems , foster city , ca , usa ) . when a mutation was detected , we confirmed that it was not presenting 363 japanese normal chromosomes by direct sequencing . the pcr conditions of these genes were modified and fragments were analyzed by electrophoresis , as described previously [ 36 , 37 ] . proband patients bearing tnnt2 mutations were additionally examined for reported hcm mutations , the coding regions of myh7 , mybpc3 , tnni3 , -tropomyosin , mly2 , mly3 , and all exons , and a promoter region of the -galactosidase a gene . primer sequences and detailed pcr conditions for these additional analyses are available upon request . reference sequences and single - nucleotide polymorphism information were obtained from the national center for biotechnology information . we genetically evaluated 173 patients ( 101 men and 72 women ; 079 years old , median age 20 years ) who were clinically diagnosed with familial hcm . written informed consent was obtained from all study subjects in accordance with the declaration of helsinki . if patients were younger than 16 years , informed consent was given by their guardians . we assessed each patient by taking their history and performing a physical examination , and reviewed their medical records . all assessments were done with the approval of the ethics committees of tokyo women s medical university . the diagnosis of hcm was determined through clinical evaluation , chest radiography , electrocardiography , echocardiography , and cardiac catheterization based on current international consensus criteria . diseases of the heart , hypertension , valvular heart disease , or metabolic disease attributable to hcm were excluded from this study by pediatric cardiologists . genomic dna was prepared from peripheral blood lymphocytes or lymphoblast cell lines transformed by the epstein tnnt2 coding regions and exon intron boundaries , including regions approximately 30100 bp upstream and downstream from the exons , were amplified from genomic dna using primers , as described in previous reports [ 34 , 35 ] . genomic dna ( 50 ng ) was amplified through the use of primers designed from flanking intron sequences ( table 1).table 1primers used for amplification of fragments from tnnt2 exonforward ( 53)reverse ( 53)2gagctcttctgaggaaggcactacccagaatccgagggac3 and 4cagggcagcgtggactccccccagggctcccaggattt5ccattctctgctctgggttcgtgcacatgggaaagctgttct6tagggcttatctgtggggaaggccttccctggaaagagcactg7gaaatggaaatccacagggatactgcaccccgttccatca8 and 9ctctaggaaggatcagggcccctcacaaaagggatggagga10gcgatgtcaccttctccctacaccgcacccggccaata11ggtttccaatcctttcccctaagctgcagtggacacctcattc12gcctttgtcttcctgccttctccagccagcccaatctcttcact13acagggagggggcaatctggcccccagagcagatgcgggcagtg14actgggtgctgccgtctggtcaagggggctgttggggaatagg15cactcagcccccttctccaagcttctccgccccacatttc16ggcaccccagtcctacccgtccccctcaacagcactttt primers used for amplification of fragments from tnnt2 amplified products were purified using a multiscreen polymerase chain reaction ( pcr ) plate ( millipore , billerica , ma , usa ) and directly sequenced using the abi - prism bigdye - terminator cycle sequencing reaction kit and abi 3130xl genetic analyzer ( applied biosystems , foster city , ca , usa ) . when a mutation was detected , we confirmed that it was not presenting 363 japanese normal chromosomes by direct sequencing . the pcr conditions of these genes were modified and fragments were analyzed by electrophoresis , as described previously [ 36 , 37 ] . proband patients bearing tnnt2 mutations were additionally examined for reported hcm mutations , the coding regions of myh7 , mybpc3 , tnni3 , -tropomyosin , mly2 , mly3 , and all exons , and a promoter region of the -galactosidase a gene . reference sequences and single - nucleotide polymorphism information were obtained from the national center for biotechnology information . to investigate mutations of tnnt2 , we performed dna analysis in 173 unrelated japanese patients with familial hcm . we identified three reported mutations and a new mutation of tnnt2 in 11 individuals from four families ( a , b , c , d ) ( table 2).table 2clinical features of patients with tnnt2 mutation in families a , b , c , and dhcm individualsa iii-2a iii-1a ii-6a ii-6 ( iii-2/mother)b iii-7b iii-2b iv-3c iv-3c iii-3c ii-3d iii-2exon9 and 109 and 109109991010108nucleotide substitutiont328a and c388tt328a and c388tt328ac388tc274c274c274c388c388c388t236camino substitutionp110i and r130cp110i and r130cp110ir130cr92wr92wr92wr92wr92wr92wi79tsexfmmffmmffffage ( years)121455463541242158025outcome ( years)12 , vf survivor14 , died suddenly24 , vf survivorclinical diagnosishcmhcmcavb , pminormaldhcm ( hcm)abnormal ecghcmhcmhcmhcmhcmelectrocardiogramsr + crbbbsr + crbbb , abnqpm rhythm ( cavb)wnlr - wave progressiondetails unknownrvh , deep qlvh , st depdetails unknowndetails unknownst dep , inv t2d echocardiogramlvh , ashlvh , ashlvhwnllvh , apical hypertrophydetails unknownlvhlvh , ashlvhdetails unknownlvh , ashcardiac catheterizationlv relaxation abnormalitieslv relaxation abnormalitieslv relaxation abnormalitiesndndndndndndndnd hcm hypertrophic cardiomyopathy , hocm hypertrophic obstructive cardiomyopathy , dhcm dilated phase of hcm , lvh left ventricular hypertrophy , vf ventricular fibrillation , cavb complete atrioventricular block , sr sinus rhythm , crbbb complete right bundle branch block , abnq abnormal q wave , lvh left ventricular hypertrophy , rvh right ventricular hypertrophy , st dep st depression , inv t inversion t , pmi pacemaker implantation , ecg electrocardiogram , ash asymmetric septal hypertrophy , aph apical hypertrophy , nd not determined , wnl within normal limits clinical features of patients with tnnt2 mutation in families a , b , c , and d hcm hypertrophic cardiomyopathy , hocm hypertrophic obstructive cardiomyopathy , dhcm dilated phase of hcm , lvh left ventricular hypertrophy , vf ventricular fibrillation , cavb complete atrioventricular block , sr sinus rhythm , crbbb complete right bundle branch block , abnq abnormal q wave , lvh left ventricular hypertrophy , rvh right ventricular hypertrophy , st dep st depression , inv t inversion t , pmi pacemaker implantation , ecg electrocardiogram , ash asymmetric septal hypertrophy , aph apical hypertrophy , nd not determined , wnl within normal limits in family a , all five members who carried or were suspected of having hcm also had arrhythmia . four of them had blocks , including a complete atrioventricular block ( cavb ) and complete right bundle branch block ( crbbb ) ( fig . 1 ; ii-1 , 6 , iii-1 , 2 ) , and one had bradycardia . two of them died suddenly ( fig . 1 ; i-2 , iii-1 ) , and one was a ventricular fibrillation ( vf ) survivor ( fig . 1 ; iii-2 ) . the proband ( fig . 1 ; iii-2 ) , aged 12 years , with sinus rhythm ( sr ) and a crbbb , had an episode of vf , and her elder brother ( fig . 1 ; iii-1 ) , the proband and her brother and father ( fig . 1 ; iii-2 , iii-1 , i-2 ) showed mild lvh ( maximum wall thickness < 20 mm ) . 1pedigree and phenotype of family a. pedigree with three generations ( roman numerals ) , the propositus is denoted by an arrow . hcm hypertrophic cardiomyopathy , cavb complete atrioventricular block , pmi pacemaker implantation , crbbb complete right bundle - branch block , vf ventricular fibrillation pedigree and phenotype of family a. pedigree with three generations ( roman numerals ) , the propositus is denoted by an arrow . hcm hypertrophic cardiomyopathy , cavb complete atrioventricular block , pmi pacemaker implantation , crbbb complete right bundle - branch block , vf ventricular fibrillation dna analysis showed that the proband and her elder brother had the double reported mutations , arg130cys and phe110ile , of tnnt2 ( fig . her father , who had arrhythmia and mild hcm , had the phe110ile mutation , and her mother ( ii-6 ) , who had no symptoms or abnormal findings , had the arg130cys mutation . in family b , 3 of 10 members who carried or were suspected of having hcm were thought to have obstructive hypertrophic cardiomyopathy . 2 ; iii-7 ) showed apical hypertrophy and apical aneurysm at a comparatively young age . her aunt ( fig . 2 ; ii-4 ) , who had a heart murmur , was thought to have hypertrophic obstructive cardiomyopathy ( hocm ) , and died when she was 19 years old . 2 ; iii-9 ) did not show obstructive hcm , but died suddenly when she was 21 years old . her grandmother ( fig . iii-7 ) , her son ( fig . 2 ; iv-3 ) , and her cousin ( fig . 2 ; iii-2 ) , who carried hcm , had an arg92trp mutation of tnnt2.fig . 2pedigree and phenotype of family b. pedigree with four generations ( roman numerals ) , the propositus is denoted by an arrow . hcm hypertrophic cardiomyopathy , hocm hypertrophic obstructive cardiomyopathy , ecg electrocardiogram pedigree and phenotype of family b. pedigree with four generations ( roman numerals ) , the propositus is denoted by an arrow . hcm hypertrophic cardiomyopathy , hocm hypertrophic obstructive cardiomyopathy , ecg electrocardiogram in family c , the proband ( fig . 3 ; iv-3 ) , her mother ( fig . 3 ; iii-3 ) , and her grandmother ( fig . 3 ; ii-3 ) showed mild cardiac hypertrophy ( maximum wall thickness < 20 mm ) and asymmetric septal hypertrophy , and her myocardial hypertrophy had been gradually increasing . her mother was suspected of having a shifting dilated phase by recent cardiac magnetic resonance imaging ( mri ) . 3 ; iv-3 ) had symptomatic west syndrome following perinatal brain damage , and was treated with corticotropin . 3 ; iv-3 ) and her mother ( fig . 3 ; iii-3 ) had the arg92trp mutation of tnnt2.fig . 3pedigree and phenotype of family c. pedigree with four generations ( roman numerals ) , the propositus is denoted by an arrow . hcm hypertrophic cardiomyopathy , wpw syndrome wolff - parkinson - white syndrome pedigree and phenotype of family c. pedigree with four generations ( roman numerals ) , the propositus is denoted by an arrow . hcm hypertrophic cardiomyopathy , wpw syndrome wolff - parkinson - white syndrome in family d , two of three members who carried or were suspected of having hcm died suddenly ( fig . 4 ; 4 ; iii-2 ) . the proband ( fig . 4 ; iii-2 ) had relatively strong cardiac hypertrophy ( maximum wall thickness > 20 mm ) , and cardiac standstill from ventricular fibrillation at the age of 24 years . her cousin ( fig . 4 ; iii-3 ) died suddenly at a young age ( 13 years ) , and her granduncle also died at the age of 40 . this transition was observed in codon 79 , and converted an isoleucine residue to a threonine residue ( fig . ile79thr occurred in conserved residues found in tnnt2 orthologues of human , mouse , rat , cat , and ox ( fig . iii-2 ) also had a 5-bp ( cttct ) deletion / deletion polymorphism of intron 3 of tnnt2 ( fig . 4pedigree and phenotype of family d. pedigree with four generations ( roman numerals ) , the propositus is denoted by an arrow . c the insertion / deletion ( i / d ) polymorphism pedigree and phenotype of family d. pedigree with four generations ( roman numerals ) , the propositus is denoted by an arrow . hcm hypertrophic cardiomyopathy , vf ventricular fibrillation a the new ile79thr mutation of tnnt2 ( exon 8) . this domain is well preserved in mammals a the deletion / deletion ( d / d ) polymorphism . c the insertion / deletion ( i / d ) polymorphism to clarify the clinical importance of this polymorphism , we performed genetic analysis in 47 hcm patients from 173 unrelated japanese patients with familial hcm . in these 47 patients , 24 had the deletion / deletion polymorphism ( 51 % , 14 men and 10 women ; 072 years old , median age 30 years old ) . five of the 24 ( 21 % ) patients who had the deletion / deletion polymorphism were diagnosed with apical hcm or hocm . in 6 of the 10 ( 60 % ) with available data of echocardiography among these 24 patients , on the other hand , in the 47 hcm patients , the remaining 23 did not have the deletion / deletion polymorphism , but had the deletion / insertion or insertion / insertion polymorphism ( 49 % , 15 men and 8 women ; 076 years old , median age 18 years ) . two of the 23 ( 9 % ) patients were diagnosed with apical hcm , and there were no hocm patients . two of the seven ( 29 % ) with available data of echocardiography among these 23 patients showed the maximum wall thickness of more than 30 mm . to investigate mutations of tnnt2 , we performed dna analysis in 173 unrelated japanese patients with familial hcm . we identified three reported mutations and a new mutation of tnnt2 in 11 individuals from four families ( a , b , c , d ) ( table 2).table 2clinical features of patients with tnnt2 mutation in families a , b , c , and dhcm individualsa iii-2a iii-1a ii-6a ii-6 ( iii-2/mother)b iii-7b iii-2b iv-3c iv-3c iii-3c ii-3d iii-2exon9 and 109 and 109109991010108nucleotide substitutiont328a and c388tt328a and c388tt328ac388tc274c274c274c388c388c388t236camino substitutionp110i and r130cp110i and r130cp110ir130cr92wr92wr92wr92wr92wr92wi79tsexfmmffmmffffage ( years)121455463541242158025outcome ( years)12 , vf survivor14 , died suddenly24 , vf survivorclinical diagnosishcmhcmcavb , pminormaldhcm ( hcm)abnormal ecghcmhcmhcmhcmhcmelectrocardiogramsr + crbbbsr + crbbb , abnqpm rhythm ( cavb)wnlr - wave progressiondetails unknownrvh , deep qlvh , st depdetails unknowndetails unknownst dep , inv t2d echocardiogramlvh , ashlvh , ashlvhwnllvh , apical hypertrophydetails unknownlvhlvh , ashlvhdetails unknownlvh , ashcardiac catheterizationlv relaxation abnormalitieslv relaxation abnormalitieslv relaxation abnormalitiesndndndndndndndnd hcm hypertrophic cardiomyopathy , hocm hypertrophic obstructive cardiomyopathy , dhcm dilated phase of hcm , lvh left ventricular hypertrophy , vf ventricular fibrillation , cavb complete atrioventricular block , sr sinus rhythm , crbbb complete right bundle branch block , abnq abnormal q wave , lvh left ventricular hypertrophy , rvh right ventricular hypertrophy , st dep st depression , inv t inversion t , pmi pacemaker implantation , ecg electrocardiogram , ash asymmetric septal hypertrophy , aph apical hypertrophy , nd not determined , wnl within normal limits clinical features of patients with tnnt2 mutation in families a , b , c , and d hcm hypertrophic cardiomyopathy , hocm hypertrophic obstructive cardiomyopathy , dhcm dilated phase of hcm , lvh left ventricular hypertrophy , vf ventricular fibrillation , cavb complete atrioventricular block , sr sinus rhythm , crbbb complete right bundle branch block , abnq abnormal q wave , lvh left ventricular hypertrophy , rvh right ventricular hypertrophy , st dep st depression , inv t inversion t , pmi pacemaker implantation , ecg electrocardiogram , ash asymmetric septal hypertrophy , aph apical hypertrophy , nd not determined , wnl within normal limits in family a , all five members who carried or were suspected of having hcm also had arrhythmia . four of them had blocks , including a complete atrioventricular block ( cavb ) and complete right bundle branch block ( crbbb ) ( fig . 1 ; ii-1 , 6 , iii-1 , 2 ) , and one had bradycardia . two of them died suddenly ( fig . 1 ; i-2 , iii-1 ) , and one was a ventricular fibrillation ( vf ) survivor ( fig . 1 ; iii-2 ) . the proband ( fig . 1 ; iii-2 ) , aged 12 years , with sinus rhythm ( sr ) and a crbbb , had an episode of vf , and her elder brother ( fig . 1 ; iii-1 ) , the proband and her brother and father ( fig . 1 ; iii-2 , iii-1 , i-2 ) showed mild lvh ( maximum wall thickness < 20 mm ) . 1pedigree and phenotype of family a. pedigree with three generations ( roman numerals ) , the propositus is denoted by an arrow . hcm hypertrophic cardiomyopathy , cavb complete atrioventricular block , pmi pacemaker implantation , crbbb complete right bundle - branch block , vf ventricular fibrillation pedigree and phenotype of family a. pedigree with three generations ( roman numerals ) , the propositus is denoted by an arrow . hcm hypertrophic cardiomyopathy , cavb complete atrioventricular block , pmi pacemaker implantation , crbbb complete right bundle - branch block , vf ventricular fibrillation dna analysis showed that the proband and her elder brother had the double reported mutations , arg130cys and phe110ile , of tnnt2 ( fig . her father , who had arrhythmia and mild hcm , had the phe110ile mutation , and her mother ( ii-6 ) , who had no symptoms or abnormal findings , had the arg130cys mutation . in family b , 3 of 10 members who carried or were suspected of having hcm were thought to have obstructive hypertrophic cardiomyopathy . 2 ; iii-7 ) showed apical hypertrophy and apical aneurysm at a comparatively young age . her aunt ( fig . 2 ; ii-4 ) , who had a heart murmur , was thought to have hypertrophic obstructive cardiomyopathy ( hocm ) , and died when she was 19 years old . 2 ; iii-9 ) did not show obstructive hcm , but died suddenly when she was 21 years old . her grandmother ( fig . iii-7 ) , her son ( fig . 2 ; iv-3 ) , and her cousin ( fig . 2 ; iii-2 ) , who carried hcm , had an arg92trp mutation of tnnt2.fig . 2pedigree and phenotype of family b. pedigree with four generations ( roman numerals ) , the propositus is denoted by an arrow . hcm hypertrophic cardiomyopathy , hocm hypertrophic obstructive cardiomyopathy , ecg electrocardiogram pedigree and phenotype of family b. pedigree with four generations ( roman numerals ) , the propositus is denoted by an arrow . hcm hypertrophic cardiomyopathy , hocm hypertrophic obstructive cardiomyopathy , ecg electrocardiogram in family c , the proband ( fig . 3 ; iv-3 ) , her mother ( fig . 3 ; iii-3 ) , and her grandmother ( fig . 3 ; ii-3 ) showed mild cardiac hypertrophy ( maximum wall thickness < 20 mm ) and asymmetric septal hypertrophy , and her myocardial hypertrophy had been gradually increasing . her mother was suspected of having a shifting dilated phase by recent cardiac magnetic resonance imaging ( mri ) . 3 ; iv-3 ) had symptomatic west syndrome following perinatal brain damage , and was treated with corticotropin . 3 ; iv-3 ) and her mother ( fig . 3 ; iii-3 ) had the arg92trp mutation of tnnt2.fig . 3pedigree and phenotype of family c. pedigree with four generations ( roman numerals ) , the propositus is denoted by an arrow . hcm hypertrophic cardiomyopathy , wpw syndrome wolff - parkinson - white syndrome pedigree and phenotype of family c. pedigree with four generations ( roman numerals ) , the propositus is denoted by an arrow . hcm hypertrophic cardiomyopathy , wpw syndrome wolff - parkinson - white syndrome in family d , two of three members who carried or were suspected of having hcm died suddenly ( fig . 4 ; 4 ; iii-2 ) . the proband ( fig . 4 ; iii-2 ) had relatively strong cardiac hypertrophy ( maximum wall thickness > 20 mm ) , and cardiac standstill from ventricular fibrillation at the age of 24 years . her cousin ( fig . 4 ; iii-3 ) died suddenly at a young age ( 13 years ) , and her granduncle also died at the age of 40 . this transition was observed in codon 79 , and converted an isoleucine residue to a threonine residue ( fig . ile79thr occurred in conserved residues found in tnnt2 orthologues of human , mouse , rat , cat , and ox ( fig . iii-2 ) also had a 5-bp ( cttct ) deletion / deletion polymorphism of intron 3 of tnnt2 ( fig . 4pedigree and phenotype of family d. pedigree with four generations ( roman numerals ) , the propositus is denoted by an arrow . c the insertion / deletion ( i / d ) polymorphism pedigree and phenotype of family d. pedigree with four generations ( roman numerals ) , the propositus is denoted by an arrow . hcm hypertrophic cardiomyopathy , vf ventricular fibrillation a the new ile79thr mutation of tnnt2 ( exon 8) . this domain is well preserved in mammals a the deletion / deletion ( d / d ) polymorphism . c the insertion / deletion ( i / d ) polymorphism to clarify the clinical importance of this polymorphism , we performed genetic analysis in 47 hcm patients from 173 unrelated japanese patients with familial hcm . in these 47 patients , 24 had the deletion / deletion polymorphism ( 51 % , 14 men and 10 women ; 072 years old , median age 30 years old ) . five of the 24 ( 21 % ) patients who had the deletion / deletion polymorphism were diagnosed with apical hcm or hocm . in 6 of the 10 ( 60 % ) with available data of echocardiography among these 24 patients , on the other hand , in the 47 hcm patients , the remaining 23 did not have the deletion / deletion polymorphism , but had the deletion / insertion or insertion / insertion polymorphism ( 49 % , 15 men and 8 women ; 076 years old , median age 18 years ) . two of the 23 ( 9 % ) patients were diagnosed with apical hcm , and there were no hocm patients . two of the seven ( 29 % ) with available data of echocardiography among these 23 patients showed the maximum wall thickness of more than 30 mm . previous studies have reported familial hcm cases where a consistent relationship between certain genetic mutations and clinical outcome was observed , allowing for the classification of benign and malignant mutations [ 1830 ] . however , in recent years it was considered that the specific gene mutation can not be the sole factor that dictates clinical phenotype . mutation , mutation type was not seen to be clinically useful in predicting prognosis in hcm given this very low incidence rate [ 32 , 3840 ] . in addition , it was reported that mutation of a sarcomeric protein gene can cause rcm , hcm , and dcm within the same family , and patients with a benign mutation experienced a very serious clinical course [ 41 , 42 ] . in the present study , the phenotype of each family was often different from reported cases , even if they had the same genetic mutation . in addition , families with the same genetic mutation showed a similar trend in the phenotype , but it was not exactly the same . however , sudden death in youth was observed in all of these families . on the other hand mutation , which did not show the phenotype of hcm or a family history of sudden death . for example , in family a , two tnnt2 mutations were found , the arg130 cys and phe110ile mutations . the arg130 cys mutation has been reported by a chinese research group , whose clinical features had an early onset , syncope , sudden death in youth , heart failure , and arrhythmia as atrial fibrillation . the phe110ile mutation has been reported to show a favorable prognosis , and have comparatively slight hypertrophy or apical hypertrophy ( in 16 individuals of 6 japanese families ) [ 19 , 20 ] . a , whereas the proband and her elder brother , who carried these two mutations , showed similar reported phenotypes of the arg130 cys mutation , her mother and mother s family members , who had the arg130cys mutation , showed no abnormal clinical features such as cardiac hypertrophy , sudden death , or abnormal electrocardiograms . furthermore , her father and father s family members , who had the phe110ile mutation , showed atrioventricular blocks , and these phenotypes have not been reported . in family b , the mutation showed comparatively slight cardiac hypertrophy or a high incidence of sudden death in males ( 19 individuals of two mixed racial families ) [ 2224 ] . however , the proband and her family , who carried hcm , showed relatively severe cardiac hypertrophy , with a high incidence of sudden death in females . in family c , an arg92trp mutation was also found . the proband and her family , who carried hcm , showed mild cardiac hypertrophy and asymmetric septal hypertrophy . the proband s myocardial hypertrophy had been gradually increasing , and her mother had a suspected shift to the dilated phase . she survived following an episode of vf , and her cousin died suddenly at the age of 13 years . previously , a mutation of the 79 residue , the ile79asn mutation , had been reported [ 24 , 25 ] , and this mutation also showed a poor prognosis . in our mutated case , even if the amino acid mutation ( ile79thr ) was different from the reported case ( ile79asn ) , the patient showed a malignant prognosis . in family d , a 5-base - pair ( cttct ) deletion / deletion ( d / d ) polymorphism in intron 3 of the tnnt2 was also found . it has been reported that this polymorphism had caused skipping of exon 4 of tnnt2 , and that the deletion allele could be associated with a predisposition for prominent lvh . although it was in a very limited range , from our genetic study on this polymorphism we gained the impression that a patient carrying the tnnt2 deletion / deletion polymorphism had a stronger tendency toward hypertrophy . to conduct further analysis , further examination including new cases is required . we considered that , at least in japanese familial hcm , only the type of genetic mutation of tnnt2 did not seem useful in distinguishing the prognosis . however , if mutations were found , there was a risk of sudden death in youth . therefore , regardless of the type of genetic mutation , it would be more important to observe the patient in detail from birth in each lineage . the development of a case - based method would be useful to treat and help each individual , and more attention needs to be paid toward searching for the modifier and environmental factors including diet , lifestyle , exercise , and the modification gene and polymorphism .

hypertrophic cardiomyopathy ( hcm ) is an autosomal dominant disorder resulting from mutations in genes for at least 15 various sarcomere - related proteins including cardiac -myosin heavy chain , cardiac myosin - binding protein c , and cardiac troponin t. the troponin t gene ( tnnt2 ) mutation has the third incidence of familial hcm , and the genotype phenotype correlation of this gene still remains insufficient in japanese familial hcm . therefore , in the present study , we focused on screening the tnnt2 mutation in 173 unrelated japanese patients with familial hcm , and found three reported mutations and a new mutation of tnnt2 in 11 individuals from four families . in these families , two individuals from one family had double mutations , arg130cys and phe110ile , six individuals from two other families had an arg92trp mutation , and one individual of another family had a new mutation , ile79thr , of tnnt2 . the phenotype of each family was often different from reported cases , even if they had the same genetic mutation . in addition , families with the same genetic mutation showed a similar trend in the phenotype , but it was not exactly the same . however , sudden death in youth was observed in all of these families . although the type of genetic mutation is not useful for predicting prognosis in hcm , the possibility of sudden cardiac death remains . therefore , the prognosis of individuals bearing the tnnt2 mutation with familial hcm should be more carefully observed from birth .

pubmed

schistosomatidae ( platyhelminthes : digenea ) includes several digenetic endoparasites with complex life cycles , whose developmental stages alternate between intermediate ( freshwater gastropods ) and definitive hosts ( birds , reptiles , fishes , and mammals ) . another important feature is the increased longevity ( over 5 years ) of the schistosoma species in the human host . schistosoma , the avian and mammalian blood flukes , is the best studied genus of schistosomatidae . several species are described , six of which infect humans causing schistosomiasis : s. haematobium , s. intercalatum , s. japonicum , s. malayensis , s. mansoni , and s. mekongi . other species are known to infect a broad range of mammals such as hippopotamus , rodents , carnivores , and ruminant animals like buffalo , cattle , goat , lechwe , and sheep . for example , s. mattheei , more commonly found in cattle , is believed to form hybrids between s. mattheei and s. haematobium thus increasing its snail and definitive host range . schistosomiasis , a chronic and debilitating disease , is considered by the world health organization as one of the most serious public health issues and the second most prevalent tropical disease with high morbidity in the world . schistosomiasis is endemic in 77 countries and its transmission is dependent on the existence and distribution of intermediate hosts . it is estimated that 237 million people require treatment worldwide and that 600 to 779 million people live in endemic areas , under infection risk . yet , exacerbating this scenario , this disease is responsible for the loss of 1.7 to 4.5 millions of years of life in the world , measured by disability - adjusted life years ( daly ) , one of the highest indexes among all the neglected tropical diseases . control of schistosomiasis represents a great challenge and it is based on drug treatment ( praziquantel ) , snail control , improved sanitation , and health education . the development of powerful and scalable methods to analyse nucleic acids and proteins has changed the way biological data is surveyed . the application of such technologies , together with the development of powerful computational tools and methods , have expanded our perspective of schistosome biology and allowed a better understanding of processes such as host - parasite interaction [ 810 ] . this paper aims to discuss some advances in schistosome research with emphasis on genomics and transcriptomics . first , we summarize the current status of sequencing projects of nuclear and mitochondrial genomes . then , we present key findings in transcriptomic analyses . finally , we point out the main challenges of the current research and suggest some future directions in schistosoma genomic and transcriptomic studies . in order to strengthen traditional methods and provide new information to improve success towards schistosomiasis control , the scientific community joined efforts to assess the schistosoma genomic information , starting in 1994 , as an initiative of who / tdr ( unicef - undp - world bank - who special programme for research and training in tropical diseases ) [ 11 , 12 ] . at that time , only a few hundred expressed sequence tags ( ests ) from s. mansoni were available . the who / tdr support opened the possibility to generate data that could be translated into new tools for schistosomiasis diagnostics and treatment , representing the kickoff of schistosoma nuclear genome studies . the karyotype of known schistosoma species comprises seven pairs of autosomes and one pair of sex chromosomes ( female = zw , male = zz ) , ranging in size from 18 to 73 megabases ( mb ) [ 14 , 15 ] . the s. japonicum ( anhui isolate ) and s. mansoni ( puerto rico isolate ) genomes were decoded and simultaneously published as an initiative of the wellcome trust sanger center institute in collaboration with the institute for genomic research ( tigr ) and the schistosoma japonicum genome sequencing and functional analysis consortium [ 16 , 17 ] . recently , the s. haematobium ( egyptian isolate ) genome was sequenced opening new possibilities in comparative genomics of schistosomes . s. haematobium , s. japonicum , and s. mansoni carry a nuclear genome of 385 , 397 , and 363 mb composed by approximately , 13,073 , 13,469 , and 10,852 protein - coding genes , respectively [ 1619 ] . it is worth to mention that the number of predicted protein - coding genes does not reflect the genome structure per se , but the actual state of analyses of the different draft genome data . the genetic linkage map of s. mansoni was obtained and allowed the refinement of the genome sequencing data providing a means for gene discovery and gene function analysis [ 20 , 21 ] . more importantly , it has allowed the identification of genetic loci that determine important traits such as host specificity , virulence , and drug resistance . in fact , although the quality and annotation of the s. mansoni genome sequence data have been greatly improved ( see below ) ; the corresponding data of s. haematobium and s. japonicum are still considered drafts [ 1619 ] . their genome sequences are distributed in a large number of contigs ; therefore extensive work is required . constant data refinement is necessary to provide a reliable comprehension of the genome and gene models and to provide a curated annotation . such strategies include new assembly of existing capillary reads supplemented with an additional ~90,000 fosmid and bac end sequences , deep sequencing of clonal dna ( nmri strain , puerto rican origin ) using 108-based paired reads on illumina genome analyzer iix platform , and rna - seq data of different parasite life stages . progress on the knowledge of s. mansoni genome features is still expanding , especially with the application of the next - generation sequencing platforms and single nucleotide polymorphism typing methods . for instance , 14 new microsatellite markers were recently identified by de novo assembly of massive sequencing reads ; these new features can be employed for pedigree studies . yet , newer sequencing technologies will allow for the development of population genomics studies of field , laboratory , and clinical isolates . as a result of the availability of the schistosoma genome sequence data , comparative analyses across different species became possible bringing together different areas of research [ 21 , 23 , 24 ] . comparative genomics of three schistosoma species revealed several conserved features such as the overall gc content , sequence identity , presence of repetitive elements , and synteny . this genome - wide analysis corroborates previous genetic studies and supports the evolutionary hypothesis of s. haematobium and s. mansoni as the most related species , followed by s. japonicum , to the exclusion of other trematoda such as clonorchis , fasciola , and schmidtea . mitochondrial genes have been used as molecular markers for species and strain identification , which is key to a variety of studies such as phylogenetics , population genetics , biogeography , and molecular ecology [ 2529 ] . besides helping to elucidate the evolutionary relationships among taxa , mitochondrial genes have been successfully applied into epidemiological studies , monitoring , and control of microbes , parasites , and vectors of medical and socioeconomic importance . the analysis of the whole mitochondrial genome of diverse taxa has changed the perspective of such studies . the mitochondrial genome of six schistosoma species was sequenced ( table 1 ) including s. haematobium ( nc_008074 ) , s. japonicum ( nc_002544 ) , s. mansoni ( nc_002545 ) , s. malayensis , s. mekongi ( nc_002529 ) , and s. spindale ( nc_008067 ) [ 27 , 3032 ] . these genomes range in size from 13,503 to 16,901 bp and encode 36 genes comprising two ribosomal genes ( large and small subunit rrna genes ) , and 22 transfer rna ( trna ) genes , as well as 12 protein - encoding genes ( atp6 , cob , cox13 , nad16 , and nad4l ) . the atp8 gene , commonly found in other phyla , is absent from platyhelminthes , whose mitochondrial genomes have been analysed . moreover , each schistosoma mitochondrial genome contains a long noncoding region that is divided into two parts by one or more trna genes , which is found in other platyhelminthes and vary in length according to each taxon . comparative mitogenomics have revealed a highly conserved gene order among distinct platyhelminthes with the exception of some schistosoma species [ 25 , 3033 ] . the mitochondrial genome of the asian schistosomes , s. japonicum and s. mekongi , displays the same gene order as other digenea and cestoda [ 25 , 32 ] . in contrast , the gene order in the mitochondrial genome of s. haematobium , s. mansoni , and s. spindale is strikingly different from other taxa [ 25 , 30 ] . the unique gene order rearrangements identified in these species constitute valuable information to the reconstruction of the phylogenetic relationships of schistosoma and other platyhelminthes ( section 3.1 ) . the use of genomic data is valuable to the understanding of the evolutionary history of schistosoma especially due to the absence of fossil records . molecular markers have been used to test hypotheses in a variety of schistosoma phylogenetic , phylogeographic and epidemiological studies [ 4 , 25 , 3436 ] . traditionally , these markers include the nuclear ribosomal rna genes ( 18s , 5.8s , and 28s ) and the internally transcribed spacer ( its ) region besides a number of mitochondrial genes ( cob , cox13 , nad16 , etc . ) . based on mitochondrial data from different studies , a standard phylogeny of 23 schistosoma species was proposed . these clades include ( 1 ) the s. japonicum complex ( s. japonicum , s. malayensis , s. mekongi , s. ovuncatum , and s. sinensium ) found in central and south eastern asia ; ( 2 ) the s. hippopotami clade ( s. edwardiense and s. hippopotami ) distributed in africa ; ( 3 ) the proto - s . mansoni clade ( s. incognitum and s. turkestanicum ) in central asia , near east , and eastern europe ; ( 4 ) the s. mansoni clade ( s. mansoni and s. rodhaini ) found in africa and south america ; ( 5 ) the s. haematobium clade ( s. bovis , s. curassoni , s. guineensis , s. haematobium , s. intercalatum , s. kisumuensis , s. leiperi , s. margrebowiei , and s. mattheei ) occurring in different parts of africa ; ( 6 ) the s. indicum clade ( s. indicum , s. nasale , and s. spindale ) found in india and south asia . there are divergent opinions concerning the origin of the genus schistosoma and its intermediate snail host [ 25 , 3739 ] . the out of asia hypothesis is the most generally accepted , indicating a migration followed by dispersion of these parasites from asia to africa . in this regard , studies of molecular phylogeny suggested that the genus schistosoma had its origin in asia and ( at least two descendants ) colonized the african continent independently , where they easily radiated , becoming exclusive parasites of planorbid snails . returning to asia , the parasites diversified into groups of species , characterized by the position of the spike in the eggs . the molecular phylogeny of seven schistosoma species suggested that schistosomes were endoparasites of rodents and ruminants and were transmitted to the first hominids in africa as a migration to savanna areas occurred . however , in order to better define the schistosoma origin , there is a need to sample a broader range of species . most of the widely accepted schistosoma phylogenies are primarily based on sequence alignments of mitochondrial genes such as cox13 , and nad16 , [ 25 , 30 ] . indeed , these gene rearrangements , considered to be rare evolutionary events , have been applied to an increasing number of studies ( cf . ) . as mentioned before , major changes in the mitochondrial gene order were identified in some schistosoma species ( section 2.2 ) , more specifically in the african clades ( s. mansoni and s. haematobium ) and the s. indicum group ( s. spindale ) . this provides further evidence for the asian ancestry of schistosomes and corroborates the concept of the reinvasion of asia by members of the s. indicum group from africa [ 27 , 30 ] . besides reconstructing the phylogenetic relationships of diverse taxa , the evolutionary framework has been applied to improve functional annotation of genes and gene products , as well as to study gene / protein family evolution . phylogenomics has allowed the identification and characterization of all eukaryotic protein kinases encoded in the s. mansoni genome and improved the functional annotation of over 40% of them , highlighting the molecular diversity of these enzymes [ 42 , 43 ] . a phylogenetic analysis was also employed in the classification of s. mansoni histone proteins , which play a key role in epigenetic modifications that might reflect the parasite complex lifestyle . by applying an evolutionary framework to analyse a large sequence dataset from a broad range of organisms , functional annotation of the s. mansoni histone proteins moreover , phylogenomics has unraveled the distinct evolutionary histories of three expanded endopeptidase families in s. mansoni . this analysis included members of the metallopeptidase m8 , serine peptidase s1 , and aspartic peptidase a1 families , which were originated from successive events of gene duplication followed by divergence in the parasite lineage after its diversification from other metazoans . other protein families such as nuclear receptors and antioxidants have been characterized in s. mansoni by applying evolutionary and functional approaches [ 46 , 47 ] . the identification of smnr4a , a member of the nuclear receptor subfamily 4 , and its relatedness to the human homologs were corroborated by phylogenetic analysis . thioredoxin and glutathione systems , involved in a variety of cellular processes including antioxidant defense , differ in parasitic and free - living platyhelminthes . by applying different phylogenetic methods and experimental testing , it has been shown that the canonical enzymes of such systems were lost in the parasitic lineages . the availability of sequencing data from mitochondrial genomes from closely related taxa to schistosoma has provided a basis for studies ranging from phylogenetic systematics to molecular ecology and so forth [ 27 , 29 , 48 , 49 ] . importantly , it allowed the assessment of molecular markers prior to broad scale sampling by comparing data from individual genes versus completely sequenced mitochondrial genomes . data from african and asian schistosomes were compared to an s. mansoni intraspecific dataset showing a positive correlation between polymorphism and species divergence . a positive correlation rather than random distribution furthermore , partial sequences of cox1 have been shown not to be the ideal marker for either species identification or population studies of schistosoma species . instead , authors have suggested the use of cox3 and nad5 sequences for both phylogenetic and population studies of such species . dna barcoding was performed for samples of s. haematobium using cox1 and nad1 as molecular markers . the results supported the identification of group 1 and 2 haplotypes based on phylogenetic analysis . moreover , no change in genetic diversity was detected across samples collected over different time points . this approach allowed the development of new assays based on group - specific pcr primers and snapshot probes to assist genetic screening of schistosome isolates . another study employing mitochondrial sequences was performed in schistosoma cercariae harvested from biomphalaria choanomphala as part of parasitological and malacological surveys for s. mansoni across lake victoria . dna barcoding of cercarial samples revealed the presence of hybrid species between s. mansoni and s. rodhaini , which is typically found in small mammals . dna barcoding studies are underway for an increasing number of schistosoma species ( table 1 ) , whose data is made available through the barcode of life database ( bold ) system ( http://www.boldsystems.org/ ) . this initiative includes contributors from the biodiversity institute of ontario , canada , and the university of new mexico , usa , among others . other schistosomatidae genera are subject to dna barcoding analysis such as allobilharzia , austrobilharzia , bilharziella , bivitellobilharzia , dendritobilharzia , gigantobilharzia , griphobilharzia , heterobilharzia , macrobilharzia , orientobilharzia , ornithobilharzia , schistosomatium , and trichobilharzia . advances in dna barcoding of a broader range of schistosomatidae will create a framework for species identification from environmental and clinical samples as well as specimens deposited in biological collections . moreover , dna barcoding analysis will help reconstructing the evolutionary relationships across different platyhelminthes and ultimately improve our understanding of parasite biology and evolution . transcriptomic analyses are valuable not only for studies of differential regulation in gene expression , but also for the identification of splicing variants . these approaches have proved to be essential in the identification of gene - coding regions during the process of genome annotation . in this section , we aim to review the achievements in schistosoma transcriptomic studies that range from the whole organism and tissue analyses to those performed at the cellular level . transcriptomics in physiological conditions and in response to a variety of treatments are investigated with respect to the interaction with the host as well as other environments . this section summarizes the accomplishments of gene function studies and the diverse techniques that are being employed . a fine tuning in gene expression of parasites must occur to enable them to survive in such a complex and extremely diverse milieu , adapting to diverse environments as evading the immune system and , at the same time , exploiting different hosts . transcriptomic studies came along allowing a temporal and quantitative analyses of gene expression , which are crucial to completely exploit different datasets and assess parasite biology at the molecular level . aiming to elucidate the differential regulation on the schistosoma transcriptomics , a large number of projects employed diverse strategies , such as the ones using expressed sequence tags ( ests ) , open reading frame expressed sequence tags ( orestes ) , and serial analysis of gene expression ( sage ) [ 52 , 53 ] . the increasing number of projects resulted in the production of a vast amount of sequence data and their availability in public databases . it is noteworthy that only s. japonicum , s. mansoni and , not until very recently , s. haematobium had some of their transcriptome explored ( table 1 ) . the large number of transcriptome projects accelerated the studies in this area by providing means to the use of complementary approaches , especially microarray analysis . together , these approaches have been used to answer a wide range of questions . in the early years , microarray datasets conceived the profile of gene expression between genders of schistosoma species such as s. japonicum and s. mansoni [ 5456 ] . this greatly extended the number of known sex - associated genes and also provided new insights into changes in gene expression promoted by parasite pairing . in addition , gender biased alternative splicing patterns were observed , which are perhaps involved in the generation and maintenance of the sexual dimorphism . later , studies profiled the s. mansoni gene expression in different life stages broadening our understanding of factors controlling schistosome development [ 8 , 53 , 58 ] . while comparing different parasite life stages , information acquired from microarray datasets highlighted putative antischistosome candidate molecules to be used as vaccine targets including dyp - type peroxidases , fucosyltransferases , g - protein coupled receptors , leishmanolysins , tetraspanins , and the netrin / netrin receptor complex . some of these candidates ( fucosyltransferases , leishmanolysins , and tetraspanins ) are members of protein families , which are expanded in the s. mansoni predicted proteome in comparison with other metazoans as inferred by phylogenomic analysis ( http://phylomedb.org/ ) . leishmanolysins , which are members of the metallopeptidase m8 family , were analysed in detail corroborating the potential of these enzymes as future therapeutic targets . another study identified upregulated genes during the first five days of schistosomula development in vitro that are involved in blood feeding , tegument and cytoskeletal development , cell adhesion , and stress responses . the highly upregulated genes included a tegument tetraspanin sm - tsp-3 , a protein kinase , a novel serine protease and serine protease inhibitor , and intestinal proteases belonging to distinct mechanistic classes . other groups have analysed immunity towards schistosoma using protein microarrays as a vaccine discovery tool . they selected a subset of proteins from genomic , transcriptomic , and proteomic data to perform experimental validation aiming at identifying novel antischistosome vaccine candidates [ 60 , 61 ] . recently , an elegant approach to investigate specific tissues such as gastrodermis and both genders reproductive tissues was performed by applying laser microdissection microscopy combined to microarray . these studies brought the perspective of expediting tissue - specific profiling [ 62 , 63 ] . additionally , expression profiles of s. japonicum were explored in studies comparing two chinese isolates ( anhui and zhejiang ) , as well as the anhui chinese and philippines isolates [ 55 , 64 ] . later , an interspecies study compared the s. japonicum and s. mansoni transcriptomes . surprisingly , despite their large phenotypic differences , there was a reduced number of differentially expressed genes based on the expression profile , which might reflect a limitation of the technique ( preferential hybridization related to species polymorphisms ) . bypassing these pitfalls , the next generation sequencing platforms allow exploiting expression data ( rna - seq ) of any organism without previous knowledge of the genome or transcriptome and , further , will reveal new features of the parasite transcriptional landscape . the most recent achievement in this area was the refinement of the genomics and transcriptomics data using a combination of sanger capillary sequencing , next generation dna sequencing , genetic markers , and rna - seq data from several s. mansoni life stages . a previous study , explored the transcriptome of s. mansoni adult males obtaining 11 novel microexon genes ( megs ) , as well as mapping 989 and 1196 contigs to intergenic and intronic genomic regions , respectively . this data could represent new protein - coding genes or noncoding rnas ( ncrnas ) . together , these approaches have significantly advanced our understanding of the dynamics parasite transcriptomes [ 9 , 65 ] . however , there is a need to polish and translate sequence data and gene expression profiles into functional information by computational predictions and experimental characterization . the vast amount of available data opened a myriad of possibilities towards the elucidation of gene functions . transcriptomics is also a key approach to study posttranscriptional regulatory mechanisms such as alternative and transsplicing [ 57 , 6668 ] . alternative splicing of transcripts of secreted proteins , like megs and polymorphic mucin isoforms , has been largely studied in schistosomes [ 51 , 66 ] . this posttranscriptional processing has been proposed as a distinct genetic system to generate protein variation that would allow parasite adaptation to the immune response of different hosts . additionally , alternative splicing has been shown to generate protein variation involved in transcriptional control and splicing . in the case of smca150 , a spliceosome interacting transcriptional cofactor , it could impact many other transcripts leading to several effects in the parasite , especially in sexual dimorphism . spliced leader ( sl ) transsplicing has been shown to be an important posttranscriptional regulation mechanism for s. mansoni . at least 11% of all transcripts has been described as undergoing transsplicing in this organism . sl - rnas are small ncrnas , products of small intronless genes , transcribed by dna polymerase ii and tandemly repeated in the genome . differently from nematodes , s. mansoni carries a single sl sequence , which is composed by 36 nucleotides with an 3-terminal aug codon . although the role of transsplicing is poorly understood , there is an evidence suggesting that sl transsplicing would function providing an initiator methionine for translation initiation of some recipient mrnas in platyhelminthes . this mechanism is also involved in the resolution of polycistronic operons in monocistronic transcripts in s. mansoni [ 19 , 70 ] . despite the great potential revealed by the increasing number of genome and transcriptome studies in schistosomes , there are still several schistosoma species that remain neglected . in contrast to the vast amount of data available in public databases , limited data has been decoded in functional studies and/or in the development of successful tools to fight schistosomes . some of them include the convolution of schistosomes life cycle , the complex interaction with their hosts , time and labor - demand functional studies , the difficulties to nurture the complete life cycle in vitro , and the absence of immortalized cell lines , which has delayed the development of transgenesis tools and models . lately , the posttranscriptional suppression of genes through rna interference ( rnai ) techniques has been a promise to fetch progress on new interventions for schistosomiasis and other helminthiases . in 2003 simultaneously gene knockdown of glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) and a glucose transporter ( sgtp1 ) in the larval stage ( sporocysts ) and cathepsin b in adult worms of s. mansoni were reported . currently , this powerful technique is widely used in schistosomes and has been employed in two studies of functional screening . the first study encompassed the exposure of sporocysts by soaking parasites to dsrna targeting 33 genes , from calcium binding proteins , transcription factors , to receptors , and antioxidant enzymes . in this work , despite the efficacy of this tool to deliver suppression of 11 different genes , it showed that , in s. mansoni , there is a gene - to - gene variability and exposure to dsrna could trigger gene specific upregulation . later , in a different study using electroporation to expose schistosomula to dsrna of 11 target genes ( expressed in distinct tissues ) , the efficiency of rnai in a target specific fashion was observed . other issues with the use of rnai have been widely discussed , not only in schistosomes , but also in other helminth parasites , showing that care should be taken when conducting and evaluating such experiments . despite the difficulties in exploring gene function by rnai in schistosomes , many proteases ( cathepsins and aminopeptidases ) [ 7779 ] , kinases , multidrug resistance transporters , and type v collagen , among other proteins have been studied in diverse life stages of s. japonicum and s. mansoni . recently , this tool has been described targeting tetraspanin 2 in diverse developmental stages of s. haematobium indicating the rnai feasibility in this species . tetraspanins belong to a family of integral membrane proteins present in the outer surface membranes of the parasite tegument . studies have shown that tetraspanins play important roles in the tegument development , maturation , or stability . importantly , the knockdown of peroxiredoxin and thioredoxin glutathione reductase , antioxidant enzymes , were amongst the first reports of lethality after gene silencing in the parasite , demonstrating the potential of rnai on drug target discovery . moreover , enzymes such as glutathione - s - transferases ( gst26 and gst28 ) , peroxiredoxins ( prx1 and prx2 ) , and superoxide dismutase have been shown to play important roles in the protection of s. mansoni sporocysts in the host - parasite interplay . knockdown of those targets increased parasite susceptibility to oxidative stress and to intermediate host immune cells as described elsewhere . recently researchers are developing new tools for the introduction and expression of homologous or heterologous gene constructs via lentiviruses , replication - defective retroviruses , transposons , retrotransposons , and dna plasmid vectors . sophisticated systems applied to assess gene function in the parasite are being achieved [ 8891 ] . to date , eggs and miracidia are indicated as the preferred life stages for successful transformation approaches in order to enter the germline . in summary , results from such independent studies have shown that schistosome transgenesis works in all the developmental stages tested and that some success has been achieved in integrating genes into the germline [ 8892 ] . altogether , the increasing information on schistosome genomes and transcriptomes is now raising the possibilities to better explore molecular techniques in the field of parasitology , genomics , and computational biology allowing the development of approaches to unravel mechanisms of parasite infection , host - parasite interaction , and so forth [ 21 , 23 , 24 ] . in order to accelerate both parasite research and development of effective tools for schistosomiasis diagnostics and treatment , user - friendly databases have been created and made publicly available to the scientific community [ 9 , 9395 ] . here , we highlight some of these major resources . schistodb integrates genome and proteome sequence data along with functional annotation of genes and gene products of s. haematobium , s. japonicum , and s. mansoni . this resource also covers results from large - scale analysis including ests , metabolic pathways , and candidate drug targets . helmcop ( helminth control and prevention ) integrates functional , structural , and comparative genomics data of helminths from different taxonomic groups and distinct hosts ( plant , animal , and human ) . helmcop offers a comprehensive suite of structural and functional annotations to support comparative analyses and host - parasite interaction studies . genedb is an annotation database that brings together data from a broad range of pathogens and closely related organisms . genedb offers database - driven annotation tools and pipelines and includes manually curated information maintained by computational and biological scientists . collectively , these computational resources support biological and biomedical research and open new frontiers in evolutionary and functional genomics , transcriptomics , and proteomics of schistosoma species and other parasites . besides biodiversity assessment at the molecular level , these resources provide a framework for the identification and prioritization of vaccine and drug targets against human diseases . as the scientific community progresses in data integration and interpretation , we will meet the goals of current and future initiatives to improve human health [ 9 , 12 , 23 , 24 ] . the -omics studies came along with the aim to fill the gaps of long - established approaches , enabling the use of a broad range of experimental and computational methods and contributing to dissect the biological basis of host - parasite interaction , diagnostics improvement , and the discovery of new drug and vaccine targets . nuclear and mitochondrial genomes of some schistosoma species were completely sequenced opening new frontiers for comparative analyses , which will improve our understanding of parasite biology and evolution . other genomes to be analysed include schistosoma species from each of the six clades currently recognized , and other representatives of the schistosomatidae . improving accuracy / quality of genome assembly and annotation will be crucial for such analyses . despite the efforts in the characterization of genomes and transcriptomes of distinct schistosoma species , many questions regarding the biology and evolution of this important taxon remain unanswered . these difficulties might be due to the size of schistosome genomes ( ~10-times larger compared to protozoan parasites ) , the complexity of its life cycle , the presence of rare features in posttranscriptional regulation ( e.g. , transsplicing ) , and little integration of the current information available , among other reasons . there is an urgent need for data integration to help interpreting the massive amount of data generated over the years in this and other areas or research . we envision that , in the future , more transcriptomic and proteomic data will be used to improve the understanding of the origin and evolution of schistosomatidae . the analysis of structural data generated by experimental or computational approaches will also shed light in the biodiversity of schistosomes at the molecular level . in conclusion , the availability of schistosome genome and transcriptome data has provided an unprecedented resource for many research areas . limitations do exist and should be addressed to allow advances in understanding schistosome biology and evolution and ultimately support vaccine and drug development against schistosomiasis .

schistosomes are digenean blood flukes of aves and mammals comprising 23 species . some species are causative agents of human schistosomiasis , the second major neglected disease affecting over 230 million people worldwide . modern technologies including the sequencing and characterization of nucleic acids and proteins have allowed large - scale analyses of parasites and hosts , opening new frontiers in biological research with potential biomedical and biotechnological applications . nuclear genomes of the three most socioeconomically important species ( s. haematobium , s. japonicum , and s. mansoni ) have been sequenced and are under intense investigation . mitochondrial genomes of six schistosoma species have also been completely sequenced and analysed from an evolutionary perspective . furthermore , dna barcoding of mitochondrial sequences is used for biodiversity assessment of schistosomes . despite the efforts in the characterization of schistosoma genomes and transcriptomes , many questions regarding the biology and evolution of this important taxon remain unanswered . this paper aims to discuss some advances in the schistosome research with emphasis on genomics and transcriptomics . it also aims to discuss the main challenges of the current research and to point out some future directions in schistosome studies .

pubmed

for patients with synchronous colorectal liver metastases ( sclm ) , hepatic resection is considered the best treatment , with reported 5-year survival rates between 23% and 37%.14 resections of both the primary colorectal lesion and the hepatic metastases are needed for patients with sclm when they are resectable . however , the optimal surgical strategy for resectable sclm still remains controversial . from the perspectives of less operation with less mental stress and simplifying perioperative treatment , simultaneous resection of the primary colorectal and liver tumors is a favorable strategy for patients with sclm.58 however , several papers reported that the morbidity rate after simultaneous resection of primary and liver tumors was high because of greater surgical stress and a longer operation time than for single - organ surgery . staged resection with initial operation for the primary lesion followed by resection of hepatic tumors is regarded as an alternative strategy to avoid excessive surgical stress for patients with sclm , though the efficacy of this strategy and the patients who could benefit from this strategy are unknown.46,9,10 thus , this study was conducted to determine which patients could benefit from staged resections instead of simultaneous resection by identifying predictive factors for postoperative morbidity and anastomotic leakage after simultaneous resection of sclm . the medical records of all consecutive patients who underwent liver resections for colorectal liver metastases from january 1992 to january 2004 at our institution were analyzed retrospectively , with institutional review board approval . all sclm patients received simultaneous resection of primary colorectal and hepatic tumors irrespective of the patient 's or the tumor 's characteristics . all 86 patients underwent contrast enhanced computed tomography ( ct ) of the chest , abdomen , and pelvis , as well as hepatic mri , preoperatively . as a control , the morbidity of 167 patients who underwent hepatectomy for metachronous liver metastasis from colorectal cancer from january 1992 to january 2004 and that of 1,728 patients who underwent only resection for colorectal cancer with colorectal reconstruction during the same period were also reviewed . of the 1,728 colorectal cancer patients , incidences of the following postoperative complications were analyzed : anastomotic leak , rectovaginal fistula , intraperitoneal or pelvic abscess , wound infection , wound dehiscence , ileus , enteroparesis , postoperative delirium , urinary tract infection , dysuria , empyema thoracis , pleural effusion , atelectasis , cholecystitis , perihepatic or subphrenic abscess , bile leak , liver failure , and others . anastomotic leakage was defined as follows : peritonitis and a dehiscence in the anastomosis , discharge of pus from the anus , vaginal fistula , or feces from the abdominal drain . leakage was confirmed by ct scan , contrast enema , re - operation , or digital rectal examination . all complications were graded according to the classification proposed by clavien et al.11 postoperative mortality was defined to include any death during postoperative hospitalization or within 30 days . correlations between postoperative morbidity and the following patient , tumor , and surgical factors were analyzed : age , sex , body mass index ( bmi ) , preoperative comorbidity , site of primary tumor , intestinal obstruction by tumor , size of primary tumor , differentiation of tumor , distribution of hepatic tumors , number of hepatic tumors , hepatic tumor size , operative methods , operation time , intraoperative blood loss , and blood transfusion . survival and follow - up were calculated from the time of the operation to the date of death or last available follow - up . multivariate analyses to evaluate the independent predictive factors for postoperative complications or anastomotic leakage were done by multiple logistic regression analysis . the medical records of all consecutive patients who underwent liver resections for colorectal liver metastases from january 1992 to january 2004 at our institution were analyzed retrospectively , with institutional review board approval . all sclm patients received simultaneous resection of primary colorectal and hepatic tumors irrespective of the patient 's or the tumor 's characteristics . all 86 patients underwent contrast enhanced computed tomography ( ct ) of the chest , abdomen , and pelvis , as well as hepatic mri , preoperatively . as a control , the morbidity of 167 patients who underwent hepatectomy for metachronous liver metastasis from colorectal cancer from january 1992 to january 2004 and that of 1,728 patients who underwent only resection for colorectal cancer with colorectal reconstruction during the same period were also reviewed . of the 1,728 colorectal cancer patients , incidences of the following postoperative complications were analyzed : anastomotic leak , rectovaginal fistula , intraperitoneal or pelvic abscess , wound infection , wound dehiscence , ileus , enteroparesis , postoperative delirium , urinary tract infection , dysuria , empyema thoracis , pleural effusion , atelectasis , cholecystitis , perihepatic or subphrenic abscess , bile leak , liver failure , and others . anastomotic leakage was defined as follows : peritonitis and a dehiscence in the anastomosis , discharge of pus from the anus , vaginal fistula , or feces from the abdominal drain . leakage was confirmed by ct scan , contrast enema , re - operation , or digital rectal examination . all complications were graded according to the classification proposed by clavien et al.11 postoperative mortality was defined to include any death during postoperative hospitalization or within 30 days . correlations between postoperative morbidity and the following patient , tumor , and surgical factors were analyzed : age , sex , body mass index ( bmi ) , preoperative comorbidity , site of primary tumor , intestinal obstruction by tumor , size of primary tumor , differentiation of tumor , distribution of hepatic tumors , number of hepatic tumors , hepatic tumor size , operative methods , operation time , intraoperative blood loss , and blood transfusion . survival and follow - up were calculated from the time of the operation to the date of death or last available follow - up . multivariate analyses to evaluate the independent predictive factors for postoperative complications or anastomotic leakage were done by multiple logistic regression analysis . from 1992 to 2004 , 86 patients were treated with simultaneous resection of primary and hepatic tumors for sclm . there were 37 female and 49 male patients , with a median age of 59 years ( range , 40 to 85 years ) . the primary tumor was staged as t3 in 54 ( 63% ) and t4 in 32 ( 37% ) according to the tnm classification . the mean diameter of the primary tumor was 55 mm ( range , 26140 mm ) . liver metastases were solitary in 29 patients and multiple in 57 patients . in 47 patients ( 55% ) , the hepatic tumor showed a unilobar distribution , while a bilobar tumor distribution was observed in 39 ( 45% ) . the mean diameter of the hepatic tumor was about 43 mm ( range , 5200 mm ) . the mean resected liver volume was 380 g ( range , 101,660 g ) . the operation for primary colorectal cancer was right ( hemi ) colectomy in 17 patients , transverse colectomy in 1 , left ( hemi ) colectomy in 4 , sigmoidectomy in 24 , high anterior resection in 7 , low anterior resection in 20 , very low anterior resection in 6 , inter - sphincteric resection in 2 , hartmann 's operation in 1 , and abdomino - perineal resection in 4 ( table 4 ) . a diverting stoma to prevent anastomotic leakage was made in 22 ( 26% ) patients at the surgeon 's discretion , and lateral lymph node dissection was performed in 20 ( 23% ) . in terms of liver tumor resection , lobectomy was performed in 11 patients , segmentectomy in 22 , bisegmentectomy in 1 , trisegmentectomy in 2 , subsegmentectomy in 3 , and partial resection in 47 . adjuvant therapy was given to only 17 patients ( 19.8% ) because adjuvant chemotherapy for colorectal cancer in stage iii or more was performed since january 2003 . no patients died within 30 days of the operation , but 55 ( 64% ) patients developed complications ( table 1 ) . eighteen patients ( 21% ) experienced leakage , of whom 6 needed urgent re - operation with ileostomy and drainage of an intra - abdominal collection caused by leakage . postoperative bleeding , wound dehiscence , and ileus were the reasons for the three other re - operation cases . the most frequent complication was wound infection.table 1postoperative complications after simultaneous resection for sclm according to clavien gradecomplicationsno . of patientsgr igr iigr iiiagr iiibgr ivacolon and rectum anastomotic leakage18 ( 21%)126 intrapelvic abscess6 ( 7%)141 intraperitoneal abscess5 ( 6%)1031 rectovaginal fistula4 ( 5%)13liver bile leakage7 ( 8%)61 hepatic abscess7 ( 8%)511 liver failure3 ( 3%)111 postoperative bleeding1 ( 1%)1other organs wound infection25 ( 29%)232 pleural effusion12 ( 14%)111 wound dehiscence6 ( 7%)321 enteroparesis5 ( 6%)5 postoperative delirium4 ( 5%)13 dysuria4 ( 5%)4 urinary tract infection3 ( 3%)3 pneumonia2 ( 2%)2 others7 ( 8%)142 postoperative complications after simultaneous resection for sclm according to clavien grade the morbidity rate of the 167 patients who underwent hepatectomy for metachronous colorectal liver metastasis during the same period was 19.8% , and that of 1,728 patients who underwent only resection for colorectal cancer was 32.1% . postoperative complications were significantly correlated with presence of diverting stoma ( p < 0.01 ) , duration of operation greater than 8 h ( p < 0.01 ) , amount of intraoperative blood loss ( p < 0.01 ) , and intraoperative blood transfusion ( p < 0.01 ) . only a greater amount of blood loss had a predictive value for increased occurrence of postoperative complications . then , the correlations between anastomotic leakage and clinicopathological factors were examined to identify risk factors for anastomotic leakage after simultaneous resection for sclm . patients who underwent abdomino - perineal resection ( n = 4 ) or hartmann 's operation ( n = 1 ) were excluded from the analysis . anastomotic leakage was significantly correlated with lateral lymph node dissection ( p < 0.01 ) , primary site of rectum ( p = 0.01 ) , duration of operation greater than 8 h ( p < 0.01 ) , and amount of intraoperative blood loss ( p = 0.02 ) . neither serum levels of tp and alb , steroid usage , nor neoadjuvant therapy showed correlation with occurrence of anastomotic leakage ( data not shown ) . multivariate analyses revealed operation time greater than 8 h ( p < 0.01 ) as the only independent predictive factor for anastomotic leakage after simultaneous resection of sclm ( table 2 ) . extent of hepatectomy , timing of anastomosis and hepatectomy , and usage of pringle maneuver did not correlate with occurrence of complication or anastomotic leakage.table 2correlation between anastomotic leakage and clinicopathological factors in patients who underwent simultaneous resection for sclmleakage ( ) ( n = 63)leakage ( + ) ( n = 18)univariate analysis p valuemultivariate analysis p value , rr ( 95%ci)patient characteristics median age ( range ) ( years)59 ( 4085)59 ( 4173)0.81 male / female33/3012/60.42 bmi ( mean sd)21.9 2.922.5 2.20.44 preoperative comorbidity absent44120.78 present196primary colorectal tumor sitecolon4260.01n.s.rectum2112 stenosisabsent5600.34present718 tumor size , mm52.058.00.25 pt stagept34190.25pt4229 pn stagepn01720.22pn+4616 histologywell , mod60150.12poor33liver metastasis distributionunilobar3890.43bilobar259 number of tumors ( range)2.3 ( 18)2.6 ( 18)0.57 tumor size , mm47330.06operative factors lateral lymph node dissection absent5510<0.01n.s . present88 diverting stoma absent48110.24 present157 liver resection partial hx , segmentectomy51160.72 lobectomy122 timing of anastomosis colectomy anastomosis hx2040.20 colectomy hx anastomosis75 hx colectomy anastomosis369 pringle maneuver absent1010.44 present5317 operation time < 8 h538<0.01<0.01 , 6.63 ( 2.0920.9 ) 8 h1010 blood loss , g ( range)1,345 ( 1626,000)2,487 ( 4306,560)0.02n.s . transfusion absent3990.37 present249 blood transfusion , ml3431,2120.05rr relative risk , ci confidence interval , hx hepatectomy , n.s . non - significant ( p > 0.05 ) correlation between anastomotic leakage and clinicopathological factors in patients who underwent simultaneous resection for sclm rr relative risk , ci confidence interval , hx hepatectomy , n.s . non - significant ( p > 0.05 ) table 3 showed the rates of complication iiia and anastomotic leakage according to operative procedures of the primary and hepatic resections which were performed in the same patient . complication iiia and anastomotic leakage were more frequently observed in patients with rectal resection ; however , extent of hepatectomy did not seem to affect occurrence of complication iiia or anastomotic leakage.table 3rates of complication gr iiia and anastomotic leakage according to the site of primary colorectal resection and extent of hepatectomyprimary colorectal resectionhepatectomycomplication gr iiiaanastomotic leakagecolectomy < lobectomy4/40 ( 10%)5/39 ( 13%)lobectomy0/7 ( 0%)1/7 ( 14%)rectal resection < lobectomy11/32 ( 34%)11/28 ( 39%)lobectomy2/7 ( 29%)1/7 ( 14%)one patient who underwent hartmann 's operation was excluded from the analysisfour patients who underwent abdomino - perineal resection were excluded from the analysis rates of complication gr iiia and anastomotic leakage according to the site of primary colorectal resection and extent of hepatectomy one patient who underwent hartmann 's operation was excluded from the analysis four patients who underwent abdomino - perineal resection were excluded from the analysis hospitalization was significantly longer in the 55 patients with postoperative morbidity ( 32.2 days ) than in the 31 patients without postoperative morbidity ( 17.6 days ) ( p < 0.01 ) . in addition , hospitalization was significantly longer in the 18 patients with anastomotic leakage ( 43.5 days ) than in the 63 patients without anastomotic leakage ( 22.2 days ) ( p < 0.01 ) . the overall survival rate after simultaneous resection for sclm of the 86 patients was 61% at 3 years and 45% at 5 years , with mst of 47 months . from 1992 to 2004 , 86 patients were treated with simultaneous resection of primary and hepatic tumors for sclm . there were 37 female and 49 male patients , with a median age of 59 years ( range , 40 to 85 years ) . the primary tumor was staged as t3 in 54 ( 63% ) and t4 in 32 ( 37% ) according to the tnm classification . the mean diameter of the primary tumor was 55 mm ( range , 26140 mm ) . liver metastases were solitary in 29 patients and multiple in 57 patients . in 47 patients ( 55% ) , the hepatic tumor showed a unilobar distribution , while a bilobar tumor distribution was observed in 39 ( 45% ) . the mean diameter of the hepatic tumor was about 43 mm ( range , 5200 mm ) . the mean resected liver volume was 380 g ( range , 101,660 g ) . the operation for primary colorectal cancer was right ( hemi ) colectomy in 17 patients , transverse colectomy in 1 , left ( hemi ) colectomy in 4 , sigmoidectomy in 24 , high anterior resection in 7 , low anterior resection in 20 , very low anterior resection in 6 , inter - sphincteric resection in 2 , hartmann 's operation in 1 , and abdomino - perineal resection in 4 ( table 4 ) . a diverting stoma to prevent anastomotic leakage was made in 22 ( 26% ) patients at the surgeon 's discretion , and lateral lymph node dissection was performed in 20 ( 23% ) . in terms of liver tumor resection , lobectomy was performed in 11 patients , segmentectomy in 22 , bisegmentectomy in 1 , trisegmentectomy in 2 , subsegmentectomy in 3 , and partial resection in 47 . adjuvant therapy was given to only 17 patients ( 19.8% ) because adjuvant chemotherapy for colorectal cancer in stage iii or more was performed since january 2003 . no patients died within 30 days of the operation , but 55 ( 64% ) patients developed complications ( table 1 ) . eighteen patients ( 21% ) experienced leakage , of whom 6 needed urgent re - operation with ileostomy and drainage of an intra - abdominal collection caused by leakage . postoperative bleeding , wound dehiscence , and ileus were the reasons for the three other re - operation cases . the most frequent complication was wound infection.table 1postoperative complications after simultaneous resection for sclm according to clavien gradecomplicationsno . of patientsgr igr iigr iiiagr iiibgr ivacolon and rectum anastomotic leakage18 ( 21%)126 intrapelvic abscess6 ( 7%)141 intraperitoneal abscess5 ( 6%)1031 rectovaginal fistula4 ( 5%)13liver bile leakage7 ( 8%)61 hepatic abscess7 ( 8%)511 liver failure3 ( 3%)111 postoperative bleeding1 ( 1%)1other organs wound infection25 ( 29%)232 pleural effusion12 ( 14%)111 wound dehiscence6 ( 7%)321 enteroparesis5 ( 6%)5 postoperative delirium4 ( 5%)13 dysuria4 ( 5%)4 urinary tract infection3 ( 3%)3 pneumonia2 ( 2%)2 others7 ( 8%)142 postoperative complications after simultaneous resection for sclm according to clavien grade the morbidity rate of the 167 patients who underwent hepatectomy for metachronous colorectal liver metastasis during the same period was 19.8% , and that of 1,728 patients who underwent only resection for colorectal cancer was 32.1% . postoperative complications were significantly correlated with presence of diverting stoma ( p < 0.01 ) , duration of operation greater than 8 h ( p < 0.01 ) , amount of intraoperative blood loss ( p < 0.01 ) , and intraoperative blood transfusion ( p < 0.01 ) . only a greater amount of blood loss had a predictive value for increased occurrence of postoperative complications . then , the correlations between anastomotic leakage and clinicopathological factors were examined to identify risk factors for anastomotic leakage after simultaneous resection for sclm . patients who underwent abdomino - perineal resection ( n = 4 ) or hartmann 's operation ( n = 1 ) were excluded from the analysis . anastomotic leakage was significantly correlated with lateral lymph node dissection ( p < 0.01 ) , primary site of rectum ( p = 0.01 ) , duration of operation greater than 8 h ( p < 0.01 ) , and amount of intraoperative blood loss ( p = 0.02 ) . neither serum levels of tp and alb , steroid usage , nor neoadjuvant therapy showed correlation with occurrence of anastomotic leakage ( data not shown ) . 0.01 ) as the only independent predictive factor for anastomotic leakage after simultaneous resection of sclm ( table 2 ) . extent of hepatectomy , timing of anastomosis and hepatectomy , and usage of pringle maneuver did not correlate with occurrence of complication or anastomotic leakage.table 2correlation between anastomotic leakage and clinicopathological factors in patients who underwent simultaneous resection for sclmleakage ( ) ( n = 63)leakage ( + ) ( n = 18)univariate analysis p valuemultivariate analysis p value , rr ( 95%ci)patient characteristics median age ( range ) ( years)59 ( 4085)59 ( 4173)0.81 male / female33/3012/60.42 bmi ( mean sd)21.9 2.922.5 2.20.44 preoperative comorbidity absent44120.78 present196primary colorectal tumor sitecolon4260.01n.s.rectum2112 stenosisabsent5600.34present718 tumor size , mm52.058.00.25 pt stagept34190.25pt4229 pn stagepn01720.22pn+4616 histologywell , mod60150.12poor33liver metastasis distributionunilobar3890.43bilobar259 number of tumors ( range)2.3 ( 18)2.6 ( 18)0.57 tumor size , mm47330.06operative factors lateral lymph node dissection absent5510<0.01n.s . present88 diverting stoma absent48110.24 present157 liver resection partial hx , segmentectomy51160.72 lobectomy122 timing of anastomosis colectomy anastomosis hx2040.20 colectomy hx anastomosis75 hx colectomy anastomosis369 pringle maneuver absent1010.44 present5317 operation time < 8 h538<0.01<0.01 , 6.63 ( 2.0920.9 ) 8 h1010 blood loss , g ( range)1,345 ( 1626,000)2,487 ( 4306,560)0.02n.s . transfusion absent3990.37 present249 blood transfusion , ml3431,2120.05rr relative risk , ci confidence interval , hx hepatectomy , n.s . non - significant ( p > 0.05 ) correlation between anastomotic leakage and clinicopathological factors in patients who underwent simultaneous resection for sclm rr relative risk , ci confidence interval , hx hepatectomy , n.s . non - significant ( p > 0.05 ) table 3 showed the rates of complication iiia and anastomotic leakage according to operative procedures of the primary and hepatic resections which were performed in the same patient . complication iiia and anastomotic leakage were more frequently observed in patients with rectal resection ; however , extent of hepatectomy did not seem to affect occurrence of complication iiia or anastomotic leakage.table 3rates of complication gr iiia and anastomotic leakage according to the site of primary colorectal resection and extent of hepatectomyprimary colorectal resectionhepatectomycomplication gr iiiaanastomotic leakagecolectomy < lobectomy4/40 ( 10%)5/39 ( 13%)lobectomy0/7 ( 0%)1/7 ( 14%)rectal resection < lobectomy11/32 ( 34%)11/28 ( 39%)lobectomy2/7 ( 29%)1/7 ( 14%)one patient who underwent hartmann 's operation was excluded from the analysisfour patients who underwent abdomino - perineal resection were excluded from the analysis rates of complication gr iiia and anastomotic leakage according to the site of primary colorectal resection and extent of hepatectomy one patient who underwent hartmann 's operation was excluded from the analysis four patients who underwent abdomino - perineal resection were excluded from the analysis hospitalization was significantly longer in the 55 patients with postoperative morbidity ( 32.2 days ) than in the 31 patients without postoperative morbidity ( 17.6 days ) ( p < 0.01 ) . in addition , hospitalization was significantly longer in the 18 patients with anastomotic leakage ( 43.5 days ) than in the 63 patients without anastomotic leakage ( 22.2 days ) ( p < 0.01 ) . the overall survival rate after simultaneous resection for sclm of the 86 patients was 61% at 3 years and 45% at 5 years , with mst of 47 months . for patients with resectable sclm , both primary tumor resection and hepatectomy for liver metastasis could lead to long - term survival , with a 5-year survival rate of 2337% . however , the optimal strategy , including surgical resection and perioperative treatment , remains controversial for resectable sclm . in terms of surgical resection for sclm , it has not been resolved whether simultaneous resection or staged resections would be preferable . a series of neoadjuvant chemotherapy or chemoradiotherapy , resection of primary tumor , chemotherapy between two operations , hepatectomy , and adjuvant chemotherapy could be the maximal total treatment for sclm , while simultaneous resection could simplify and shorten the treatment schedule by eliminating one operation . completion of the two resections and initiation of adjuvant chemotherapy occur earlier with simultaneous resection than with staged resections . considering survival , comparable survival for simultaneous resection was shown in comparison with that for staged resections.13 furthermore , simultaneous resection could relieve patients from a considerable degree of mental and physical stress and decrease total treatment cost by preventing a second resection for hepatic metastases . recent advances in colorectal and hepatic surgery have enabled simultaneous resection to be performed more safely . the overall complication rate was reduced , and length of hospital stay was shortened , with no change in operative mortality.7,8 however , at present , staged resections with initial resection of the primary tumor followed by hepatic resection have been frequently performed in patients with sclm for several reasons.4,5,9,10 first , the perioperative risk of staged resections has been thought to be less than that of simultaneous resection.4,13,14 sheele et al . reported 13 anastomotic leakages of 90 simultaneous procedures in their series , and two of them led to death.4 thelen et al . proposed the criteria for simultaneous liver resection according to the age and extent of liver resection , because death after simultaneous liver resection ( n = 4 ) occurred after major hepatectomies , and three of these four patients were 70 years of age or older.15 second , staged resections might offer a chance to evaluate liver or extrahepatic metastases between the two operations . lambert et al . reported that staged resections of synchronous hepatic metastases with an interval of 3 to 6 months might allow occult disease to become clinically detectable and could potentially identify patients for whom a hepatic resection would offer no survival benefit.10 fujita recommended an interval resection to assess the metastatic status of the regional lymph nodes , because the presence of six or more lymph node metastases was an independent poor prognostic factor in patients with resected sclm and a relative contraindication for hepatic resection.9 some authors proposed chemotherapy between primary tumor resection and liver resection to select patients that could benefit from hepatectomy.13,16 alternatively , a liver - first approach of doing liver resection first and primary resection second was newly proposed as a strategy for sclm.17,18 the liver - first approach might avoid needless radical colorectal surgery by confirming curability of hepatic metastases first and also might increase resectability compared with the ordinary staged resections especially in patients with progressive hepatic metastases . this study evaluated morbidity , especially anastomotic leakage , after simultaneous resection for sclm in order to assess the safety of simultaneous resection . anastomotic leakage is sometimes fatal and can cause a difficult situation with physical and mental discomfort or pain . the morbidity rate of patients who underwent simultaneous resection for sclm seemed to be higher than that of patients with resected metachronous colorectal hepatic metastasis or that of patients who underwent only resection for colorectal primary cancer . predictive factors for postoperative morbidity and for anastomotic leakage were intraoperative blood loss and operation time greater than 8 h , respectively . the overall morbidity rate and the rate of anastomotic leakage were 91% and 50% , respectively , in patients with operation time greater than 8 h , and 54% and 13% , respectively , in patients with operation time less than or equal to 8 h. blood loss and operation time usually represent the amount of surgical stress . in particular , the average hospitalization of the 18 patients with anastomotic leakage was more than 43 days . retrospective studies have also indicated that the occurrence of anastomotic leakage is associated with increased morbidity , mortality , and prolonged hospital stay . additionally , anastomotic leakage may be associated with an increased risk of local recurrence.19 various risk factors for anastomotic leakage have been analyzed by several investigators . age , sex , obesity , level of anastomosis , smoking , blood transfusion , tumor diameter , preoperative ( chemo ) radiotherapy , physical status , obstruction , and coronary heart disease have been shown to be significant risk factors for leakage.2024 in simultaneous resection for sclm , not only the factors related to the tumor , the patient , or the colorectal operation , but factors related to the hepatectomy could affect the occurrence of anastomotic leakage . however , the extent of hepatic resection , sequence of colectomy , hepatectomy , anastomosis , use of the pringle maneuver , and total time of the pringle maneuver were not predictive factors for anastomotic leakage or postoperative complications in patients with resected sclm . recently , a diverting stoma has been often used to prevent anastomotic leakage in patients who undergo low anterior resection by diverting the fecal stream and keeping the anastomosis free of material.19,25,26 in this study , the presence of a diverting stoma was not a predictive factor for absence of postoperative anastomotic leakage . however , the analysis estimating efficacy of a diverting stoma in this study was not accurate , because a diverting stoma was basically used in patients whose risk for anastomotic leakage was considered to be high by the surgeons . the site of primary tumor that has been reported as a strong predictive factor in previous studies was not a predictive factor for anastomotic leakage in this series . use of diverting stoma might affect the result of analyses of predictive factors for anastomotic leakage . a randomized , controlled trial is needed to elucidate the efficacy of a temporary diverting stoma . although several rationales for the simultaneous resection for sclm are clear , staged resections should be selected to prevent anastomotic leakage or serious complications when the scheduled operation would result in considerable surgical stress , i.e. , predicted operation time greater than 8 h according to the results of the present study . predicted operation time should be calculated by considering various factors , such as characteristics of the patient , primary and metastatic tumor , extent of operation , difficulty of the procedure , and so on . based on the results of this study , we now select staged resections when operation time is expected to be greater than 8 h ; otherwise , we select simultaneous resection . a prospective study of sclm to evaluate the efficacy and safety of the operation time - based decision model is in progress . then , comparison of ratio of patients who could receive adjuvant chemotherapy will be essential when comparing the efficacy of simultaneous resection and that of staged resections in a future study of sclm . furthermore , in staged resections , there is a risk that some patients could not undergo a second resection after the first resection due to tumor progression or complication of first surgery . resection rate of patients who could undergo both primary and hepatic resections should be assessed when comparing simultaneous resection and staged resections in sclm . the limitations of our study are its retrospective design and the relatively small number of patients studied . the morbidity rate and the frequency of anastomotic leakage were high with simultaneous resection for sclm , especially in patients with greater intraoperative blood loss or operation time greater than 8 h. for patients with sclm , staged resections should be considered when simultaneous resection would involve excessive surgical stress . this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use , distribution , and reproduction in any medium , provided the original author(s ) and source are credited .

backgroundthe optimal surgical strategy for resectable , synchronous , colorectal liver metastases remains unclear . the objective of this study was to determine which patients could benefit from staged resections instead of simultaneous resection by identifying predictive factors for postoperative morbidity and anastomotic leakage after simultaneous resection of synchronous , colorectal liver metastases and the primary colorectal tumor.methodsthis study involved 86 patients with synchronous colorectal liver metastases who underwent simultaneous resection of the primary colorectal tumor and the hepatic tumor . postoperative mortality , morbidity , and other surgical outcomes , including survival and hospitalization , were assessed . predictive factors for postoperative morbidity and for anastomotic leakage were evaluated.resultspostoperative morbidity and anastomotic leakage were found in 55 ( 64% ) and 18 ( 21% ) patients . predictive factors for postoperative morbidity and for anastomotic leakage were intraoperative blood loss and operation time > 8 h , respectively . the overall 5-year survival rate was 45%.conclusionsthe frequency of morbidity and that of anastomotic leakage seemed to be high after simultaneous resection for synchronous colorectal liver metastases , especially when intraoperative blood loss or operation time increased greatly . staged resections should be considered in cases in which excessive surgical stress from simultaneous resection of synchronous colorectal liver metastases would be expected .

pubmed

asthma is a chronic inflammatory disease that causes airflow limitation and affects the performance of activities of daily living.1 pharmacological treatment is essential2 and is aimed at achieving and maintaining clinical asthma control.1 inhalation is the most widely used approach for the treatment of asthma because it allows drugs to reach the lungs in a selective manner , increasing drug concentration in the airways and reducing systemic adverse effects . only half of all asthma patients actually use the prescribed medication and do so correctly . 5 low treatment adherence is related to the fact that patients do not know how to use or have difficulty in using inhaler devices correctly.3 asthma education can remedy that , being a key component of asthma management.6 incorrect inhaler use can lead to treatment failure by reducing drug concentration in the airways and contribute to treatment nonadherence , making clinical asthma control difficult.9 the frequency of incorrect use of metered dose inhalers ( mdis ) ranges from 14% to 90% , with an estimated mean of 50%,10 incorrect mdi use reducing lung drug deposition to less than 20% . in a study of patients using inhaled or oral corticosteroids , increased bronchodilator use , nebulizer use , and hospitalizations were observed in more than 50% of the patients who did not adhere to treatment.11 instruments ( or scoring systems ) assessing the difficulties that patients face in using inhaled drugs might be useful to reduce those difficulties . by using such instruments , health professionals can devise an educational program targeting the most common errors made by patients , thus improving treatment adherence . there are some scoring systems that assess inhaler technique in patients with lung disease.12 although some assess inhaler technique errors , there is no one system that is considered the gold standard for this purpose . such an instrument could play an important role in the evaluation of patients using inhalers.13 leal13 developed a scoring system to assess the difficulties that patients face in using mdis ; the system scores correct and incorrect mdi use , assessing the errors made during mdi use . on the basis of that instrument , santos et al.3 developed scoring systems for the assessment of dry powder inhaler ( dpi ) and mdi technique . although the aforementioned instruments have yet to be validated for use , they are used in our hospital in order to assess inhaler technique in patients with asthma3 and in those with copd . instrument validation allows determination of the congruence between the scoring system used and the reality being measured , thus increasing the reliability of the instrument.14 the use of validated instruments in order to assess inhaler technique in asthma patients will yield results that are more reliable and will ensure data quality . the objectives of the present study were to validate two scores quantifying the ability of patients to use mdis or dpis ; to identify the most common errors made during their use ; and to identify the patients in need of an educational program for the use of these devices . the inclusion criteria were as follows : having been diagnosed with asthma in accordance with the global initiative for asthma criteria15 ; having been in outpatient treatment for at least two years ; being in the 15- to 65-year age bracket ; having had at least four years of schooling ; having no hearing impairment ; and agreeing to participate in the study by giving written informed consent . patients who had previously participated in educational programs regarding the use of asthma medications were excluded . the study was conducted in three phases : validation of the reliability of the inhaler technique scores ( phase 1 ) ; assessment of the content validity of the two scores ( phase 2 ) ; and assessment of the criterion validity and discriminant validity of the instruments ( phase 3 ) . in phase 1 , two guest pharmacists ( who were blinded to the study methodology ) simultaneously evaluated inhaler technique . they used the inhaler technique scores developed by santos et al.3 in order to identify the errors made during mdi and dpi use . the instruments provide step - by - step descriptions of mdi and dpi techniques . for each step that is performed correctly , patients receive a score of 1 ; for each step that is performed incorrectly , they receive a score of 0 . for each serious inhaler technique error , the final score determines whether a patient is able or unable to use an mdi or a dpi . the pharmacists were trained in the correct use of the aforementioned scoring systems , in accordance with the guidelines established in the third brazilian consensus on asthma management.16 the following drugs were used in the evaluation : for mdis , 250 g of beclomethasone dipropionate and 100 g of albuterol ; for dpis , 200 g of budesonide and formoterol + budesonide ( 6/200 or 12/400 g ; inhalation capsules ) ; and 50 g of salmeterol xinafoate ( diskus . in phase 2 , an expert panel evaluated the instruments from the previous phase in order to assess their contents . the expert panel comprised three pulmonologists , two pediatric pulmonologists , and two allergists , all of whom had extensive experience in the use of inhalers . the proposed changes were judged in terms of the relevance of the items assessed by the preliminary instruments , the need for additions , deletions , or changes to improve the accuracy of the modified inhaler technique scores being evaluated . after the changes had been systematized by consensus , new mdi and dpi technique scores were developed . in phase 3 , a new sample of patients using dpis , mdis , or both were randomly selected to participate in the study , the eligibility criteria being the same as those used in the previous phases . they were given placebo - containing inhalers and no instructions on how to use them , being asked to use them in the same manner as they did their own inhalers . each patient was simultaneously evaluated by two other pharmacists , who were blinded to the changes that had been made . they used the instruments derived from phases 1 and 2 and were not allowed to communicate their decisions to one another . after the evaluation , all patients were instructed on how to use inhalers correctly . the median and interquartile range of the scores obtained in phase 3 allowed us to establish cut - off values and divide the patients into two groups : the group of patients who were able to use inhalers and that of those who were not . the latter were then enrolled in our pharmaceutical care program . in phases 1 and 2 , we used a convenience sample based on previous validation studies.14 17 19 the sample size for phase 3 was calculated on the basis of the hypothesis of an association of at least 60% between the scores given by the pharmacists . considering a power of 0.95 and a type i error of 0.05 , we calculated that a sample size of 31 assessments was required for each inhaler type . the results obtained in phases 1 and 2 were described qualitatively . in phase 3 , the mean scores were compared between the observers , agreements and disagreements being evaluated individually . the major errors made during inhaler use ( as assessed by the observers ) were described by frequency . in phase 3 , descriptive analysis of the absolute values of mdi and dpi technique scores by observer was performed with the statistical package for the social sciences , version 18 ( spss inc . , the degree of correlation between the scores by observer was assessed by cronbach 's alpha coefficient . after assessment of data normality , the scores by observer were compared by means of the mann - whitney rank sum test , their correlation being determined by spearman 's test , with sigmastat software , version 3 ( systat software inc . , the chi - square test was used in order to determine interobserver agreement in the classification of patients as being able or unable to use mdis and dpis . pharmacists together identified the need for changes in the scoring systems and proposed two preliminary changes : removal of the overall score ; and removal of the suggestions section in order to reduce the number of sheets . this resulted in a scoring system consisting of a single table for assessment of inhaler technique , including examples of correct / incorrect inhaler use . interobserver disagreement in phase 1 was found in 19% and 25% of the dpi and mdi scores , respectively , indicating not only inhaler technique errors made by the patients but also interpretation errors made by the observers ( the pharmacists ) . that level of disagreement was considered acceptable and was of relatively little significance , being clarified and resolved in the subsequent phase . the expert panel suggested that two changes be made to the dpi technique score , and both suggestions were accepted : the inclusion of an item regarding patient head position ( at an angle of more than 90 or less than 90 ) , patients receiving 1 point for " an angle of less than 90 " and 0 points for " an angle of more than 90 " ; and the definition of tlc , which was included at the bottom of the page . with regard to the mdi technique score , the expert panel suggested that eight changes be made , and all of their suggestions were accepted : the item " shaking the inhaler " was changed from " 3 times " to " twice or more " ; the item " mode of use " was changed to " inhaler use " ; the score for the item " in the mouth , with a spacer " was changed to 1 because it provides a correct example of mdi use , as does the item " out of the mouth , without a spacer " ; the item " time point " was changed to " time point ( rapid inhalation ) " ; the items " upon actuation " and " immediately after actuation " have the same weight and were therefore merged ; the item " only sprays it into the mouth and does not inhale " was changed to " the device is actuated directly into the mouth , and the patient does not inhale " ; the item " not completely " was changed to " insufficient " , whereas the item " adequately " was changed to " sufficient " ; and the item " interval " was changed to " time between mdi actuations " . according to all observers and experts , the resulting scoring systems were more practical ( chart 1 ) . although the maximum total score remained the same ( i.e. , nine points ) for the instrument assessing mdi technique , it increased ( to six points ) for the instrument assessing dpi technique . according to the literature , a 20% variation in the total score is acceptable for a patient to be considered able to use an inhaler correctly . for the present study , this means a minimum mdi technique score of 7.2 and a minimum dpi technique score of 4.8 ; however , it is not clear whether the aforementioned variation ( standard deviation ) takes into account whether a given patient received previous training in inhaler use . of those patients , 39 were assessed with the mdi technique score and 50 were assessed with the dpi technique score , a total of 89 assessments being performed . those assessments allowed us to identify the most common difficulties encountered / errors made during inhaler use . the most relevant difficulty encountered during the use of both types of devices was the maintenance of tlc after a deep inhalation . the number of errors made was higher during mdi use than during dpi use ( table 1 ) . errors identified in more than 50% of all tests performed included & lt ; 60 s between mdi actuations , maintenance of tlc for & lt ; 10 s after inhalation , incorrect speed of inhalation , incorrect mdi use without a spacer , and not exhaling prior to inhalation . among the patients who used a dpi , the most common errors were maintenance of tlc for & lt ; 10 s , not exhaling prior to inhalation , and incorrect speed of inhalation ( in 75% , 62% , and 57% , respectively ) , as shown in table 2 . of a sample of 72 patients , 60 did not hold their breath correctly , 58 inhaled too quickly , and 6 did not synchronize inhaler actuation with inhalation . median mdi technique scores in phase 3 of the study were 4 and 5 for observers 1 and 2 , respectively ( table 3 ) . median dpi technique scores were 4.5 and 5.0 for observers 1 and 2 , respectively . the degree of correlation of the scores by observer was 0.97 , as assessed by cronbach 's alpha coefficient . the correlation was statistically significant ( p & lt ; 0.0001 ) , as assessed by spearman 's correlation coefficient ( figure 1 ) . there were no significant differences between the scores given by each observer , as assessed by the mann - whitney test . all patients whose mdi and dpi technique scores were & lt ; 4 were enrolled in an educational program . with regard to discriminant validity , there was good interobserver agreement in the classification of patients as being able / unable to use a dpi ( 50%/50% and 52%/58% ; p & lt ; 0.01 ) and an mdi ( 49%/51% and 54%/46% ; p & lt ; 0.05 ) . the present study allowed assessment of the content validity , criterion validity , and discriminant validity of the dpi and mdi scores . it also allowed identification of the major difficulties that patients encounter when using inhaler devices for the treatment of asthma . finally , it allowed determination of cut - off scores to classify patients as being able or unable to use their inhalers , the latter patients being enrolled in an educational program . to the best of our knowledge , there is only one validation study of mdi technique scores ( in patients with asthma)12 and one of dpi technique scores ( in patients with copd).21 in addition , according to basheti et al.,22 although the use of device - specific checklists is the most feasible method for assessing inhaler technique , little evidence is available to assess the relative importance of different criteria . divergence between the scoring systems for the same inhaler device in different studies makes direct comparison of results difficult . in our study , mdi and dpi technique scores were validated in asthma patients , and the results allowed us to identify mdi and dpi technique errors and address them in a personalized manner , meaning that the instructions provided to patients were based on their own inhaler technique errors . chart 1scoring systems modified and approved by an expert panel ( phase 2 ) and validated for use in brazil ( phase 3 ) . evaluation criteriaerrors , % interval of & lt ; 60 s between doses ( patients are instructed to allow 60 s between doses)89maintenance of tlc for & lt ; 10 s after inhalation ( patients are instructed to hold their breath for 10 s after inhalation)83inadequate inspiratory flow rate ( patients inhaled too quickly , shallowly , or both)81incorrect mdi use without a spacer ( for patients who used a spacer)73not exhaling prior to inhalation ( patients did not exhale before using their mdis)59shaking the inhaler only once ( patients are instructed to shake their mdis at least twice)32not shaking the inhaler ( patients are instructed to shake their mdis at least twice)31incomplete inhalation ( patients did not perform a deep inhalation maneuver)18incorrect mdi position ( patients are instructed to sit down in such a way that their legs and the floor form a 90 angle)10lack of synchronization between mdi actuation and inhalation ( the device was actuated before the beginning of inhalation)8nasal breathing ( patients breathed through their nose during inhalation)5the device is actuated directly into the mouth , and patients do not inhale ( patients are instructed to place their mdis at a distance of three fingers ' breadth from their mouth and inhale after actuation)5others9.5mdi : metered dose inhaler . our findings corroborate those of other studies.9 23 24 the inability to hold their breath for more than 10 s after inhalation was the major difficulty that our patients encountered during mdi and dpi use ( 83% and 75% , respectively ) . manzella et al.12 assessed inhaler technique and reported that 69% of the patients studied did not hold their breath for at least 10 s. a review compiling the results of 12 studies ( including 955 patients ) identified the most common errors made by patients using inhaler devices , less than 10 s of breath - holding after inhalation having been observed in 26% . a period of 10 s of breath - holding increases drug deposition in the lungs by allowing more time for the particles to settle.25 the author of the aforementioned review reported that 50% of the patients were able to hold their breath for 10 s between doses . in our study , less than 10 s of breath - holding after inhalation was the most common error made during mdi use , having been made by 89% of those who used mdis . a period of 10 s of breath - holding is important to ensure that a greater quantity of drug reaches the airways.12 table 2proportion of inhaler technique errors as assessed by the dry powder inhaler score . evaluation criteriaerrors , % maintenance of tlc for & lt ; 10 s after inhalation ( patients are instructed to hold their breath for 10 s after inhalation)75not exhaling prior to inhalation ( patients did not exhale before using their dpis)62inadequate inspiratory flow rate ( patients inhaled too quickly , shallowly , or both)57incomplete inhalation ( patients did not perform a deep inhalation maneuver)21incorrect dose preparation ( patients did not place the capsule inside their dpis , did not puncture the capsule before inhalation , or both)*18inhaling shallowly ( patients did not perform a deep inhalation maneuver)5exhaling into the device ( patients exhaled while their dpis were in their mouth)3nasal breathing ( patients breathed through their nose during inhalation)2inhaling irregularly ( patients were unable to inhale continuously)2dpi : dry powder inhaler . * for the dpi type used in the present study ( adaptation ) . incorrect speed of inhalation was the third most common mdi technique error in our study and was reported by mcfadden as the third most common error in 12 studies , inappropriately rapid inspiratory flow rates having been observed in 19% of the total of patients . this is a serious error , given that a slower inspiratory flow rate translates to higher lung drug deposition with the use of mdis.25 not exhaling prior to inhalation was an error made by 62% of the patients using dpis , being the second most common dpi technique error . the lung volume at the beginning of inhalation interferes with drug deposition , which is why patients should exhale before inhaling.26 figure 1interobserver agreement . in a , metered dose inhaler ( mdi ) scores . in b , incorrect speed of inhalation was the third most common dpi technique error ( 57% ) . in order to deliver medication , dpis depend on patient inspiratory flow . if the flow rate ( volume / time ) is lower than required , the inhaled doses will be lower , and this contributes to treatment failure.8 27 sandrini et al.28 analyzed mdi use in a sample of patients , classifying inhaler technique as correct , slightly incorrect , moderately incorrect , or clearly incorrect . the most common errors were placing the inhaler in the mouth ( 68.0% ) , inhaling before actuation ( 15.5% ) , inhaling too quickly ( 11.0% ) , and inhaling through the nose ( 9.0% ) . although our method of evaluation differed from that used in the aforementioned study , our patients made the same errors as did those in that study . dalcin et al.24 and souza et al.29 used checklists or forms in order to determine whether patients were using their inhalers correctly at work . souza et al.29 found that 54.5% of the patients who used dpis did not exhale properly before inhaling the medication . this finding is consistent with those of the present study , because that was the second most common error made by our patients . dalcin et al.24 found that the patients who used mdis made more errors than did those who used other types of inhalers , a finding that is also consistent with those of the present study . scoresdpi techniquemdi techniqueobserver 1observer 2observer 1observer 2median ( ir)4.5 ( 4.0/6.0)5.0 ( 3.0/5.0)4.0 ( 0.5/6.0)5.0 ( 1.0/6.5)minimum / maximum1/71/712/913/10dpi : dry powder inhaler ; mdi : metered dose inhaler ; and ir : interquartile range . dpi : dry powder inhaler ; mdi : metered dose inhaler ; and ir : interquartile range . in the aforementioned studies , the instruments used allowed the authors to quantify and classify the errors made by patients . we found no technical differences between the mdi and dpi techniques used at our institution and those described in the third brazilian consensus on asthma management6 or in the 2012 brazilian thoracic association guidelines for asthma management.1 however , in addition to providing a step - by - step description of inhaler technique , the scoring systems used in the present study are tools that health professionals can use in order to assess objectively and mathematically the errors that patients make when using inhaler devices . in addition , patients can be classified as able or unable to use their inhalers on the basis of their cut - off scores . this allows health professionals to monitor improvements in inhaler technique in an objective manner . our results show that the proportion of mdi technique errors was higher than that of dpi technique errors . this difference might be related to the fact that the correct dpi technique is more easily understandable to patients than is the correct mdi technique . the results showed that incorrect inhaler use affects drug efficacy , and the authors reported that assessment of inhaler technique is still considered irrelevant by many health professionals.21 in a study conducted in 2011 in the state of bahia , brazil , inhaler technique errors were assessed , and the proportion of errors was found to be low . this was attributed to the fact that the patients in that study were monitored at an asthma referral center ; they periodically received instructions and refresher training on inhaler technique from a multidisciplinary team.5 one of the limitations of the present study is the lack of follow - up . a follow - up evaluation might have allowed us to determine whether there were improvements in inhaler technique . the present study was biased by the fact that the proportion of mdi technique errors was high , with median scores of 4 and 5 for observers 1 and 2 , respectively ; these scores are significantly different from the score of 7.2 suggested in the literature . given that interobserver agreement was high when a cut - off score of less than 4 was used - a finding that suggests that the mdi score has good accuracy - it can be inferred that many patients were classified as being able to use an mdi despite the fact that they did not obtain the minimum score predicted in theory , i.e. , 20% of a maximum total score of 9 . this underscores the need for reassessing inhaler technique at each visit and for sequential use of the mdi score in a large population sample . the new mdi and dpi scores will allow the implementation of educational programs proposed in asthma management guidelines and strategies , which recommend that patients receive ongoing training in inhaler technique to ensure correct inhaler use . by providing pharmaceutical care , pharmacists can instruct patients on how to use their inhaler devices correctly . by using our inhaler technique scores , pharmacists can assess inhaler technique errors and determine whether patients are able to achieve an ideal level of technical correctness ( of 80% ) and maintain it throughout the educational program . the present study showed that our inhaler technique scores can reveal exactly what it is that patients are doing incorrectly when using mdis or dpis so that the educational program during follow - up can focus on their errors rather than repeating what they already know . inhalation is the most widely used approach for the treatment of asthma , and correct inhaler technique is directly related to the therapeutic efficacy of the drug . therefore , correct inhaler technique is essential for the efficacy of pharmacological treatment.3 we believe that the present study is clinically relevant because it validated objective mdi and dpi technique scores that can reveal the major difficulties encountered by patients using mdis or dpis and provided cut - off scores that can be used in order to classify patients as being able or unable to use their inhalers . by using the validated scores , health professionals will be able to identify and correct inhaler technique errors during visits and , as a result , improve inhaler use . validar dois escores para medir a habilidade de pacientes em utilizar inaladores pressurizados ( ips ) ou inaladores de p ( ipos ) , verificar os erros mais comuns na sua utilizao e identificar os pacientes que necessitam de um programa educacional para o uso desses dispositivos . este estudo foi realizado em trs etapas : validao da confiabilidade dos escores de uso dos dispositivos inalatrios ; validao do contedo dos escores utilizando - se uma amostra de convenincia ; e realizao de testes para a validao de critrio e a validao discriminante desses instrumentos em pacientes que preenchiam os critrios de incluso do estudo . a amostra de convenincia foi composta por 16 pacientes , e houve discordncia interobservador em 19% e 25% para os escores de ipo e ip , respectivamente . aps a anlise de expertos no assunto , os escores sofreram modificaes e foram aplicados em 72 pacientes . a dificuldade mais relevante no uso de ambos os dispositivos foi a manuteno da capacidade pulmonar total aps inspirao profunda . o grau de correlao dos escores por observador foi de 0,97 ( p & lt ; 0,0001 ) . houve boa concordncia interobservador na classificao dos pacientes como aptos / no aptos para uso de ipo ( 50%/50% e 52%/58% ; p & lt ; 0,01 ) e de ip ( 49%/51% e 54%/46% ; p & lt ; 0,05 ) . os escores validados permitem identificar e corrigir os erros da tcnica inalatria ao longo das consultas e , em consequncia , melhorar o manejo dos dispositivos para inalao . a asma uma doena crnica inflamatria que limita o fluxo areo e pode impedir que os pacientes exeram suas atividades dirias adequadamente.1 o tratamento farmacolgico essencial2 e tem como objetivo alcanar e manter o controle clnico da doena.1 a inalao a via mais utilizada para o tratamento da asma porque permite que os medicamentos alcancem seletivamente os pulmes , com maior concentrao do frmaco nas vias areas , reduzindo os efeitos adversos sistmicos.1 3 4 apenas metade dos asmticos utiliza , de fato e corretamente , a medicao prescrita.5 destaca - se que o baixo ndice de adeso est relacionado ao desconhecimento e / ou dificuldade de administrao dos medicamentos para inalao.3 tais aspectos podem ser melhorados se a educao em asma ocorrer , sendo ela um componente estratgico para o controle da doena.6 a utilizao incorreta dos inaladores pode acarretar o insucesso teraputico por diminuir a concentrao do frmaco nas vias areas e contribuir para a no adeso ao tratamento , dificultando o controle clnico da asma.9 a frequncia de uso incorreto de inalador pressurizado ( ip ) varia entre 14% e 90% , com uma mdia estimada de 50%,10 acarretando deposio pulmonar do medicamento inferior a 20% . um estudo com pacientes que faziam uso de corticoterapia inalatria ou oral apontou que mais de 50% dos pacientes que no eram aderentes ao tratamento apresentavam maior uso de broncodilatadores e nebulizaes , alm do aumento no nmero de hospitalizaes.11 a utilizao de instrumentos ( ou escores ) que meam e pontuem as dificuldades dos pacientes durante o uso dos seus medicamentos para inalao pode ser til para reduzi - las . ao utilizar esses instrumentos , o profissional de sade pode elaborar um programa educacional direcionado para os principais erros cometidos pelo paciente , revertendo assim a baixa adeso ao tratamento . existem alguns escores que avaliam o uso correto desses inaladores em portadores de molstias pulmonares.12 alguns deles avaliam os erros na utilizao desses dispositivos para inalao ; porm , no existe um escore considerado padro - ouro com essa finalidade . tal instrumento poderia ser um diferencial na avaliao de pacientes que utilizam esses dispositivos.13 leal13 desenvolveu um escore para avaliao das dificuldades dos pacientes no uso de ips ; tal escore pontua acertos e erros na utilizao dos dispositivos , promovendo a valorao dos erros . a partir daquele instrumento , santos et al.3 desenvolveram escores para a avaliao de inaladores de p ( ipo ) e ip . esses instrumentos ainda no esto validados e so utilizados em nosso hospital para a mensurao da qualidade da tcnica de uso dos dispositivos para inalao por pacientes asmticos3 e portadores de dpoc . a validao de um instrumento permite verificar a congruncia entre o escore que utilizado para realizar uma mensurao e a realidade que medida , aumentando a confiabilidade do mesmo.14 ao se utilizar instrumentos validados para medir a tcnica de inalao de pacientes asmticos , sero obtidos resultados mais fidedignos e com maior garantia da qualidade dos dados . os objetivos do presente estudo foram validar os escores para a avaliao da tcnica de uso de ips e ipos em portugus do brasil , verificar quais erros so mais comuns durante a utilizao desses dispositivos e identificar quais pacientes necessitam de sesses de treinamento para essa utilizao . trata - se de um estudo aberto e prospectivo desenvolvido em um hospital universitrio de referncia . os pacientes foram selecionados de acordo com os seguintes critrios de incluso : apresentar diagnstico de asma de acordo com os critrios da global initiative for asthma,15 estar em seguimento ambulatorial por pelo menos dois anos , ter idade entre 15 e 65 anos , ter cursado no mnimo quatro anos do ensino fundamental , ter sua capacidade auditiva preservada e concordar em participar do estudo , assinando o termo de consentimento livre e esclarecido . pacientes que j haviam participado de programas educacionais sobre a utilizao de medicamentos para asma foram excludos . inicialmente , foi realizada a validao da confiabilidade dos escores de uso dos inaladores ( etapa 1 ) e a de seu contedo ( etapa 2 ) . na etapa 3 foram realizados os testes para a validao de critrio e a validao discriminante desses novos instrumentos . na etapa 1 , dois farmacuticos convidados ( cegos em relao metodologia do estudo ) avaliaram simultaneamente a tcnica de uso dos inaladores . aplicaram os escores para ipo e ip , elaborados por santos et al.,3 para elencar os erros apresentados . esses escores detalham o passo a passo da tcnica para cada tipo de inalador . o paciente recebe um ponto a cada etapa executada de modo correto , e , quando a tcnica feita de modo incorreto , o paciente no recebe pontos . se o paciente comete erros graves , esses so subtrados . a pontuao determina se o paciente est apto ou no para utilizar o dispositivo para inalao . os farmacuticos foram treinados quanto correta utilizao dos escores acima referidos , seguindo as diretrizes do iii consenso brasileiro de manejo da asma.16 para essa avaliao foram utilizados os seguintes medicamentos : a ) ip : dipropionato de beclometasona 250 g e salbutamol 100 g ; b ) ipo : budesonida 200 g e formoterol + budesonida ( 6/200 ou 12/400 g ; cpsulas inalatrias ) ; e xinafoato de salmeterol 50 g ( diskus . na etapa 2 , os escores oriundos da etapa anterior foram avaliados por expertos com o objetivo de mensurar o contedo desses instrumentos . o grupo de expertos foi constitudo por trs pneumologistas , dois pneumopediatras e dois alergistas , todos com slida experincia no uso de inaladores . as modificaes propostas foram julgadas quanto pertinncia dos itens medidos pelos escores preliminares , avaliando - se a necessidade de incluses , excluses ou alteraes para melhorar a acurcia dos escores modificados . depois que as alteraes foram sistematizadas por consenso , procedeu - se elaborao de novos escores para ips e ipos . na etapa 3 , novos pacientes utilizando ipo , ip ou ambos foram convidados a participar do estudo , de forma aleatria , para uma avaliao individual , obedecidos os mesmos critrios de elegibilidade das etapas anteriores . foi - lhes solicitado , sem nenhuma orientao prvia , que utilizassem inaladores contendo placebo e que realizassem a mesma tcnica da sua rotina de tratamento . cada paciente foi avaliado simultaneamente por outros dois farmacuticos , cegos em relao s modificaes realizadas , utilizando os escores derivados das etapas 1 e 2 , sendo vetado qualquer tipo de contato entre os avaliadores sobre os valores emitidos . aps as avaliaes , todos os pacientes foram orientados sobre o modo correto do uso dos inaladores . a mediana e o intervalo interquartil dos escores obtidos nessa fase permitiram a determinao dos valores de corte e a diviso dos pacientes em dois grupos : pacientes aptos e pacientes no aptos a utilizar os dispositivos para inalao . os pacientes no aptos foram ento encaminhados ao programa de ateno farmacutica existente em nossa instituio . para as etapas 1 e 2 foi utilizada uma amostra de convenincia baseada em estudos de validao anteriores . para a etapa 3 calculamos a amostra baseados na hiptese de que a associao entre os escores pontuados pelos farmacuticos seria de pelo menos 60% . considerando um poder de 0,95 e um erro tipo i de 0,05 , a amostra indicada na etapa 3 , os valores mdios dos escores foram comparados entre os observadores , avaliando - se as concordncias e divergncias individualmente . na etapa 3 , a anlise descritiva dos valores absolutos dos escores para cada dispositivo , por observador , foi realizada usando o programa statistical package for the social sciences , verso 18 ( spss inc . , chicago , il , eua ) . os graus de correlao entre os escores e por observador foram avaliados utilizando - se o coeficiente alfa de cronbach . aps a avaliao de normalidade , a comparao entre os escores por observador foi realizada atravs do teste de mann - whitney ( rank sum ) e sua correlao pelo teste de spearman , utilizando o programa sigmastat , verso 3 ( systat software inc . , san jose , ca , eua ) . o teste do qui - quadrado foi utilizado para avaliar a concordncia dos grupos de pacientes ( aptos ou no aptos para utilizao dos inaladores ) entre os avaliadores . farmacuticos em conjunto identificaram a necessidade de alteraes em determinados campos dos escores e propuseram duas modificaes preliminares : a retirada da pontuao geral dada ao paciente e a retirada do campo de sugestes para diminuir o nmero de folhas do escore . foi criado um escore com uma nica tabela para a avaliao da tcnica do uso dos inaladores , com os acertos / erros que os pacientes poderiam cometer . a discordncia entre os avaliadores na etapa 1 ocorreu em 19% e 25% em relao aos escores para ipo e ip , respectivamente , indicando no somente erros essas discordncias foram consideradas aceitveis na aplicao dos escores e proporcionalmente pouco significativas , sendo esclarecidas e dirimidas na etapa seguinte . na avaliao dos escores pelos expertos , foram sugeridas e acatadas duas modificaes no escore para ipo : acrescentar um campo sobre a posio da cabea do paciente ( ngulo maior ou menor que 90 ) , sendo atribudo um ponto para " ngulo menor que 90 " e zero para " ngulo maior que 90 " ; e o significado da sigla cpt , adicionado ao final do escore . em relao ao escore para ip , os especialistas fizeram oito sugestes de modificaes que foram acatadas : o item " agitao do dispositivo " foi alterado de " 3 vezes " para " 2 vezes ou mais " ; o campo " modo " foi alterado para " modo de uso " ; a pontuao do item " dentro da boca com espaador " foi alterada para um ponto por ser to correto quanto forma de utilizao do inalador no item " fora da boca sem espaador " ; no campo " momento " , foi adicionado o item " inspirao rpida " ; os itens " junto ao acionamento " e " logo depois do acionamento " possuem o mesmo peso e foram unificados ; o item " s jogar na boca ou no inspirar " foi alterado para " acionamento direto na boca e no inspirar " ; no campo " profundidade " o item " no completamente " foi modificado para " insuficiente " , enquanto o item " adequadamente " foi alterado para " suficiente " ; e o campo " intervalo " foi alterado para " intervalo entre os acionamentos " . os escores resultantes foram considerados mais prticos por avaliadores e expertos ( quadro 1 ) . o novo escore para ip manteve a pontuao total mxima ( nove pontos ) , mas houve um aumento na pontuao do escore novo para ipo ( seis pontos ) . de acordo com a literatura , para se considerar que um paciente conhece e domina a utilizao correta do tipo de inalador , o escore obtido por ele pode apresentar uma variao aceitvel de 20% . isso significa uma pontuao mnima de 7,2 e de 4,8 para os escores para ip e ipo , respectivamente ; entretanto , no est definido se essa variao ( desvio - padro ) leva em considerao se o paciente foi treinado anteriormente para o uso do inalador . participaram da etapa 3 do estudo 72 pacientes , com mdia de idade de 41 anos , sendo 82% do sexo feminino . esses pacientes geraram 89 avaliaes , sendo 39 pacientes avaliados com o escore para a tcnica de uso de ip e 50 pacientes com o escore para a de ipo . essas avaliaes permitiram elencar as principais dificuldades / erros dos pacientes no uso de dispositivos para inalao . o erro mais relevante entre os pacientes avaliados quanto ao uso de ambos os tipos de inaladores foi a falta de manuteno da cpt aps a inspirao profunda . observou - se um maior nmero de erros entre os pacientes com o uso do ip ( tabela 1 ) . o intervalo entre as doses & lt ; 60 s , a manuteno da cpt & lt ; 10 s aps a inalao , a velocidade da inspirao inadequada , o uso inadequado do ip sem espaador e a ausncia de realizao de expirao antes da inalao foram erros cometidos em mais de 50% dos testes realizados . entre os pacientes que utilizaram ipo , os erros mais comuns foram a manuteno da cpt & lt ; 10 s , a ausncia de expirao antes da inalao e a velocidade inadequada da inspirao ( em 75% , 62% e 57% , respectivamente ) , conforme demonstrado na tabela 2 . de uma amostra de 72 pacientes , 60 pacientes no realizaram a pausa inspiratria de maneira correta ; 58 pacientes realizaram a inspirao muito rpida ; e 6 pacientes no sincronizaram o incio da inspirao com o acionamento do dispositivo . nas mensuraes para o escore para ip realizadas na etapa 3 , os valores encontrados por avaliador , em mediana , foram 4 e 5 pontos para os observadores 1 e 2 , respectivamente , conforme mostrado na tabela 3 . o grau de correlao entre os escores avaliados por observador , utilizando - se o coeficiente alfa de cronbach , foi de 0,97 . essa correlao foi estatisticamente significativa usando - se a correlao de spearman ( p & lt ; 0,0001 ) , como se observa na figura 1 . o teste de mann - whitney confirmou que os valores atribudos aos escores pelos dois avaliadores no foram estatisticamente diferentes . foram encaminhados ao programa de educao pacientes com valor de escore & lt ; 4 pontos tanto para os escores para ip quanto para ipo . esse foi o menor valor inteiro da mediana de avaliao concordante entre os dois observadores . para a validao discriminante , encontrou - se uma concordncia entre os grupos de pacientes aptos e de no aptos ( necessitando de um programa de educao ) indicados pelo observador 1 e pelo observador 2 , tanto para o escore para ipo ( 50%/50% e 52%/58% ; p & lt ; 0,01 ) quanto para o escore para ip ( 49%/51% e 54%/46% ; p & lt ; 0,05 ) . o presente estudo possibilitou a validao de contedo , de critrio e de discriminante dos escores para ipo e ip , permitindo tambm elencar as maiores dificuldades encontradas pelos pacientes no uso dos dispositivos para inalao no tratamento da asma , alm de determinar uma nota de corte , classificando os pacientes como aptos a utilizar seus inaladores ou identificar aqueles que necessitavam ser encaminhados para um programa educacional . no melhor do nosso conhecimento atual , encontramos na literatura pesquisada um estudo que validou escores para ip para pacientes asmticos12 e , no caso de escores para ipo , somente um estudo com pacientes com dpoc.21 alm disso , segundo basheti et al.22 apesar de o uso de check - lists para um inalador especifico ser o mtodo mais vivel de avaliao , existem poucas evidncias para avaliar a importncia relativa dos diferentes critrios . as divergncias dos sistemas de pontuao para o mesmo dispositivo para inalao em diferentes estudos dificultam a comparao direta dos resultados . considerando que o nosso estudo validou escores tanto para ip quanto para ipo para pacientes asmticos , os resultados obtidos nos permitiram pontuar erros em ambos e trat - los de forma personalizada , de modo que todos os pacientes puderam ser orientados sobre o modo correto de utilizao desses dispositivos com base em seus erros de execuo da tcnica . quadro 1escores modificados e aprovados pelos expertos ( etapa 2 ) e validados para uso em portugus do brasil ( etapa 3 ) . tabela 1proporo de erros realizados pelos pacientes na avaliao do escore da tcnica do inalador pressurizado.critrios avaliadoserros , % intervalo entre as doses & lt ; 60 s ( paciente orientado a aguardar 60 s entre uma dose e outra)89manuteno da cpt & lt ; 10 s aps a inalao ( paciente orientado a prender a respirao por 10 s aps a inalao)83fluxo inspiratrio inadequado ( inspirao rpida e / ou curta)81uso inadequado do ip sem o espaador ( para os pacientes que utilizavam o espaador)73ausncia de expirao antes da inalao ( no expirar antes do uso do ip)59agitao do inalador apenas uma vez ( paciente orientado a agitar o ip no mnimo 2 vezes)32no agitar o inalador ( paciente orientado a agitar o ip no mnimo 2 vezes)31manobra inspiratria incompleta ( paciente no realiza uma inspirao profunda)18posio do ip inadequada ( paciente orientado a sentar - se de modo que suas pernas formem um ngulo de 90 com o cho)10ausncia de sincronizao entre o acionamento do ip e incio da inspirao ( paciente no sincroniza o disparo do ip com o incio da inspirao)8respirao nasal ( paciente realiza a respirao nasal durante a inalao)5acionamento do inalador direto na boca e ausncia de inspirao ( paciente orientado a distanciar o ip trs dedos da boca e realizar a inspirao aps o acionamento)5outros9,5ip : inalador pressurizado tabela 2proporo de erros realizados pelos pacientes na avaliao do escore da tcnica do inalador de p.critrios avaliadoserros , % manuteno da cpt & lt;10 s ( paciente orientado a prender a respirao por 10 s aps a inalao)75ausncia de expirao antes da inalao ( no expirar antes do uso do ipo)62fluxo inspiratrio inadequado ( inspirao rpida e / ou curta)57profundidade incompleta ( paciente no realiza uma inspirao profunda)21preparo da dose incorreta ( paciente no coloca a cpsula dentro do ipo e / ou no fura a cpsula antes de realizar a inalao)*18inspirar levemente ( paciente no realiza uma inspirao profunda)5expirar dentro do ipo ( paciente realiza a expirao com o ipo na boca)3respirao nasal ( paciente realiza a respirao nasal durante a inalao)2inspirar irregularmente ( paciente no consegue realizar a inspirao de modo contnuo)2ipo : inalador de p . * relativo ao tipo de ipo utilizado no estudo ( adaptao ) . a capacidade de prender a respirao por mais de 10 s aps a inalao foi a dificuldade mais importante entre os pacientes do nosso estudo , tanto para ip como para ipo ( 83% e 75% , respectivamente ) . manzella et al.12 avaliaram a tcnica inalatria e relataram que 69% dos pacientes avaliados no prendiam a respirao por no mnimo 10 s. uma reviso que compilou resultados de 12 estudos ( 955 pacientes includos ) classificou os erros mais cometidos pelos pacientes , sendo que a dificuldade de prender a respirao por 10 s aps a inalao ocorria em 26% da populao . esse tempo determinado de prender a respirao importante para que ocorra uma maior sedimentao das partculas do medicamento nos pulmes.25 o autor da reviso tambm relatou que 50% dos pacientes faziam a pausa de tempo necessria entre as doses . no nosso estudo , esse foi o erro mais frequentemente ( 89% ) encontrado em relao ao escore para ip . esse passo da tcnica importante para garantir que uma maior quantidade do medicamento alcance as vias areas.12 tabela 3valores dos escores encontrados por instrumento de acordo com o observador.valoresipoipavaliador 1avaliador 2avaliador 1avaliador 2mediana ( ii)4,5 ( 4,0/6,0)5,0 ( 3,0/5,0)4,0 ( 0,5/6,0)5,0 ( 1,0/6,5)mnimo / mximo1/71/712/913/10ipo : inalador de p ; ip : inalador pressurizado ; e ii : intervalo interquartil . ipo : inalador de p ; ip : inalador pressurizado ; e ii : intervalo interquartil . a velocidade de inalao inadequada , terceira dificuldade mais comumente encontrada em nosso estudo para ip , foi igualmente relatada por mcfadden como a terceira dificuldade mais frequentemente observada em 12 estudos , sendo que , no total , 19% dos pacientes realizavam a inalao de forma muito rpida . esse erro grave , pois quanto mais devagar a taxa de medicamento inalado , maior ser a deposio pulmonar desse medicamento com o uso de ip.25 em relao ao ipo , a ausncia de expirao antes da inalao foi verificada em 62% dos casos , sendo a segunda dificuldade mais frequente para esse tipo de inalador . o volume pulmonar existente no incio da inspirao interfere na deposio da droga e por isso que se recomenda a realizao da expirao antes do incio da inalao.26 a velocidade de inspirao inadequada foi o terceiro erro mais comumente encontrado ( 57% ) no uso de ipo . no caso desses dispositivos para inalao , a sada do medicamento decorrente do fluxo inspiratrio que o paciente realiza . se o fluxo ( volume / tempo ) for menor que o necessrio , as doses inaladas sero reduzidas , e esse um fator importante para o insucesso teraputico . sandrini et al.28 analisaram o uso de ip em uma amostra de pacientes , classificando a tcnica de inalao em correta , pouco errada , com erro intermedirio e muito errada . os autores classificaram a tcnica de 48% dos pacientes como no correta . os erros mais encontrados foram colocao do inalador dentro da boca ( 68,0% ) ; inspirao antes de acionar o jato ( 15,5% ) ; inspirao rpida ( 11,0% ) ; e inspirao pelo nariz ( 9,0% ) . esses erros tambm foram encontrados em nossos pacientes , mesmo com o modo de avaliao diferente do utilizado naquele estudo . dalcin et al.24 e souza et al.29 utilizaram listas ou fichas de controle para verificar a utilizao adequada dos inaladores pelos pacientes em seus trabalhos . souza et al.29 encontraram que 54,5% dos pacientes avaliados que faziam uso de ipo no realizavam a expirao antes da inalao de forma adequada . esse achado corrobora o nosso , pois esse foi o segundo erro mais comumente encontrado na avaliao de nossos pacientes . j dalcin et al.24 encontraram um maior nmero de erros nos pacientes que utilizavam ip quando comparados aos que usavam outros inaladores , sendo esse achado tambm igual ao nosso . fato que , nos trabalhos acima citados , os instrumentos utilizados permitiram aos autores quantificar e classificar os erros cometidos pelos pacientes . comparando as tcnicas de inalao para o uso de ip e ipo publicadas no iii consenso de asma16 e nas diretrizes para o manejo de asma de 2012,1 verifica - se que as orientaes no diferem tecnicamente das utilizadas em nossa instituio . entretanto , verificamos que os escores utilizados no presente estudo , alm de especificarem o passo a passo da tcnica de inalao , so uma ferramenta para que o profissional de sade possa mensurar , de forma objetiva e matemtica , os erros cometidos pelos pacientes . alm disso , eles possibilitam verificar se o paciente est apto ou no a utilizar seu inalador atravs da especificao de uma nota de corte . isso tambm permite que o profissional de sade acompanhe objetivamente a evoluo do paciente quanto a sua tcnica de inalao . nossos resultados demonstram que a proporo de erros no escore para ip foi maior do que no para ipo . essa diferena na proporo de erro entre os dispositivos pode estar associada maior facilidade na compreenso da tcnica de uso de ipo em detrimento quela de ip por parte dos pacientes . destacamos que lavorini et al.21 realizaram uma reviso da literatura com 47 artigos que analisavam a tcnica do uso do ipo . os resultados demonstram que o uso incorreto de inaladores influencia a eficcia da droga , e os autores destacam que a avaliao do uso correto dos inaladores ainda considerada irrelevante por muitos profissionais de sade.21 em um estudo realizado em 2011 na bahia , foram avaliados os erros que os pacientes cometiam ao utilizar os seus inaladores ; encontrou - se baixa proporo de erros na tcnica inalatria . tais resultados foram associados ao acompanhamento em um centro de referncia em asma que os pacientes daquele estudo recebiam ; eles participavam de peridicas orientaes e revises acerca da tcnica inalatria , realizadas por uma equipe multiprofissional.5 como limitao do estudo , pode - se indicar a falta de seguimento dos pacientes avaliados aps o mesmo . tal acompanhamento poderia permitir que se determinasse a evoluo da tcnica do uso dos inaladores pelos pacientes . um vis do presente estudo ocorreu porque os pacientes apresentaram um alto ndice de erros ao utilizarem ip ( mediana de 4 e 5 pontos para os avaliadores 1 e 2 , respectivamente ) , distantes dos valores tericos de 7,2 pontos sugeridos pela literatura . como os resultados obtidos pelos dois avaliadores foram altamente concordantes usando uma nota de corte menor que 4 pontos , sugerindo boa acurcia do escore , pode - se inferir que os observadores classificaram muitos pacientes como aptos para o uso do ip apesar de no terem atingido a pontuao mnima prevista em teoria ( 20% de 9 pontos mximos ) . isso refora a necessidade de reavaliao do uso do inalador a cada consulta e a utilizao sequencial desse escore em uma grande amostra populacional . estes novos escores permitiro a execuo de programas educacionais , propostos em diretrizes e estratgias para o tratamento da asma . segundo esses documentos , os pacientes precisam receber treinamento contnuo sobre a tcnica de inalao para que se possa garantir a utilizao correta do inalador . o farmacutico , por meio da ateno farmacutica , pode orientar o paciente quanto ao manejo dos dispositivos para inalao e , ao utilizar nossos escores , pode quantificar os acertos / erros e mensurar se o paciente alcana e mantm o ideal de 80% de correo na tcnica ao longo do programa de educao . os escores no presente estudo demonstraram ser capazes de especificar em qual passo os pacientes esto errando , permitindo direcionar o programa educativo durante o acompanhamento sem repetir o que paciente j sabe . a inalao a via mais utilizada para o tratamento da asma , e a realizao de uma tcnica correta na utilizao dos dispositivos est diretamente relacionada eficcia teraputica do medicamento . sendo assim , a execuo correta da tcnica de inalao essencial para que o tratamento farmacolgico seja eficaz.3 acreditamos que o presente estudo , ao validar escores objetivos para o uso de ip e de ipo que especificam as maiores dificuldades encontradas pelos pacientes no uso dos dispositivos e uma nota de corte , assim como classifica os pacientes como aptos a utilizar seus inaladores ou no , pode ser de relevncia clnica . o profissional de sade ser capaz , por meio desses escores validados , de identificar e corrigir os erros do paciente ao longo das consultas e , consequentemente , melhorar o manejo dos inaladores .

abstractobjective : to validate two scores quantifying the ability of patients to use metered dose inhalers ( mdis ) or dry powder inhalers ( dpis ) ; to identify the most common errors made during their use ; and to identify the patients in need of an educational program for the use of these devices . methods : this study was conducted in three phases : validation of the reliability of the inhaler technique scores ; validation of the contents of the two scores using a convenience sample ; and testing for criterion validation and discriminant validation of these instruments in patients who met the inclusion criteria . results : the convenience sample comprised 16 patients . interobserver disagreement was found in 19% and 25% of the dpi and mdi scores , respectively . after expert analysis on the subject , the scores were modified and were applied in 72 patients . the most relevant difficulty encountered during the use of both types of devices was the maintenance of total lung capacity after a deep inhalation . the degree of correlation of the scores by observer was 0.97 ( p & lt ; 0.0001 ) . there was good interobserver agreement in the classification of patients as able / not able to use a dpi ( 50%/50% and 52%/58% ; p & lt ; 0.01 ) and an mdi ( 49%/51% and 54%/46% ; p & lt ; 0.05 ) . conclusions : the validated scores allow the identification and correction of inhaler technique errors during consultations and , as a result , improvement in the management of inhalation devices .

pubmed

bacteriocins are ribosomally synthesized antimicrobial peptides produced by bacteria that deserve considerable interest for their use as natural and nontoxic food preservatives as well as for their potential use in human and veterinary applications , and in the animal production field . in 1976 tagg et al . defined bacteriocins as proteinaceous compounds that kill closely related bacteria but it is now recognized that bacteriocins can also have activity across genera ( broad spectrum ) [ 24 ] . the antimicrobial sensitivity of a target strain to any given bacteriocin may depend on the ecological conditions under which it is grown as variations in salt concentration , ph , the presence of membrane disrupting molecules , or inducing cultures , and a large number of other environmental parameters can have a significant impact [ 5 , 6 ] . the classification of gram - positive bacteriocins is rendered difficult because of their heterogeneity and thus , as the number of gram - positive bacteriocins identified has continued to increase , classification schemes have had to continuously evolve . klaenhammer suggested four classes of bacteriocins including class i or lantibiotics , which are small membrane - active peptides ( < 5 kda ) containing the unusual amino acids lanthionine or -methyl lanthionine ( hence the name lantibiotics ) and dehydrated residues . class ii is defined as small heat - stable nonlanthionine containing membrane active peptides characterized by the presence of a gly - gly processing site in the bacteriocins precursor , the presence of amphiphilic helices with varying amounts of hydrophobicity and moderate to high - heat stability . these were further subdivided into three subgroups named subclass iia , which are listeria active peptides and ygngv box in the n - terminal ; subclass iib : poration complexes consisting of two proteinaceous peptides for activity and subclass iic , which contains thiol activated peptides requiring reduced cysteyl residues for activity . class iii is large heat labile proteins , often with enzymatic activity and finally class iv , which is composed of complex proteins . many enterococcus strains from various ecosystems were characterized as antagonists of broad range of foodborne pathogens [ 12 , 13 ] . the bacteriocins produced by enterococcal strains displayed heterogeneous structures and differences in their mode of action . most of bacteriocin producers enterococcal strains were isolated not only from different types of food , but also from broilers [ 2 , 4 ] and animal secretions . as reported in the literature , some of these bacteriocins exhibited high activities against broad spectrum of bacteria including gram - positive bacteria , like staphylococcus and streptococcus and gram - negative bacteria like campylobacter or salmonella , strains of medical interest [ 2 , 4 , 16 ] . the ability to produce such a biocompound may play a role in providing an ecological advantage on nonbacteriocin producer strains . previously , we have isolated a strain of e. faecalis from chicken drop which appeared to be a bacteriocin producer . in this work , we have undertaken the purification and characterization of the mode of action of this peptide named enterocin s37 . e. faecalis s37 was isolated from poultry feces collected in a french farm located in ancenis ( west of france ) . e. faecalis s37 showed in first instance an antagonism directed against strains belonging to listeria and campylobacter species . the antibacterial activity of enterocin s37 was assessed against the gram - negative and gram - positive bacteria listed in table 1 . the cell - free supernatant ( cfs ) and partially purified enterocin s37 , by ammonium sulfate precipitation and two successive reversed phase - high performance liquid chromatography ( rp - hplc ) steps , were used in order to assess the antagonism toward the target strains listed in table 1 . e. faecalis s37 , e. faecalis jh2 - 2 , lactobacillus brevis f1.114 , lb . enterica serotype enteritidis cip 81.3 , staphylococcus epidermidis cip 68.21 , bacillus cereus cip 78.3,and leuconostoc mesenteroides subsp . mesenteroides dsm 20240 were grown on brain heart infusion broth ( bhi ) medium ( aes , bruz france ) , whilst l. monocytogenes egde and l. innocua f were grown on elliker medium ( aes ) . for this purpose , 100 l from overnight culture of targeted strains was added to 20 ml of elliker or bhi soft agar medium already containing 0.8% ( w / v ) of agar ( biokar diagnostics , beauvais , france ) . wells were performed in solid agar and 50 l of each sample tested was poured into the wells . the petri dishes were left at room temperature , in sterile conditions , for 1 h before incubation for 18 h at adequate temperature according to the target strain to be tested . after this period of incubation , the antibacterial activity was detected by observing the inhibition zones around the well containing the cfs . it should be noted that supernatant was obtained by centrifuging at 15000 g during 30 min at 4c , an overnight culture of e. faecalis grown at 37c for 18 to 24 h , on de man rogosa sharpe ( mrs ) medium ( aes ) . the cfs was neutralized by addition of 1 n naoh and filtered through 0.2 m membrane ( sartorius , goettingen , germany ) . serial two fold dilution of samples was performed with phosphate buffer ( 25 mm k2hpo4 , 25 mm kh2po4 , ph 7 ) filtered through 0.2 m membrane ( sartorius ) . these samples were tested by agar diffusion test on l. monocytogenes egde strain , as described previously . one arbitrary unit was defined as the reciprocal of the lowest dilution that did not show growth of the target strain . the cfs neutralized and filtered ( 400 ml ) was precipitated with ammonium sulfate to 80% saturated solution . three distinct fractions were recovered and were resuspended in 40 ml of phosphate buffer ( 25 mm k2hpo4 , 25 mm kh2po4 , ph 7 ) ; the active one was applied onto rp - hplc column ( atoll 15 mp3 ; 250 mm 10 mm interchim , montluon , france ) previously equilibrated with solvent a ( 0.05% , v / v , trifluoroacetic acid [ tfa ] ) . elution was performed by using a gradient from 0% solvent b ( 0.045% , v / v , tfa , 80% acetonitrile ) to 100% solvent b in 45 min . the flow rate was adjusted to 3 ml.min and the absorbance was recorded at 220 and 280 nm the acetonitrile was removed from the active fractions by speed - vac concentrator ( sc110a , savant ) and ph was adjusted to 6.8 using phosphate buffer ( 50 mm ; ph 7 ) . the active fractions were pooled and the purification of the bacteriocin was continued by rp - hplc on a waters alliance apparatus with millennium software ( millford , ma , usa ) . concentrated bacteriocin ( 80 l ) was injected into an analytical c18 column ( symmetry 300 ; 5 m ; spherical 300 ; 150 4.6 mm , waters , guyancourt , france ) equilibrated in 0% solvent c ( 0.045% tfa in h2o ) and 50% solvent d ( 80% acetonitrile , 20% h2o , 0.05% tfa ) . the elution was performed with a linear gradient from 30% to 100% solvent d in 30 min . the eluted peaks were detected by spectrophotometry measuring the absorbance between 210 and 300 nm with a photo diode array detector ( pda 996 ; waters ) and collected manually . the effect of -chymotrypsin ( 1 mg ml ) , papain ( 1 mg ml ) , proteinase k ( 0.5 mg / ml ) , lysozyme ( 2 mg ml ) , and lipase ( 1 mg ml ) ( all enzymes were from sigma - aldrich , germany ) on the antibacterial activity of enterocin s37 was checked by the agar diffusion assay as described by batdorj et al . . thus , samples ( cfs ) of 10 ml were adjusted to ph values ranging from 3 to 10 with 1 n hcl or 1 n naoh . the obtained fractions were incubated for 2 h at 37c . after this period of incubation , the antibacterial activity of each treated sample was determined by the agar well diffusion assay . the stability of enterocin s37 was studied at high temperature by heating 100 l of supernatant at 80c for 15 min , 80c for 1 h and 90c for 15 min . after cooling in ice , each sample was tested for its antagonism by agar well diffusion assay . in order to determine the apparent molecular mass of enterocin s37 the part of the gel containing the molecular markers and the samples were stained by silver nitrate coloration method , and the remaining part , containing only samples , was extensively washed with regularly replaced sterile milliq water . second part of the gel was used for direct detection of antimicrobial activity by overlaying with soft agar ( 0.8% ) seeded with indicator strain l. monocytogenes egde and incubated overnight at 37c . resistant variants to enterocin s37 were developed by stepwise culture of strain of l. monocytogenes egde at 30c in tryptic soy broth ( biokar diagnostics ) supplemented with 0.6% ( w / v ) yeast extract ( organotechnie sa , la courneuve , france ) and called tsye broth ( tsyeb ) . enterocin s37 was added at 2- 4- 6- 8- and 10-fold the minimal inhibitory concentration ( mic ) . the mic was determined for l. monocytogenes egde according to naghmouchi et al . using microplate reader elx808 ( biotek instruments , bad friedrichshall , germany ) . after growth of 50 generations in tsyeb at 30c , the strain was inoculated in the tsyeb containing enterocin s37 at 10-fold mic . l. monocytogenes egde and its bacteriocin - resistant variant were grown at 30c for 24 h in tsyeb in the presence of enterocin s37 at 5 g mland k channel modulators such as pinacidil ( pi ) at 10 g ml , cromakalim ( cro ) at 10 g ml and glipizide ( gli ) at 10 g ml combination of enterocin s37 with each of k channel modulator was also realized in similar conditions . growth was monitored by measuring the optical density at 595 nm ( od595 ) of 250 l of freshly inoculated medium placed into microplate wells . the inhibitory activity ( ia ) of enterocin s37 in presence or absence of each k channel modulator was calculated as described by naghmouchi et al . clearly , a percentage of ia = 100 100 ( od595(x)/od595(i ) ) ( during exponential growth ) . the value ( x ) was the culture containing inhibitor and ( i ) was the uninhibited control culture . data were expressed as the percent change in ia obtained with the enterocin s37/k channel modulator . cfs of e. faecalis s37 displayed activities against e. faecalis jh2 - 2 , lb . brevis f145 , l. monocytogenes egde , and l. innocua f. however , none antibacterial activity was detected against the remaining strains listed in table 1 . surprisingly , the anti - campylobacter activity previously observed was not recovered in this study arguing on instability trait ( table 1 ) . furthermore , the activity observed against strains of enterococcus genus is interesting in order to generate novel knowledge on immunity proteins and cross - resistance within this genus . cfs of e. faecalis s37 displayed activity against the target strains e. faecalis jh2 - 2 , lb . brevis f145 , l. monocytogenes egde and l. innocua f even after treatments with lipase ( 1 mg ml ) and lysozyme ( 2 mg ml ) . the antibacterial activity observed upon lipase and lysozyme treatments was similar to that observed with the untreated supernatant used as positive controls . however , treatment with proteolytic enzymes , such as -chymotrypsin , papain , and proteinase k resulted in the loss of the bacteriocin activity ( table 2 ) . the stability of antibacterial activity of enterocin s37 remained intact after heating treatments ( 80c for 1 h and 90c for 15 min ) and at ph values ranging from 4 to 9 ( table 2 ) . attempts to reduce putative disulfide bridge ( s ) of enterocin s37 were performed with dithiothreitol ( sigma , germany ) ; the experiment was not conclusive because of toxicity of this compound , even at low concentrations , towards the target strains . the bacteriocin was purified by a three - step method including ammonium sulfate precipitation and two rp - hplc . the first stage consisted in an ammonium sulfate precipitation at 80% , which allowed concentrating the active fraction 10-fold ( v / v ) ; subsequently , active fractions ( those exhibiting antibacterial activity ) were eluted at 60% of elution buffer containing 80% acetonitrile , 20% h2o , and 0.05% tfa . the purity of each active fraction collected after each step was checked on sds - page ( table 3 , figure 1 ) . bacteriocin appeared to have a molecular weight comprised between 4 and 6 kda ( figure 1(a ) ) , and fitting thereof with the inhibition zone observed on sds - page ( figure 1(b ) ) . for this purpose , the electrophoresis gel obtained after migration under nondenaturing conditions was plated directly on elliker soft agar ( 0.9% ) inoculated with l. monocytogenes egde with 1% volume transfer ( v / v ) . the antimicrobial activity of each sample , obtained during purification process , was also checked by agar diffusion test on petri dish ( figure 1(c ) ) both k channel activators ( pinacidil and cromakalim ) and inhibitor ( glipizide ) affected differently the growth of l. monocytogenes egde and its bacteriocin - resistant variant to enterocin s37 obtained in this research . as shown in figure 2 , the highest effect was attributed to the combination of enterocin s37 ( 5 g ml ) and pinacidil ( 10 g ml ) . effect of the combinations of enterocin s37 with cromakalim ( 10 g ml ) and glipizide ( 10 g ml ) was less important on l. monocytogenes egde bacteriocin - resistant variant . the predominant habitat is the gastrointestinal tract of animals and humans where they can be found in numbers as high as 10 cfu / g of feces . enterococci were found in foods of animal origin ( milk , cheese , and fermented sausages ) , vegetables and plant materials . many enterococci were reported to produce bacteriocins called enterocins , which are various , having great diversity in their structure , and active against numerous microorganisms , especially foodborne pathogens and against microorganisms of environmental and medical interests [ 2 , 4 , 14 , 23 ] . have studied a set of 139 strains of e. faecium and e. faecalis for their capabilities to produce enterocins and they concluded that 51% of isolates were producers of enterocins and 46% of them were able to produce more than one enterocin . the strains producing enterocins were carrying at least one structural gene coding for the most known enterocins ( enta , entp , entl50ab and cyll ) . other studies pointed out the importance of e. faecalis as sources of enterocins [ 24 , 25 ] . taken together , these studies indicate the distribution and importance of enterococcal strains . it should be pointed out that most of enterocins so far studied exhibited activity towards gram - negative and gram - positive bacteria . we have undertaken isolation and characterization of enterocin named enterocin s37 , which is produced by e. faecalis recently isolated from chicken feces . enterocin s37 appeared to be interesting regarding its activities against the food - borne pathogen l. monocytogenes egde and casually against campylobacter jejuni nctc 11168 ( table 1 ) . however the anti - campylobacter activity remained unstable and less important than that observed for other bacteriocins recently reported in the literature [ 24 ] . the data gathered from this study let us think of the possibility of enterocin s37 to be a class iia bacteriocin , as ( i ) this peptide was anti - listeria , ( ii ) has a molecular weight < 10 kda , ( iii ) was sensitive to proteases , and ( iv ) was stable at different ph and high temperatures . the activity increased several folds ( from 400 au ml to 51,200 au ml ) during the purification process ( table 3 ) , in parallel with an increase of specific activity , which reaches 1,557.17 au g . this phenomenon was largely described previously as the result of removing inhibitory compounds [ 2628 ] . the spectrum of partially purified enterocin s37 was the same to that observed with cfs , but with stronger activities ( data not shown ) . for the first time , the effect of k channel modulators on enterocin activity was revealed in this research . to this aim , we have utilized two k channel activators ( cromakalim and pinacidil ) and one k channel blocker ( glipizide ) . cromakalim was shown to act on atp - sensitive potassium channels and caused membrane hyper polarization , pulls their membrane potential away from the threshold . in the pharmacology sector , the use of cromakalim to treating hypertension was reported . finally , glipizide was described to affect the cell by partially blocking the k channels , reducing k conductance and causing depolarization of the membrane , which leads to ca ions influx through voltage - sensitive ca channels , causing rising of the intracellular concentrations of ca ions [ 31 , 32 ] . furthermore , the impact of cromakalim and glipizide on growth of sensitive and resistant l. monocytogenes strains was studied and resulted to be overall weak . thus , the inhibition of wildtype ( sensitive phenotype ) to cromakalim and glipizide was estimated to 14.29% 1.38 and 17.14% 4.08 while the inhibition of the mutant strain ( resistant phenotype ) was evaluated to 4.46% 3.53 and 8.93% 1.06 . pinacidil showed high - inhibitory activity on the wildtype ( 86.79% 1.78 ) and interestingly not on the mutant strain ( 19.64% 4.02 ) . combinations of enterocin s37 and different k channel modulators suggested an implication of enterocin s37 in a depletion of intracellular k level , by an action on katp channels rather than kv channels . these data are in good agreement with those obtained formerly with nisin a , whose antibacterial activity was connected to katp channels with concomitant loss of all intracellular k and atp . as for enterocin as-48 and enterocin p [ 33 , 34 ] , enterocin s37 is certainly responsible of uncontrolled efflux of intracellular k and affecting sensitive cells growth . the resistant variant was able to grow but was slightly affected by the combination of enterocin s37/k modulators , except for pinacidil , where the inhibitory effect has reached 49.79% 3.36 , instead of 23.84% 7.73 and 24.11% 7.07 when cromakalim and glipizide were added , respectively ( figure 2 ) . it was also well established that many bacteriocins , including enterocins , interact with membrane lipids leading to the formation of pores and subsequently to the loss of intracellular components , resulting thereof in the cell death [ 3539 ] . the lipid composition of membrane appeared to play an important role in the efficiency of bacteriocin activity [ 9 , 35 , 36 , 38 ] . enterocin s37 alone did not affect the growth of bacteriocin - resistant variant of l. monocytogenes egde . however , the combination of enterocin s37/k channel modulators impacted strongly the growth of resistant variant . otherwise , the k channel modulators enhanced the inhibitory activity of enterocin s37 on sensitive strain of l. monocytogenes egde , with values reaching , 92.86% 5.90 and 94.29% 4.55 when enterocin s37 was combined with glipizide and pinacidil , respectively . combination of cromakalim and enterocin s37 resulted in 84.29% 2.5 inhibition of the growth of sensitive strain ; this data is equal to that obtained with enterocin s37 alone 84.21% 5.87 ( figure 2 ) . however , in the case of resistant strain , when enterocin s37 was combined to pinacidil , a high - inhibitory activity ( 49.79% 3.36 ) was observed as compared to combinations of cromakalim + enterocin s37 ( 23.84% 7.73 ) and enterocin s37 + glipizide ( 24.11% 7.07 ) ( figure 2 ) . the mode of action of enterocin s37 could be triggered by potential interaction with cell membrane .

the aim of this research was to purify and characterize the mode of action of enterocin s37 , a bacteriocin produced by enterococcus faecalis s37 , a strain recently isolated from the chicken feces . enterocin s37 has a molecular weight comprised between 4 and 5 kda . it remained active after 1 h at 80oc and at ph values ranging from 4.0 to 9.0 . furthermore , cell - free supernatant of enterococcus faecalis s37 and purified enterocin s37 were active against gram - positive bacteria including listeria monocytogenes egde , l. innocua f , enterococcus faecalis jh2 - 2 , and lactobacillus brevis f145 . the purification of enterocin s37 was performed by ammonium sulfate precipitation followed up by hydrophobic - interaction chromatography procedures . treatment of enterocin s37 with proteinase k , -chymotrypsin , and papain confirmed its proteinaceous nature , while its treatment with lysozyme and lipase resulted in no alteration of activity . enterocin s37 is hydrophobic , anti - listeria and likely acting by depletion of intracellular k+ ions upon action on katp channels . this study contributed to gain more insights into the mode of action of enterocins .

pubmed

these trials were conducted in compliance with the declaration of helsinki , the international conference on harmonisation good clinical practice guidelines , the european union clinical trials directive , and local regulatory requirements . approval for the study protocols and all amendments were obtained from columbia university medical center institutional review board ( approval # aaai6758 and # aaak5458 ) . written informed consent and assent ( if applicable ) were obtained before any evaluations were conducted for eligibility . the phase 1 study was a first - in - human , open - label , escalating - dose study to assess the safety , tolerability , and pharmacokinetics of a single intrathecal dose ( 1 , 3 , 6 , or 9 mg ) of nusinersen in children with spinal muscular atrophy . each dose cohort had 6 to 10 participants ( n = 28 ) . upon completion , all participants had the opportunity to enroll in a subsequent extension study and receive additional dosing with nusinersen . the methods and results of the phase 1 study and its extension are detailed elsewhere . briefly , medically stable spinal muscular atrophy participants 2 to 14 years of age were enrolled in 4 sites in the united states ( boston children s hospital , boston , ma ; columbia university medical center , new york , ny ; ut southwestern medical center children s medical center dallas , dallas , tx ; and university of utah school of medicine , salt lake city , ut ) . eligible participants had to be able to complete all study procedures , meet age - appropriate institutional guidelines for lumbar puncture procedures , and have a life expectancy of > 2 years . participants were excluded for serious respiratory insufficiency , hospitalization for surgery or pulmonary event within the past 2 months , active infection at screening , history of brain or spinal cord disease or bacterial meningitis , presence of implanted cerebrospinal fluid drainage shunt , clinically significant laboratory abnormalities , any ongoing medical condition that would interfere with the conduct and assessments of the study , or treatment with another investigational drug within 1 month of screening . patients with scoliosis were allowed to participate if , in the opinion of the investigator , a lumbar puncture could be performed safely . a total of 3 lumbar punctures were scheduled during the 2 trials for drug delivery and/or follow - up collection of cerebrospinal fluid for safety and pharmacokinetic analyses . drug was administered via intrathecal injection of a 5-ml bolus over 1 to 3 minutes . the protocol recommended a 22- to 25-gauge spinal anesthesia needle ( 21-gauge needle allowed if participant s weight or condition dictated ) and that the lumbar punctures were performed at the l3-l4 disc space or 1 level above or 1 to 2 levels below , as needed . in all cases , anesthesia and/or sedation and fluoroscopy or ultrasonography were permitted to facilitate the procedure and varied by institution at the discretion of the investigators at each site . participants underwent the first lumbar puncture on day 1 for cerebrospinal fluid collection and nusinersen dosing , the second lumbar puncture on day 8 or day 29 for cerebrospinal fluid collection , and the third lumbar puncture during the extension study for cerebrospinal fluid collection and redosing with nusinersen 9 to 14 months after the initial lumbar puncture . in the phase 1 single - dose study , participants were initially monitored for safety and tolerability for 29 days ( 1-mg and 3-mg cohorts ) or 85 days ( 6-mg and 9-mg cohorts ) post dosing . safety reporting included adverse events related to lumbar puncture , and a subgroup analysis was performed to compare reported lumbar puncture related adverse events by needle size , participant age , and spinal muscular atrophy type . these trials were conducted in compliance with the declaration of helsinki , the international conference on harmonisation good clinical practice guidelines , the european union clinical trials directive , and local regulatory requirements . approval for the study protocols and all amendments were obtained from columbia university medical center institutional review board ( approval # aaai6758 and # aaak5458 ) . written informed consent and assent ( if applicable ) were obtained before any evaluations were conducted for eligibility . the phase 1 study was a first - in - human , open - label , escalating - dose study to assess the safety , tolerability , and pharmacokinetics of a single intrathecal dose ( 1 , 3 , 6 , or 9 mg ) of nusinersen in children with spinal muscular atrophy . each dose cohort had 6 to 10 participants ( n = 28 ) . upon completion , all participants had the opportunity to enroll in a subsequent extension study and receive additional dosing with nusinersen . the methods and results of the phase 1 study and its extension are detailed elsewhere . briefly , medically stable spinal muscular atrophy participants 2 to 14 years of age were enrolled in 4 sites in the united states ( boston children s hospital , boston , ma ; columbia university medical center , new york , ny ; ut southwestern medical center children s medical center dallas , dallas , tx ; and university of utah school of medicine , salt lake city , ut ) . eligible participants had to be able to complete all study procedures , meet age - appropriate institutional guidelines for lumbar puncture procedures , and have a life expectancy of > 2 years . participants were excluded for serious respiratory insufficiency , hospitalization for surgery or pulmonary event within the past 2 months , active infection at screening , history of brain or spinal cord disease or bacterial meningitis , presence of implanted cerebrospinal fluid drainage shunt , clinically significant laboratory abnormalities , any ongoing medical condition that would interfere with the conduct and assessments of the study , or treatment with another investigational drug within 1 month of screening . patients with scoliosis were allowed to participate if , in the opinion of the investigator , a lumbar puncture could be performed safely . a total of 3 lumbar punctures were scheduled during the 2 trials for drug delivery and/or follow - up collection of cerebrospinal fluid for safety and pharmacokinetic analyses . drug was administered via intrathecal injection of a 5-ml bolus over 1 to 3 minutes . the protocol recommended a 22- to 25-gauge spinal anesthesia needle ( 21-gauge needle allowed if participant s weight or condition dictated ) and that the lumbar punctures were performed at the l3-l4 disc space or 1 level above or 1 to 2 levels below , as needed . in all cases , anesthesia and/or sedation and fluoroscopy or ultrasonography were permitted to facilitate the procedure and varied by institution at the discretion of the investigators at each site . participants underwent the first lumbar puncture on day 1 for cerebrospinal fluid collection and nusinersen dosing , the second lumbar puncture on day 8 or day 29 for cerebrospinal fluid collection , and the third lumbar puncture during the extension study for cerebrospinal fluid collection and redosing with nusinersen 9 to 14 months after the initial lumbar puncture . in the phase 1 single - dose study , participants were initially monitored for safety and tolerability for 29 days ( 1-mg and 3-mg cohorts ) or 85 days ( 6-mg and 9-mg cohorts ) post dosing . safety reporting included adverse events related to lumbar puncture , and a subgroup analysis was performed to compare reported lumbar puncture related adverse events by needle size , participant age , and spinal muscular atrophy type . a total of 28 children were enrolled and received dosing in the phase 1 study ; 15 children had spinal muscular atrophy type 2 and 13 had spinal muscular atrophy type 3 ( table 1 ) . at baseline , participant mean ( range ) age was 6.1 ( 2.0 - 14.0 ) years , 17 were female , 10 were ambulatory , and 13 had scoliosis . one child had vertical expandable prosthetic titanium rods inserted and the pedicle screws were inserted into t2 , t3 , and t4 with fixation of the growing rods to the pelvis , leaving the lumbar spine area spared . the first and second lumbar punctures were performed on 28 and 27 children in the parent study , respectively , and the third on 18 children who re - enrolled in the extension study . of the 10 children not re - enrolling , 6 re - enrolled in a multiple - dose study of nusinersen and 4 decided not to enroll . of these , 1 child did not re - enroll for reasons related to the lumbar puncture procedure . this was a 12-year - old girl with spinal muscular atrophy type 2 who had severe scoliosis at baseline and in whom the second fluoroscopically guided lumbar puncture procedure was not performed successfully at day 8 in the phase 1 study . participants were enrolled and treated in 4 sites ; the institutional lumbar puncture procedures used in each site are reported in table 2 . both cutting - tip and atraumatic ( pencil - point ) spinal needles were used and the general practice was to perform the procedure in the lateral decubitus or prone position by pediatric anesthesiologists , neurologists , or neuroradiologists in either inpatient or outpatient settings . all sites performed the procedure under intravenous ( midazolam , ketamine , fentanyl , remifentanil , and/or propofol ) or inhaled anesthesia / sedation ( sevoflurane and/or nitrous oxide ) . some sites also reported the use of topical anesthesia ( emla cream ) or locally injected lidocaine ( table 2 ) . 21 , 22 , 25 , or 27 gauge whitacre 3.0-inch spinal needle whitacre spinal needles sprotte 35-mm 24-gauge spinal needle quincke 25-mm 22-gauge spinal needle quincke 1.5- to 3-in . spinal needle inhalational anesthesia : sevoflurane nitrous oxide iv sedation : remifentanil , midazolam , propofol , fentanyl inhalational anesthesia : sevoflurane nitrous oxide iv sedation : midazolam ketamine inhalational anesthesia : sevoflurane and/or nitrous oxide iv sedation : propofol inhalational anesthesia : sevoflurane and nitrous oxide iv sedation : propofol neuroradiologist ( lumbar puncture ) neurologist ( drug administration ) pediatric anesthesiologist ( anesthesia / sedation ) neurologist ( lumbar puncture / drug administration ) anesthesiologist , nurse practitioner , or sedation nurses ( anesthesia / sedation ) pediatric anesthesiologist ( entire procedure ) pediatric anesthesiologist ( entire procedure ) prone position on the angiography table left lateral decubitus position or supporting sitting lateral decubitus position left lateral position interventional diagnostic suite ( part of the department of radiology ) rapid treatment unit or outpatient clinic suite within the hospital setting ecg , pulse oximetry , non - invasive blood pressure , end - tidal co2 , temperature ecg , oxygen saturation , and expired co2 oxygen saturation and heart rate monitoring ecg , pulse oximetry , oscillometric blood pressure , and end - tidal co2 ventilation was spontaneous or assisted ecg , pulse oximetry , blood pressure , temperature , and end - tidal co2 abbreviations : ecg , electrocardiogram ; iv , intravenous . n s indicate the number of lumbar punctures performed at each site ( total 73 ) . fluoroscopy was used routinely per institutional practice . only in some participants . in participants with severe scoliosis only . for children receiving lumbar puncture . for children receiving iv sedation the majority of lumbar punctures were carried out using either a 22- ( 48% ) or 25-gauge ( 37% ) needle inserted in the l3-l4 ( 44% ) or l4-l5 ( 29% ) space . nearly half ( 44% ) of the lumbar punctures were guided using fluoroscopy ( table 3 ) . participants were treated for post lumbar puncture headache with acetaminophen , ibuprofen , and/or caffeine citrate . of the 73 lumbar punctures performed , the majority ( n = 50 ; 68% ) had no complications ; lumbar puncture related adverse events were reported in 23 ( 32% ; table 4 ) . the most common adverse events were headache ( 9 events ) , back pain ( 9 events ) , and post lumbar puncture syndrome ( 8 events ; post dural puncture headache with or without vomiting ) , and all events resolved without long - term complications . the timing of the adverse events varied ; 67% ( n = 6 ) of the headaches occurred 12 to 72 hours post lumbar puncture , whereas back pain was reported 0 to 48 hours post procedure ( figure 1 ) . fifty percent ( n = 4 ) of the post lumbar puncture syndrome events occurred between 12 and 48 hours , with the remaining occurring after 72 hours ( figure 1 ) . all 8 incidences of post lumbar puncture syndrome were managed with acetaminophen , ibuprofen , and/or caffeine citrate for headache . all 8 cases of post lumbar puncture syndrome resolved with conservative therapy , and none of the 7 participants needed an epidural blood patch . in 1 case , a blood patch was performed prophylactically during the second lumbar puncture procedure in a patient who had experienced a post dural puncture headache following the first procedure . resolution of the post lumbar puncture syndrome occurred between 4 hours and 5 days , with the majority of the events ( 50% of cases ) lasting 1 to 2 days . participants reporting > 1 adverse event after each lumbar puncture were counted only once for the lumbar puncture . time of onset of headache , back pain , and post lumbar puncture syndrome . most common lumbar puncture associated adverse events ( n = 73 ) and their time of onset are shown . a subgroup analysis compared lumbar puncture complications with needle size , participant age , and spinal muscular atrophy type and demonstrated that headache , back pain , and post lumbar puncture syndrome were observed more frequently when a 21- or 22-gauge needle was used , in older children ( 8 - 14 years of age ) , and in children with spinal muscular atrophy type 3 ( table 5 ) . lumbar puncture related adverse events are well documented in children and infants , with the most common complications being back pain and headache with an incidence of 11% to 40% and 12% to 33% , respectively . although the incidence of post lumbar puncture syndrome in children is not well reported , 4% to 11% of the reported headaches are classified as post dural puncture headaches . in addition to headache , post lumbar puncture syndrome can also include transient effects of backache , dizziness , nausea with or without vomiting , numbness , and lower extremity weakness . in rare cases , more severe symptoms have been reported , such as intracranial hypotension , epidural hematoma , and cauda equina syndrome . in this phase 1 study and its extension , 73 lumbar punctures were performed for cerebrospinal fluid collection and/or intrathecal drug administration in children with spinal muscular atrophy . approximately one - third of the procedures were associated with adverse events , most commonly with headache , back pain , and post lumbar puncture syndrome , and the majority occurred within 72 hours after the procedure . a subgroup analysis demonstrated that the highest incidence of adverse events was reported in older children , children with spinal muscular atrophy type 3 , and when a larger 21- or 22-gauge needle was used . using a 24-gauge needle or smaller atraumatic needle inserted with the bevel parallel to the dura fibers had been suggested to considerably reduce damage to the dura and consequently decrease the risk for cerebrospinal fluid leak after lumbar puncture , including in children . needle type ( atraumatic or traumatic ) may have a greater impact on the reported incidence of post dural puncture headache than needle size . turnbull et al found the incidence of post dural puncture headache varied from 0.6% to 4% ( 22 gauge ) and 0% to 14.5% ( 25 gauge ) with an atraumatic ( whitacre ) needle versus 36% ( 22 gauge ) and 3% to 25% ( 25-gauge ) with a traumatic cutting - tip ( quincke ) needle , respectively . although using an atraumatic needle may reduce the incidence of post lumbar puncture syndrome , some studies have identified increased failure rate and paresthesia when using an atraumatic needle versus a cutting - tip spinal needle . nearly half of the lumbar punctures were successfully carried out in children with spinal muscular atrophy using a 24-gauge spinal needle or smaller and , consequently , were associated with reduced incidence of headache and post lumbar puncture syndrome compared with procedures performed using a 21- or 22-gauge needle . after initial experiences , all investigational sites switched to using a smaller needle size , except when use of a larger needle size was required ( eg , scoliosis ) . the higher incidence of adverse events , particularly headaches , observed among older children and/or in children with spinal muscular atrophy type 3 were likely related to the use of larger bore / gauge spinal needles , cutting - tip needles ( eg , quincke type ) , multiple attempts , and/or due to technical difficulties resulting from increased body weight and the presence of scoliosis or excessive lumbar lordosis . in most cases , post dural puncture headache can be successfully treated with conservative therapy , consisting of bed rest in prone or lateral position , hydration , oral or intravenous caffeine , anti - nausea or antiemetic therapy , and/or analgesics . however , in children with post dural puncture headaches persisting > 48 hours or that worsen despite the use of conservative therapy , a therapeutic epidural blood patch may be indicated . although there is conflicting evidence , some studies also support the use of a prophylactic blood patch to prevent post dural puncture headache ; however , none of these studies included children . in this study , all post dural puncture headaches resolved with conservative treatment of bed rest , adequate hydration , and administration of caffeine and other analgesics , and therapeutic epidural blood patches proved unnecessary . other considerations that may facilitate lumbar puncture success in children include patient positioning and the use of spinal ultrasound . few studies in children and infants have compared the feasibility of lumbar puncture in upright versus lateral recumbent position . both the seated and lateral decubitus position with hip flexion increased the interspinous space compared with hip neutral positions and may increase lumbar puncture success rate . the use of spinal ultrasound may facilitate the lumbar puncture procedure , and is favored over fluoroscopy in children , particularly if repeated procedures are planned , to minimize radiation exposure and cost . in this study , lumbar punctures were performed in the lateral decubitus or prone position , and nearly half of the lumbar punctures were successfully done without ultrasound and/or fluoroscopy . however , in patients with spinal muscular atrophy with scoliosis , spinal rods , or other hardware , the use of imaging may be warranted to facilitate the procedure and increase the success of intrathecal medication delivery . lumbar punctures were successfully performed in the participants with scoliosis ( 13 in the first procedure , 12 in the second procedure , and 11 in the third procedure ) and in the one child who had spinal rods . the second fluoroscopy - guided procedure was unsuccessful in only 1 patient because of scoliosis . this suggests that in some cases scoliosis might hinder lumbar puncture success in children with spinal muscular atrophy , but the frequency needs to be confirmed in larger studies . general anesthesia and/or sedation are routinely performed in children to reduce procedure - related anxiety , pain , and distress , and to increase the overall success rate of lumbar puncture . however , increasing concerns about the impact of repeated anesthesia exposure on the developing nervous system in infants and young children must be carefully considered when repeated procedures are planned . all 73 lumbar punctures performed in this study were performed using either intravenous ( midazolam , ketamine , fentanyl , remifentanil , and/or propofol ) or inhaled anesthesia / sedation ( sevoflurane , nitrous oxide ) , with no associated complications reported . children with spinal muscular atrophy who have moderate to severe muscle weakness and/or severe scoliosis are at higher risk for hypoventilation and respiratory compromise . thus , if deep sedation or general anesthesia is used , assisted ventilation may be required by either non - invasive or invasive means . the use of local topical or subcutaneous anesthesia may help decrease the requirements for sedation . based on our experience in this study , we recommend the most minimal use of sedative medications or anesthesia to permit safe and effective completion of the procedure . the limitations of this study include a small sample size and the limited number of lumbar puncture procedures performed . further experiences with lumbar puncture in children and infants with spinal muscular atrophy are needed to add to this knowledge . in summary , repeated lumbar punctures were successfully performed in children with spinal muscular atrophy in the initial nusinersen clinical studies . the frequency of lumbar puncture related adverse events in children with spinal muscular atrophy was similar to that previously reported in children and infants , and were mainly limited to headache , back pain , and post lumbar puncture syndrome . a 24-gauge needle or smaller was successfully used in children with spinal muscular atrophy to perform lumbar puncture with lower incidence of complications , suggesting that using a 24-gauge or smaller needle would likely reduce the chance of adverse events . bed rest , adequate hydration , oral caffeine , and/or analgesics were sufficient to resolve post lumbar puncture headache / syndrome without the need of a therapeutic blood patch in all patients . provisions for ultrasound guidance may be warranted in future research protocols given the potential benefit of reduced radiation exposure compared to fluoroscopy , especially in cases of serial procedures for repeated drug delivery . overall , we conclude that intrathecal delivery of medication is feasible , safe , and well tolerated . our experience may prove useful for guiding the development of best practice strategies for safe and effective intrathecal delivery of nusinersen and/or other promising emerging therapies for spinal muscular atrophy .

nusinersen ( isis - smnrx or isis 396443 ) is an antisense oligonucleotide drug administered intrathecally to treat spinal muscular atrophy . we summarize lumbar puncture experience in children with spinal muscular atrophy during a phase 1 open - label study of nusinersen and its extension . during the studies , 73 lumbar punctures were performed in 28 patients 2 to 14 years of age with type 2/3 spinal muscular atrophy . no complications occurred in 50 ( 68% ) lumbar punctures ; in 23 ( 32% ) procedures , adverse events were attributed to lumbar puncture . most common adverse events were headache ( n = 9 ) , back pain ( n = 9 ) , and post lumbar puncture syndrome ( n = 8) . in a subgroup analysis , adverse events were more frequent in older children , children with type 3 spinal muscular atrophy , and with a 21- or 22-gauge needle compared to a 24-gauge needle or smaller . lumbar punctures were successfully performed in children with spinal muscular atrophy ; lumbar puncture related adverse event frequency was similar to that previously reported in children .

pubmed

the main concern is to ensure the highest possible standard for the services provided and to meet the needs of individual service users and communities ( 1 ) . in 1997 , the uk department of health introduced clinical governance ( cg ) as a strategy for improving quality of health care services ( 2 ) . the classic definition of cg is provided by scally and donaldson as a system through which [ health ] organizations are accountable for continuously improving the quality of their services and safeguarding high standards of care by creating an environment in which excellence in clinical care will flourish ( 2 , 3 ) . the iranian ministry of health and medical education ( mohme ) has applied cg as a framework for improving quality and safety in all hospitals since 2009 . mohme used the definition cited above as a guide to implement the policy . the cg model developed in iran consists of seven interlocking components including : clinical effectiveness , clinical audit , risk management , patient and public involvement , education and training , staff and staff management , and use of information ( 4 ) . systems awareness , leadership , ownership , teamwork , and communication are considered as a foundation of this model . mohme required curative deputy of medical universities and hospital managers to work together to implement such initiatives in iranian hospitals . the deputies for curative affairs in each medical university have the role of leadership in planning , implementing , monitoring and following up ministry of health policies particularly in quality improvement programs including cg ( 5 ) . a number of studies have assessed the implementation of cg in different health systems and health care settings ( 611 ) . insufficient knowledge and attitude toward cg , lack of resources , inadequate information technology systems , resistance to change , necessity of cultural change and professional boundaries were the main factors explored by lathman et al . another study using qualitative research identified some barriers such as speed of cg implementation , workload and earmarked funding in cg implementation ( 7 ) . ( 2002 ) considered lack of adequate senior management support as well as resources , structural and cultural issues as obstacles ( 8) . the scarcity of resources was one of the most important barriers in implementation of cg noted by walsh et al . a number of factors were identified which ultimately could affect the success of quality improvement activities . raising awareness of cg among managers , supportive culture and sufficient resources declared that state level accountability in clinical governance implementation could be addressed by allocating proper resources and empowering policy implementers with proper performance control system ( 11 ) . previous literatures mainly provide inadequate understanding about senior managers viewpoint toward facilitators and barriers in implementation of cg . in the current study an effort was done to capture both facilitators and barriers in cg implementation from the viewpoint of curative deputies in iranian medical universities . to obtain a comprehensive understanding about senior managers viewpoint toward cg barriers and facilitators , a qualitative research was employed . to do so , two main information sources were used : face to face interviews and relevant document reviews . deputies for curative affairs of all types of iranian medical universities were purposefully selected to maximize the sample diversity and provide a comprehensive view toward cg implementation . the sampling continued until reaching data saturation . finally , forty three deputies were interviewed . in the first step , an interview topic guide was developed on the basis of findings of literature review and expert opinions ( table 1 ) . it covered the concept of clinical governance , key factors relating to clinical governance implementation process , facilitators and barriers that hospitals were experiencing . some relevant documents were also analyzed such as cg annual reports , audit reports and minutes of meetings . the qualitative thematic framework analysis was used to analyze the data with the assistance of the atlas - ti , qualitative data analysis software . data analysis process includes five stages : familiarization , developing a thematic framework , indexing , charting , and mapping and interpretation ( 12 ) . to increase the validity , it helped to ensure that the findings were congruent with participants perceptions , beliefs and opinions ( 13 ) . the five main themes were explored and presented according with their sub - themes in ( table 2 ) . most senior managers accepted that improving quality of health care should be integral to their role and essential to safeguard patient care . they believed that quality improvement is a strategic goal , to achieve central government targets . the findings demonstrated the willingness to support and positive attitude towards clinical governance .. senior managers also declared that having adequate knowledge about clinical governance also positive attitude toward the necessity of the program are the main factors affecting clinical governance implementation . the level of enthusiasm expressed by senior managers seemed to be related in particular to their knowledge and attitude about cg concept and components . those managers who had gained such knowledge were more optimistic toward success of the program . continuous training about clinical governance concept and principles can make a positive attitude toward the program . main themes of senior managers viewpoint toward cg barriers and facilitators it was stated that such quality improvement program may facilitates the development of a culture focusing on continuous improvement . most interviewees declared that appropriate culture for improving quality in the organization ; team work and readiness toward change are the main factors which influence cg implementation . culture of openness in which staffs are willing to bring ideas related to service quality development was the other important factor mentioned by participants . it was believed that culture which promotes alignment of clinical governance goals at both managerial and staff level should be developed . they also stated that clinical governance components needed to be embedded in day to day work . at first , we should culturalise cg in our work place . interviewees believed that adequate organizational commitment toward clinical governance should be created in order to increase the likelihood of program progress . when continuous meetings are hold in high managerial levels of medical university about reporting our progress in implementing cg , it makes me sure that there is an organizational commitment . effective organizational interactions both within and between departments was the other organizational key factor highlighted by senior managers . they emphasized that clinical governance activities would be more effective if all departments in organization accept the importance of cg and participate in the implementation process . everywhere in our organization you can see something about cg definition , process , implementation and so on . they stated that most of staff working in this program does not have official position in the organizational chart which negatively affects their work . they suggested that creation of such position can guarantee stability and legitimacy of staff in their workplace and have positive effect . senior managers accepted that allocating adequate staff to undertake the responsibilities required by program had a critical role in pushing forward the program . the other important factor recognized as an organizational factor was the necessity for development and dissemination of national standards . interviewees believe that establishment of such standards and presence of guidelines is prerequisites of such huge quality improvement program . in addition , it can help to conduct evaluation against determined standards which shows gaps in delivery of services . one of the factors was managerial commitment toward clinical governance and their executive ability in implementing the program . most of senior managers mentioned themselves ultimately accountable for clinical activities and quality of care in their organization . they also believed that successful outcome of quality activities depends on managers participation in quality procedures and the level of their commitment . interviewees added that senior managers themselves should demonstrate executive capabilities in order to motivate departments to implement clinical governance principles , provide adequate resources , facilitate staff training and remove any obstacle in the way . i feel positive about the future of clinical governance because of my involvement with the program . furthermore , managers should use appropriate incentive mechanisms to sustain staff motivation and participation . the matter is that how top managers will motivate us in implementing quality programs besides doing our regular organizational task . interviewees highlighted that stability in managerial and executive positions is crucial to maintain the consistency and continuity of the program . such unnecessary managerial position changes not only waste money and reduce the program progress , but also , ruin the managers motivation . major changes in top managerial positions threaten the stability of cg and other quality improvement programs . most of the senior managers agreed that shortage of staff and limited dedicated resources to implement clinical governance were main barriers in implementing clinical governance . they believed that such barriers leave many managers feel beleaguered and faced with problems to effectively perform the program . i think a major challenge in implementation of cg is lack of resources especially staff and money . senior managers mostly emphasized that fostering a sense of engagement among medical staff especially physicians is important . one of the reasons for resistance was that the staff is overwhelmed by high workload and different responsibilities . they are seeing the program as being imposed and as policing their performance , rather than supporting quality improvement . there is somehow resistance to clinical governance among some physicians and medical staff which i think it is because they are overwhelmed with the responsibilities they have regarding to care giving . some managers had a concern that this program might be temporary and act as a wave . in addition some issues such as lack of support from physicians and medical staff , legal challenges , parallel quality improvement models running in the hospitals , increased workload , parallel functions in different domains of curative deputy and inadequate supporting systems developed for the staff in the way of clinical governance implementation were mentioned as barriers . much of work i perform on clinical governance is done on my own limited time competing with other activities on my time . i have lots of responsibilities to undertake and this encounters me with lack of time . some activities were also addressed by senior managers in order to mitigate problems in five afore - mentioned domains . most senior managers accepted that improving quality of health care should be integral to their role and essential to safeguard patient care . they believed that quality improvement is a strategic goal , to achieve central government targets . the findings demonstrated the willingness to support and positive attitude towards clinical governance .. senior managers also declared that having adequate knowledge about clinical governance also positive attitude toward the necessity of the program are the main factors affecting clinical governance implementation . the level of enthusiasm expressed by senior managers seemed to be related in particular to their knowledge and attitude about cg concept and components . those managers who had gained such knowledge were more optimistic toward success of the program . continuous training about clinical governance concept and principles can make a positive attitude toward the program . it was stated that such quality improvement program may facilitates the development of a culture focusing on continuous improvement . most interviewees declared that appropriate culture for improving quality in the organization ; team work and readiness toward change are the main factors which influence cg implementation . culture of openness in which staffs are willing to bring ideas related to service quality development was the other important factor mentioned by participants . it was believed that culture which promotes alignment of clinical governance goals at both managerial and staff level should be developed . they also stated that clinical governance components needed to be embedded in day to day work . at first , we should culturalise cg in our work place . interviewees believed that adequate organizational commitment toward clinical governance should be created in order to increase the likelihood of program progress . when continuous meetings are hold in high managerial levels of medical university about reporting our progress in implementing cg , it makes me sure that there is an organizational commitment . effective organizational interactions both within and between departments was the other organizational key factor highlighted by senior managers . they emphasized that clinical governance activities would be more effective if all departments in organization accept the importance of cg and participate in the implementation process . everywhere in our organization you can see something about cg definition , process , implementation and so on . they stated that most of staff working in this program does not have official position in the organizational chart which negatively affects their work . they suggested that creation of such position can guarantee stability and legitimacy of staff in their workplace and have positive effect . senior managers accepted that allocating adequate staff to undertake the responsibilities required by program had a critical role in pushing forward the program . the other important factor recognized as an organizational factor was the necessity for development and dissemination of national standards . interviewees believe that establishment of such standards and presence of guidelines is prerequisites of such huge quality improvement program . in addition , it can help to conduct evaluation against determined standards which shows gaps in delivery of services . one of the factors was managerial commitment toward clinical governance and their executive ability in implementing the program . most of senior managers mentioned themselves ultimately accountable for clinical activities and quality of care in their organization . they also believed that successful outcome of quality activities depends on managers participation in quality procedures and the level of their commitment . interviewees added that senior managers themselves should demonstrate executive capabilities in order to motivate departments to implement clinical governance principles , provide adequate resources , facilitate staff training and remove any obstacle in the way . i feel positive about the future of clinical governance because of my involvement with the program . furthermore , managers should use appropriate incentive mechanisms to sustain staff motivation and participation . the matter is that how top managers will motivate us in implementing quality programs besides doing our regular organizational task . interviewees highlighted that stability in managerial and executive positions is crucial to maintain the consistency and continuity of the program . such unnecessary managerial position changes not only waste money and reduce the program progress , but also , ruin the managers motivation . major changes in top managerial positions threaten the stability of cg and other quality improvement programs . most of the senior managers agreed that shortage of staff and limited dedicated resources to implement clinical governance were main barriers in implementing clinical governance . they believed that such barriers leave many managers feel beleaguered and faced with problems to effectively perform the program . i think a major challenge in implementation of cg is lack of resources especially staff and money . senior managers mostly emphasized that fostering a sense of engagement among medical staff especially physicians is important . one of the reasons for resistance was that the staff is overwhelmed by high workload and different responsibilities . they are seeing the program as being imposed and as policing their performance , rather than supporting quality improvement . there is somehow resistance to clinical governance among some physicians and medical staff which i think it is because they are overwhelmed with the responsibilities they have regarding to care giving . some managers had a concern that this program might be temporary and act as a wave . in addition some issues such as lack of support from physicians and medical staff , legal challenges , parallel quality improvement models running in the hospitals , increased workload , parallel functions in different domains of curative deputy and inadequate supporting systems developed for the staff in the way of clinical governance implementation were mentioned as barriers . much of work i perform on clinical governance is done on my own limited time competing with other activities on my time . i have lots of responsibilities to undertake and this encounters me with lack of time . some activities were also addressed by senior managers in order to mitigate problems in five afore - mentioned domains . in the present study , we tried to explore senior managers viewpoint about the facilitators and barriers in cg implementation . we found that sufficient knowledge and clear understanding about the principles and practice of cg have major roles in achieving desired improvement in service quality and patient safety in health care settings . this study has also highlighted the importance of supporting culture , appropriate organizational structure and managerial commitment as perceived facilitators in cg implementation . there was also a strongly accepted view that staff at all levels should be consulted , involved in planning and implementation of cg programs . the main identified obstacles were lack of adequate managerial support as well as resource , structural and cultural issues and professional boundaries . our results are parallel with the findings of many other studies . a model developed by o brien et al the model outlines four dimensions in successfully implementation of clinical governance : cultural , technical , structural and strategic . the cultural dimension related to beliefs , values , norms and behaviors in the organization which either suppress or support quality improvement activities . an organization with strong and clear vision and goals , stable managerial leadership , supportive structures for team work , effective interactions and inter professional relationships and continuous learning culture is more likely to successfully implement clinical governance ( 14 ) . in our study it is clear that senior managers perception about the important factors in implementing clinical governance were classified in five main domains . the domains were knowledge and attitude , culture , organizational factors , managerial factors and barriers . most of the senior managers believed in knowledge and attitude toward clinical governance and culture as the most important factors in cg implementation . this was thought to be a major factor in achieving improvement in service quality and patient safety mentioning in another study . similar to o brien study , our findings showed that culture is also an important factor and many organizations expend much effort to shape their culture in a way to improve quality . a sense of ownership toward quality improvement and a positive attitude to contribute new ideas also provide an organizational climate necessary to allow alignment of attitudes and values with a continuous quality improvement . in hogan study about consultants attitudes to clinical governance , quality improvement was considered as an integral part of consultants role and they accepted that maintaining service standards , monitoring and improving outcomes for patients were activities they should undertake . there was also recognition about the importance of team based approaches to quality improvement ( 15 ) . this supports the findings of our study which focuses on the importance of being involved in quality improvement activities by all staff especially physicians and medical staff . these include structures and processes with clear vision and goals toward quality improvement , involving staff in the process of change , rewarding positive behaviors , improving the effectiveness of communication across the organization and providing opportunity for team work . campbell study on the role of cg as a strategy for quality improvement in primary care found significant barriers in the way of cg implementation . these included in appropriate culture , too few staff , limited resources , disengagement by some practices and staff , lack of time to perform quality activities ( 8) . our study supported the above findings and declared that some senior managers felt powerless with the volume of work and shortage of resources . meaningful engagement and commitment at all levels of managers and staff has been highlighted as a major factor in implementing cg . the managerial level needed to match its commitment to a program of change with realistic timetables to secure the cultural and organizational changes needed to improve quality of care . the need for top management support is the most frequently cited imperative for success of any program . wilkinson and witcher in an examination of factors important in successfully implementation of clinical governance stressed on the importance of quality committed senior managers and staff effectively involved in all levels of organization ( 16 ) . fenton o creevy suggests that the most consistently barrier to the success of every quality improvement program is resistance from managers ( 17 ) . dawson found that one of the major problems encountered in implementing quality program is lack of commitment at the middle and supervisory management . they suggest that many of the problems of survivor syndrome arise from the breakdown of traditional psychological contract where managers promised job security ( 18 ) . in our study , the necessity of physicians and medical staff participation in clinical governance program has been emphasized . lawler , mohrman and ledford have demonstrated the close relationship between success of quality programs and employee involvement initiatives ( 19 ) . another issue is the importance of having an employee recognition and rewards system with supporting mechanisms providing adequate salaries for the staff being involved in the implementation of clinical governance program . encouraging workers to become involved in continuous improvement activities is relatively an important factor ( 20 ) . wilkins and witcher suggest that employees who are highly skilled , with adequate salaries and incentives are typically more likely to accept the program ( 16 ) . master produced a list of eight barriers in the way of implementing a quality improvement program includinglack of management commitment , lack of training , inability to adopt organizational culture suitable for quality improvement , lack of employee involvement , lack of resources , improper planning , in compatible organizational structure and inadequate use of team work ( 21 ) . he declares that quality improvement activities , team work , effective communication and supporting the quality program in all organizational levels are of great importance ( 22 ) . sohal , samson and ramsay also investigated the barriers of successful implementation of quality plan from the viewpoint of organizational management . they categorized the barriers in a number of groups : organizational culture ( top management support and effective involvement , changing values and culture to align with quality improvement requirements ) , strategic planning issues ( lack of planning for quality , inappropriate organizational structural ) , resource management issues ( lack of resources , inadequate number of personnel and additions to normal working load ) ( 23 ) . another study conducted by terziovski , sohal and moss showed that a successful quality organization would include the following characteristics : managers and staff with positive attitude toward quality , employment of quality management practices , dissemination of responsibility of quality to all staff at all levels , , leadership commitment , having strategic planning , providing adequate resources , focusing on training and adoption of appropriate culture ( 24 ) . in our study , senior managers stated some recommendations for implementing clinical governance more effectively such as : creating a suitable culture for implementing quality programs , evaluating the quality of organization , determining the existing deficiencies , setting up appropriate strategic and functional plan to achieve determined objectives , following the programs , evaluating the results and encouraging quality improvement activities continuously ( table 3 ) . the interviews reflect only top managerial perspectives at medical university level and not managers working in hospitals who should play important role in cg implementation . in addition , due to the nature of qualitative studies the results can not be generalized although we are looking for theoretical generalization . although this study has provided the first evaluation of senior managers viewpoint about facilitators and barriers in cg implementation in iran , further research is required to track the progress of the cg policy as it unfolds over time . this qualitative paper explores main facilitators and barriers perceived by deputies in curative affairs of iranian medical universities . identifying facilitators and barriers from the viewpoint of senior managers can have an effective role in successful progress of cg program . the reason is that these managers are directly responsible for piloting such quality programs and are the most familiar with challenges existing in the way of implementing cg . the authors conclude that one of the possible solutions is developing educational courses and workshops with the purpose of raising staff awareness toward cg concept and practice . developing a supportive culture , having all levels of staff commitment and involvement , effective communication , developing clinical guidelines , using incentive tools and overcoming legal challenges are other resolutions mentioned in this regard . by successfully implementing the program , patients will benefit from quality services . also health professionals take an advantage of working in a safer and more supportive system . evidence suggests that governance needs to match its commitment to a program of change with realistic timetables to secure the cultural and organizational changes needed to improve quality of care . ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc . ) have been completely observed by the authors .

abstractbackgroundhealth care systems should assign quality improvement as their main mission . clinical governance ( cg ) is a key strategy to improve quality of health care services . the iranian ministry of health and medical education ( mohme ) has promoted cg as a framework for safeguarding quality and safety in all hospitals since 2009 . the purpose of this study was to explore perceived facilitators and barriers to implementing cg by deputies for curative affairs of iranian medical universities.methodsa qualitative study was conducted using face to face interviews with a purposeful sample of 43 deputies for curative affairs of iranian medical universities and documents review . thematic analysis was used to analyze the dataresultsfive themes were explored including : knowledge and attitude toward cg , culture , organizational factors , managerial factors and barriers . the main perceived facilitating factors were adequate knowledge and positive attitude toward cg , supporting culture , managers commitment , effective communication and well designed incentives . pe rceived barriers were the reverse of facilitators noted above in addition to insufficient resources , legal challenges , workload and parallel quality programs.conclusionssuccessful implementation of cg in iran will require identifying barriers and challenges existing in the way of cg implementation and try to mitigate them by using appropriate facilitators .

pubmed

hydatid cyst disease is one of the most important zoonotic diseases , which occur , in larval stage of echinococcus granulosus parasite . in this disease , canines such as dog , wolf , fox , and jackal ( the final host ) are infected by eating contaminated viscera , and small adult worms with size of 27 mm are formed in their intestines and begin to spawn . canines repel the eggs of adult worms along with feces and infect the soil , water , and vegetables . herbivores as the main intermediate host are infected by eating forage contaminated with the larvae of this parasite . besides , human as the intermediate host is infected accidentally either by eating contaminated water and vegetables , or direct contact with contaminated dogs is infected to parasite egg of this disease ; and hydatid cyst is usually involved the lung and liver in human ( 1 , 2 ) . epidemiological , clinical , and pathological symptoms of this disease depend on some factors such as age , gender , infection severity , cyst size , and the involved organ in the body ( 1 , 2 ) . this disease is common in most parts of the world especially in countries where animal husbandry is current . contamination to this parasite is spreading in most tropical regions of the world and it is globally distributed in many countries such as argentina , peru , east africa , central asia , china , and south america ( 3 ) . in endemic regions , the incidence rate of disease can be reached over 50 per 100,000 person - years with the prevalence rate of 5%10% in human ( 3 , 4 ) . on the other hand , hydatidosis is among the most important human diseases with the high health and economic burden ( 5 , 6 ) . iran is one of the areas that has been considered as hyper - endemic area by who in terms of close relationship of a high proportion of society with animals , traditional animal husbandry , and then contact with the sources of infection ( 7 , 8) . the prevalence rate of this disease in the intermediate host has reported as 11.5%34.6% ( 9 ) , and the prevalence rate of human hydatid cyst has been reported from 1.1% to 13.7% in different parts of iran ( 1012 ) . assessment of the epidemiological and clinical aspects of the disease can help health policy - makers to focus on the main public health problems in the community and to assess the efficacy of control and preventive programs to limit the spread and transmission of the parasite in humans . hence , the present study aimed to determine the epidemiological and clinical status of human hydatidosis in khorasan razavi province during 201114 . this cross - sectional study was conducted in khorasan razavi province , the northeast of iran , in 2014 . the study population was all cases with hydatid cyst who diagnosed in governmental and private laboratories , hospitals and health centers in khorasan razavi province during 201114 . the data collection was performed using a checklist including age , sex , nationality , place of residence , occupation , infected organ , the number of cysts , the type of diagnosis ( ct scan , mri , radiology , sonography , immunofluorescence , other ) , clinical symptom , and having risk factors such as how to wash vegetables and contact with dog . descriptive statistics including frequency tables and diagrams as well as analytical statistics including chi square and t - student tests were used for data analysis . all statistical analyses were performed at 0.05 significance levels using stata software , version 12 ( stata corp , college station , tx , usa ) . in addition , mapping the percentage of hydatid cyst cases was created using gis software by cities . for considering ethical issues in data collection , it should be noted that all information of the patients has been confidential and the information has been recorded with code and without mentioning name . in total , 357 hydatid cyst cases had been registered in the health centers during the periods 201114 in all parts of province . the most percentage of cases was related to the cities mashhad ( 51.8% ) , ghoochan ( 7.8% ) , neishabour ( 7.0% ) , and fariman ( 4.8% ) , respectively . percentage of hydatid cyst cases by county in khorasan razavi province in 201114 demographic characteristics of studied patients by sex are shown in table 1 . of 357 cases , 54.9% were women , 40.3% rural , 45.8% housewives , and 3.4% were afghan . the mean age of women was higher than that of men ( 39.1318.9 compared to 34.717.9 , respectively , p - value=0.025 ) . demographic characteristics of patients with hydatid cyst by sex in khorasan razavi province in 201114 data are presented as number ( % ) . the most common symptom of cases was related to the cities mashhad ( 51.8% ) , ghoochan ( 7.8% ) , neishabour ( 7.0% ) , and fariman ( 4.8% ) , respectively . moreover , 45.9% of patients had domestic dog and hygiene principles of washing the vegetables was adhered by 6.7% of patients and 64.4% were washed the vegetables with water alone ( table 2 ) . risk factors of hydatid cysts in the patients in khorasan razavi province in 201114 the majority of patients ( 70.5% ) were infected with a single cyst , and 24.5% of them had multiple cysts . one hundred fifty one ( 42.3% ) of patients were diagnosed by radiology method and 37.8% by ct scan method . the most common clinical symptom in the hydatidosis patients was abdominal pain ( 42.3% ) , after that hepatomegaly ( 27.4% ) , chest pain ( 21.6% ) , and cough ( 8.7% ) were other common symptoms . liver involvement was reported in 59.4% of patients , and 8.4% had multiple organ involvement ( fig . 2 ) . status of organ involvement in the patients with hydatid cysts in khorasan razavi province during 201114 previous studies conducted from different parts of iran found the annual incidence of 1.33 in hamadan ( 13 ) , and 0.54 in kermanshah ( 14 ) per 100,000 population . in addition , the rate of infection varied from 1.79% in ardabil ( 11 ) to 13.8% in khozestan ( 12 ) . our findings showed that the highest proportion of hydatidosis ( 39.2% ) in the age group of 2140 years old . according to some factors such as geographical region , culture , and workforce ; age can play an important role on the occurrence of the infection . in this regard , previous studies have reported the highest proportion of hydatidosis in the different age groups . for example , the age group of 6069 in isfahan ( 10 ) , 4049 year in ardabil ( 11 ) , and 3060 year in qom ( 15 ) . based on a review study , the range of 2040 years old as the age group of the highest proportion has been reported ( 7 ) . the findings of this study showed the most percentage of infection in women than men ( 54.9% compared with 45.1% , respectively ) . this might be due to more contribution of women to agricultural and animal husbandry in this region . the high frequency of the disease in women than men is consistent with the obtained results of other iranian studies ( 16 , 17 ) and other countries ( 18 , 19 ) . however , some studies reported the higher rate of infection in men ( 10 , 11 , 15 ) . moreover , as stated in the previous studies conducted in iran ( 7 , 20 ) , the housewives had the highest proportion of infection . it may be due to the more likely to contact with the source of contamination especially vegetables contaminated to parasitic eggs . in our study , for example , the most percentage of the province s population is urbanite and another might be a good - quality disease reporting . there are conflicting results in this regard ; some studies reported the same result with our study ( 11 , 15 , 20 , 21 ) , and other reported inconsistent result ( 8 , 22 ) . we found that abdominal pain was the main symptom with a proportion of 42.3% in the patients . studies conducted in turkey ( 23 ) and iran ( 24 ) has reported a proportion of 77.6% and 66% for this symptom , respectively . in our study in line with previous reports ( 24 , 25 ) , liver was the most common localization of hydatid cyst . about 8.4% of the patients in this study had multiple organs involved and it was 12.3% in another study in iran ( 24 ) . the common diagnosis methods of the disease were radiology ( 42.3% ) followed by ct scan ( 37.8% ) . in a study to determine the pattern of hydatid cyst during 200010 in tehran revealed that ultrasonography and ct scan were the most used imaging methods ( 24 ) . our findings demonstrate that these methods are the available tools for detecting hydatid cyst in iran . concerning risk factors of infection with hydatidosis , our results found that 45.9% of individuals were in contact with dog , 64.4% of them have washed the vegetables with the water alone and 6.7% have washed the vegetables by hygiene principles of washing . consequently , focus of improvement in education level of the society could be better . in bulgaria ( 25 ) 90% of the affected children the prevalence of human hydatidosis , as a most important neglected disease , should be considered by health policy - makers in public health domain . in addition , educational programs to better recognition of the disease symptoms , and to identify the infection sources are needed in high risk group of population .

background : this study aimed to investigate the epidemiological and clinical aspects of patients with hydatid cyst during 2011 to 2014.methods:this cross - sectional study was conducted in khorasan razavi province , the northeast of iran , from 2011 to 2014 . the study population was all cases with hydatid cyst who diagnosed in governmental and private laboratories , hospitals and health centers ( hc ) in khorasan razavi province during 201114.results:the prevalence rate of hydatidosis was 1.44 per 100000 individuals . of 357 cases , 54.9% were women , 40.3% rural , 45.8% housewives , and 3.4% were afghan . the mean age of women was higher than that of men ( 39.1318.9 compared to 34.717.9 yr , respectively , p - value=0.025 ) . the highest proportion of cases ( 39.2% ) was in the age group of 2140 yr old . abdominal pain was reported in 42.3% of cases . liver involvement was the most common localization of hydatid cyst reported in 59.4% of patients , and 8.4% had multiple organ involvement . the common diagnosis methods of the disease were radiology ( 42.3% ) followed by ct scan ( 37.8% ) . 45.9% of patients had domestic dog and hygiene principles of washing the vegetables was adhered by 6.7% of patients.conclusion:the prevalence of human hydatidosis , as a most important neglected disease , should be considered by health policy - makers in public health domain . in addition , educational programs to better recognition of the disease symptoms , and to identify the infection sources are needed in high risk group of population .

pubmed

friederich is credited with describing the first case of a ciliated hepatic foregut cyst ( chfc ) in 1857 and positing their congenital origin . wheeler and edmonson were the first to use the term chfc to describe the lesion . they also identified specific characteristics , which differentiate it from other hepatic cysts , such as the presence of four typical layers : a pseudostratified columnar epithelium layer with interspersed mucus cells , a sub - epithelial connective tissue layer , a smooth muscle layer and an outer fibrous capsule . this lesion is thought to be the result of evagination of the foregut during embryonogenesis . in the last 150 years , approximately 103 cases have been reported in the literature world - wide , only four of which describe laparoscopic excision . we report 5 case of a patient who recently underwent successful laparoscopic excision of a chfc and the rationale for attempting laparoscopic removal of these lesions and review of the literatures . this paper reports a case of a 32-year - old woman presented with a 6-month history of right upper quadrant pain . the ca 19 - 9 serum level was 18 iu / l ( normal 0 - 37 an abdominal ultrasound scan reported thin walled 10 cm 7.6 cm 5.8 cm cyst in the superior part of the right lobe of liver raising a suspicion of a simple cyst of liver . the cystic lesion did not enhance postcontrast and appeared to be well circumscribed without evidence of hepatic invasion suggestive of a simple cyst . therefore , a laparoscopic deroofing of the cyst was planned . at the time of surgery , port placement was initiated with a 10-mm infraumbilical trocar followed by two 5-mm ports inferior to the right costal margin along the anterior and midaxillary lines . intraoperatively [ figure 2 ] , the 10 cm 8 cm cyst was found adherent to the superior surface of liver compressing segments vii and viii . on further dissection , it was noted that the cyst was actually free from the liver parenchyma and was adherent to the liver by a small pedicle . since there was only a pedicle , it was decided to do a complete excision instead of a de - roofing . pathologic review of the cyst showed an inner layer of ciliated pseudo stratified mucin secreting columnar epithelium with scattered goblet cells surrounded by loose connective tissue , a discontinuous smooth muscle layer and an outer fibrous capsule these findings were consistent with a ciliated foregut cyst of hepatic origin without evidence of epithelial dysplasia or invasive malignancy . contrast enhanced computed tomography shows non - enhancing cystic lesion in segment viii of right lobe of liver laparoscopic view of ciliated hepatic foregut cyst in segment viii chfcs are rare , with only 103 cases reported since first described as a congenital malformation by friederich in 1857 . the majority ( 55 ) of these cases have been reported in the last 25 years as imaging techniques have improved . the term chfc was coined to indicate the origin of this lesion as being a detached hepatic diverticulum or detachment and migration of buds from the oesophageal and bronchial regions of the foregut and subsequent entrapment by the liver during the early embryonic development of the foregut . it is found most commonly in segment iv of the left lobe just beneath the hepatic capsule . the cyst size ranges from < 1 cm to 24 cm in greatest diameter , the mean greatest dimension is 4 cm and the median is 3 cm . the average age of presentation is 52 years , ranging from 3 months to 82 years , with a slight female predominance . nearly 40% of the reported cases have been found incidentally on imaging studies , 26% incidentally at autopsy , 6% incidentally at surgery and 22% presented with abdominal symptoms . chfc are most frequently located superficially in the median segments of the liver ( segments iv , v , viii ) , are rarely multi - loculated or septated and are mostly asymptomatic . they also identified specific characteristics , which differentiate it from other hepatic cysts , such as the presence of four typical layers : a pseudostratified columnar epithelium layer with interspersed mucus cells , a sub - epithelial connective tissue layer , a smooth muscle layer and an outer fibrous capsule . friederich initially suggested a congenital origin , but currently it is suspected that the chfc is a detached hepatic diverticulum or abnormal tracheobronchial bud that may have migrated caudally to be included with the liver during the early embryological development of the foregut . although , sludge - like bile and viscid mucoid content of the cyst have been described , no communication with the biliary tree could be demonstrated in any reported cases . the imaging appearances of chfc 's are variable and appear as anechoic or hypoechoic cysts with spotty hyperechoic areas on ultrasound . ct findings are of a non - enhancing , rounded lesion of varying density depending on the contents of the cyst , which can include calcium crystals and cholesterol . typical magnetic resonance imaging features are a hyperintense cyst on t2-weighted images and a variable appearance on t1-weighted images . pathologically , the cysts are typically solitary and unilocular , and have four layers : an outer fibrous rim , a layer of smooth muscle ( often incomplete ) , subepithelial connective tissue and a lining of ciliated , pseudo - stratified , mucinsecreting columnar epithelium .. presence of unique histological features indicates chfc with a reasonable degree of confidence , although a rare morphologic variant of chfc without smooth muscle layer has reported . histologically , the differential diagnosis of a hepatic cyst included simple cyst , hydatid cyst , biliary cystadenoma , cystadenocarcinoma and bronchogenic cyst . the main differential diagnosis can be bronchogenic cyst because both cystic lesions share a common embryonic origin . simple cysts are usually located in the right hepatic lobe and are more prevalent in women . serologic tumour markers can be confounding , as elevated ca19 - 9 levels have been found in association with benign chfc . an aspirate of hepatocytes admixed with ciliated cells and mucus cells distinguish chfc from other radiologically benign - appearing entities among many of the considered lesions , which are easily differentiated by their typical epithelial lining , cyst . of concern are reports of chfc harbouring squamous cell carcinoma ( 4.4% in reported cases ) . there is also a single report of portal vein compression secondary to the mass - effect of the chfc . until date , the recommended treatment of chfc described in the literature has varied from observation to aspiration to surgical excision however , given the recent cases of chfc malignant transformation , serial imaging may be required for those patients undergoing cyst sclerosis . most of the authors agree that chfcs should be surgically excised for cysts larger than 4 cm to 5 cm , symptomatic or enlarging lesions and asymptomatic lesions with wall abnormalities on imaging or in patients with otherwise unexplained abnormal liver function tests . until date several authors have also noted that laparoscopic approach of aspiration and injection of a sclerosing agent can be utilised effectively to stop cyst growth , but the definitive approach is excision of cyst but minimally invasive approach . this technique is best suited to lesions in segments ii to vi , the anterolateral portion of the liver where visualisation and access are easiest . lesions located centrally or in the posterior aspects of segments vi , vii , or iva are less accessible and render laparoscopic therapy more challenging . in general , the patient is positioned in lithotomy , allowing the surgeon to stand between the legs with assistants at the sides . the cyst wall is excised with harmonic scalpel ; hemostasis may be obtained with electrocautery . for patients with lesions in segment vii or viii , positioning and port placement similar to that used for laparoscopic right adrenalectomy may afford improved exposure . first , the small size and anterior subcapsular location allow for easy access to the cyst with minimal dissection to isolate the lesion . second , the benign nature of the process allows the cyst to be enucleated from is hepatic bed with little concern for adequate circumferential margins . third , the chfc traditionally has a thick cyst wall that permits easy handling with laparoscopic instruments . the subcapsular location and the presence of thick outer fibrous layer in the cyst wall makes it amenable to laparoscopic excision with minimal morbidity . the rarity of chfc underscores the importance of re - porting individual cases , as illustrated by the case presented here , which may be few reported cases of chfc presenting with abdominal pain in her early 30 s. it should be noted that although the size and location of the cystic mass were characteristic of chfc , this patient 's cyst demonstrated imaging features that are less common for a chfc , leading to the decision to excise the cyst . by utilising a minimally invasive approach to excise the chfc , the patient benefited from an effective ambulatory procedure for the removal of a lesion with risk for malignant transformation . chfc should be considered as a diagnosis for a superficially located unilocular hepatic cystic mass that presents incidentally or asymptomatically and laparoscopic excision should be considered as a first - line treatment to remove this benign cyst .

ciliated hepatic foregut cysts ( chfcs ) are rare congenital cysts of the liver that originate from the embryologic foregut . despite an increase in incidence , they remain rare and several key characteristics remain poorly understood , including the range of presentation and the risk of malignant conversion . large , symptomatic cysts and small asymptomatic , atypical cysts should be resected . we present a patient who recently underwent laparoscopic excision of a chfc , review the literature and propose the rationale for attempting removal of these cysts through a laparoscopic approach .

pubmed

oral route is one of the most preferred routes of drug administration due to its safety , ease of administration , and acceptability by patients . about 60% of conventional dosage forms are available as the oral solid dosage forms . the low bioavailability , longer onset of action , and dysphasia patients turned the manufacturer towards the parenterals and liquid dosage forms . but the liquid dosage forms ( syrup , suspension , emulsion , etc . ) have the problem of accurate dosing and parenterals are painful drug delivery systems , so they result in patient incompliance . the most popular oral dosage forms are tablets and capsules ; one major drawback of these dosage forms is the difficulty to swallow . drinking water plays an important role in the swallowing of oral dosage forms . people experience inconvenience in swallowing tablet dosage forms when water is not available particularly in the case of traveling ( motion sickness ) and sudden episodes of coughing during the common cold , allergic condition , and bronchitis . under such circumstances , tablets that can rapidly dissolve or disintegrate in the oral cavity known as fast dissolving tablets have attracted a great deal of attention . fast dissolving tablets are also known as mouth - dissolving tablets , orodispersible tablets , rapidmelts , and porous tablets . fast dissolving tablets dissolve or disintegrate within 60 seconds when placed in the mouth without drinking water or chewing . the active ingredients are absorbed through mucous membranes in the mouth and git and enter the blood stream . but due to certain disadvantages like their physical solid form , psychological fear of swallowing , chewing , or chocking , friability of wafer like porous and low pressure moulded tablet , and expensive packaging cost of these dosage forms to protect them , a new technology was developed as orally dissolving strip . orally dissolving strips are the most advanced form of oral solid dosage form due to more flexibility and comfort . it improves the efficacy of apis by dissolving within a minute in oral cavity after the contact with saliva without chewing and need of water for administration . it gives quick absorption and instant bioavailability of drugs due to high blood flow and permeability of oral mucosa which is 41000 times greater than that of skin . orally dissolving strips are useful in patients such as pediatrics , geriatrics , bedridden , and emetic patients and conditions such as sudden episodes of allergic attacks or coughing . there is an increasing interest in the development of orally dissolving strips as an alternative to fast dissolving tablets , due to their faster dissolution rate , higher flexibility , and better patient compliance . presently , research work on the use of orally dissolving strips as promising carriers for the delivery of multiple active pharmaceutical ingredients has emerged [ 611 ] . marketed orally dissolving strips products have also become available including listerine , chloraseptic , triaminic , and multivitamins . the backbone of an orally dissolving strip is generally formed of a plasticizer and film forming polymer or a mixture of polymers that provide the necessary elasticity and shape to the film . examples of polymers that have been used in the formulation of orally dissolving strips include hydrocolloids or povidone k-90 , maltodextrin ( mdx ) , hydroxypropyl ethylcellulose ( hpmc - e15 , 5 ) , pectin , sodium alginate , or blends of polymers . orally dissolving strips can be prepared using a solvent - casting , rolling , hot melt extrusion , or solid dispersion methods . epileptic seizures can occur in nonepileptic patients subjected to a variety of stresses and stimuli . epilepsy is a neurological disorder which requires quick management of seizures in order to avoid the risk of permanent brain damage . management of epilepsy differs from the treatment of other diseased conditions in that a single epileptic attack has a major negative effect on quality of life . clobazam is a newer 1 , 5-benzodiazepine derivative which is a well tolerated , safe , and very effective antiepileptic drug having a broad spectrum of antiepileptic activity and minimal side effects and being relatively inexpensive . thus , to control the epileptic seizures in the shortest possible time , an attempt has been made to develop , evaluate , and optimize orally dissolving strips of clobazam with improved bioavailability and palatability . clobazam is an ideal drug candidate for an orally dissolving strip formulation because of its indication in children and its low - dose requirement . the formulation of clobazam as an orally dissolving strip , required to be placed on the patient 's tongue without swallowing for dose administration , would significantly facilitate dose administration , with subsequent improvement in patient compliance . thus , the aim of this work was to design , characterize , and optimize orally dissolving strip of clobazam using two polymers : ssg ( disintegrant ) and pva ( film former ) . a 3 factorial design was used to evaluate the influence of film forming polymer pva and disintegrating agent ssg on the film 's mechanical properties , disintegration time , and dissolution rate . this study also assessed the in vivo performance and ivivc of the optimum formulation by administration to healthy rabbits . sodium starch glycolate , peg-400 , and directly compressible mannitol were received from loba chemie pvt . the pure drug clobazam and physical mixture of clobazam and polymers were mixed with ir grade kbr pellets in the ratio of 100 : 1 and corresponding pellets were prepared in a hydraulic press . the pellets were scanned over a wave number range of 4000500 cm in using perkin emler spectrum 400usa , ftir instrument . differential scanning calorimetry ( dsc ) analysis was undertaken to visualize the changes , if any , observed during the preparation of the orally dissolving strip using mettler toledo model dsc 821e instrument . dsc of clobazam ( pure drug ) , physical blend of pva , clobazam and ssg ( polymer ) , and optimized film formulation f6 were carried out over a temperature range of 30 to 300c at a scanning rate of 5c / min . solubility studies were carried out by taking an excess amount of drug in 10 ml of different solvents and ph 6.8 buffers in conical flask , closed with aluminum foil and constantly agitated at room temperature for 24 hrs , using orbital shaking incubator ( remi instruments , c-24 bl , and mumbai , india ) . further , the solutions were filtered and the amount of drug solubilised was estimated at a wave length of 232 nm by using systronics pc based double beam spectrophotometer 2202 , mumbai , india . the orally dissolving strips of clobazam using film forming polyvinyl acetate ( pva ) and peg 400 as plasticizer were prepared by solvent - casting method . an aqueous solution of the polymer was prepared in warm distilled water and was kept aside for 4 hours for swelling of polymer . clobazam was added to the aqueous polymeric solution after levigation with required volume of peg 400 . this was followed by the addition of mannitol as a sweetener as well as a solubilizer and sodium starch glycolate as a superdisintegrant . the solution was casted on a plastic petri dish and dried at room temperature for 24 hr . the strip was carefully removed from the petri dish , checked for any imperfections , and cut into the required size ( 2 2 cm ) to deliver the equivalent dose of 5 mg per strip . preliminary trials were undertaken for designing the orally dissolving strips where the effect of various concentrations of the film forming agent and superdisintegrants on the characteristics of the strips was noted . in addition , the prepared strips were also checked for surface perfection , smoothness , and ease of removal from petri dish without rupturing , folding , or cracking . a 3 full factorial design was employed to study the effect of independent variables x1 ( pva ) and x2 ( ssg ) over the dependent variables like tensile strength ( n / cm ) , disintegration time ( sec ) , and in vitro drug release ( % ) as shown in design layout tables 1 and 2 . in this design , two factors were evaluated each at three levels ( 1 , 0 , + 1 ) and all possible nine experimental batches were formulated . composition of all nine possible combinations of orally dissolving strip of clobazam using 3 full factorial designs is shown in table 3 . the data was subjected to contour and 3d response surface plot using design - expert software version 8.0.7.1 . a multiple linear regression equation incorporating interactive and polynomial terms was used to calculate the response as follows : ( 1)y = b0+b1x1+b2x2+b12x1x2+b11x12+b22x22 , where y is the dependent , that is , response variable , namely , disintegration time , tensile strength , and in vitro drug release ; b0 is the arithmetic mean response of the nine runs ; and b1 and b2 are the estimated coefficients for the factors x1 and x2 , respectively . the main effects ( x1 and x2 ) represent the average result of changing one factor at a time from its low to high value . the interaction term ( x1x2 ) shows how the response changes when two factors are simultaneously changed . the polynomial equations can be used to draw conclusions after considering the magnitude of the coefficient and the mathematical sign it carries ( i.e. , positive or negative ) . the high values of the correlation coefficients for the dependent variables indicate a good fit . the thickness of each strip was measured at five different locations ( centre and four corners ) using calibrated digital vernier caliper ( mituotoyo , japan ) . a strip of ( 2 2 cm ) was cut and repeatedly folded at the same place till it broke . the number of times the strip could be folded at the same place without breaking was taken as a measure of folding endurance . the oral strip of size 4 cm was dissolved in 10 ml of phosphate buffer ph 6.8 and solution was filtered and drug content was estimated at 232 nm using double beam uv / visible spectrophotometer ( systronics 2202 , mumbai , india ) . the experiments were carried out in triplicate for the strips of all formulations and average values were recorded . the surface ph of orally dissolving strips was determined in order to investigate the possibility of any side effects in vivo . as an acidic or alkaline ph may cause irritation to the oral mucosa , it is determined to keep the surface ph as close to neutral as possible . the ph was measured by bringing the electrode in contact with the surface of the oral strip . this study was performed on three strips of each formulation and mean sd calculated . in vitro disintegration time was measured by placing the film ( 2 2 cm ) on stainless steel wire mesh placed in a petri dish containing 10 ml of phosphate buffer ph 6.8 . time required for the oral strip to break was noted as in vitro disintegration time . the test was performed on three strips of each formulation batch and mean sd was calculated . in vitro dissolution test was performed according to the usp type ii paddle apparatus ( labindia ds 8000 , mumbai , india ) . test was performed by fixing the oral strip ( 2 2 cm ) to rectangular glass plates so as to prevent it from floating and it was placed at the bottom of dissolution vessel containing 900 ml of phosphate buffer ph 6.8 at 37c with a rotation speed of 50 rpm . a 5 ml of sample was taken at time intervals from 1 to 30 min , and the same volume was replenished with fresh buffer solution maintained at 37c . the samples were filtered and analyzed at 232 nm using double beam uv / visible spectrophotometer ( systronics 2202 , mumbai , india ) ; the content of drug was calculated using equation generated from standard calibration curve of clobazam . the release mechanism of clobazam from oral strip was also determined by fitting the release data to different kinetic models , zero order , first order , and higuchi . the surface morphology of the optimized orally dissolving strip formulation was observed with scanning electron microscope ( hitachi s-3400n type ii model , japan ) . pictures were taken at an excitation voltage of 1.0 kv and a magnification of 1000x . films were cut into 2 2 cm square strips ( 4 cm ) . the moisture uptake by the strip ( n = 3 ) was determined by exposing them to an environment of 75% relative humidity ( rh ) at room temperature ( 25 2c ) for 1 week [ 26 , 27 ] . the uptake of moisture by the strips was measured and calculated as percent increase in weight . tensile strength testing was determined at central institute of post harvesting engineering and technology ( ciphet ) , ludhiana , india , using a texture analyzer tahdi ( stable microsystem ) , equipped with a 5 kg load cell . the strip was cut into 100 12.5 mm strips and equilibrated at 25c for 1 week . initial grip separation was 60 mm and crosshead speed was 50 mm min . tensile strength , elongation at break , was computed to evaluate the tensile properties of the strips . tensile strength ( ts ) was calculated by dividing the maximum load by the original cross - sectional area of the strip and it was expressed as ( n / cm ) . percent elongation at break ( e% ) was calculated by dividing the length at the time of break of the strip by the initial length of the strip and multiplying by 100 using the following equation : ( 2)e%=ll0l0100 , where l0 is the initial length of the strip and l is the length at the time of break . the calibration curve was performed with standards of the final concentrations of 5 , 10 , 25 , 50 , and 100 ng / ml in rabbit plasma . the intraday , interday precisions and accuracy of the method were determined with three replicates spiked plasma samples at different concentrations of clobazam . the intraday and interday variation was calculated in terms of percent relative to standard deviation . six rabbits of either sex ( weighed 2.5 0.2 kg ) were selected for the study . all the rabbits were fasted overnight before the administration of the selected fast dissolving film and marketed formulation but had free access to water . the rabbits were randomly divided into two groups with each group containing three rabbits ( n = 3 ) . the rabbits were positioned on a table with lower jaw supported in a horizontal position and orally dissolving strip was carefully placed on the rabbit tongue in one group as shown in figure 12 . blood samples for pharmacokinetic analysis were collected from marginal ear veins of rabbits immediately before drug administration and at 5 , 10 , 15 , 30 , 60 , 120 , 180 min , 6 hrs , 12 , and 24 hrs interval . the plasma samples were prepared by mixing 0.1 ml of plasma with 0.05 ml ibuprofen as internal standard ( from the stock 6000 ng / ml ) in a clean eppendorf polypropylene tube and then extracting with 1.5 ml of acetonitrile after vertical agitation ( 1 min ) and centrifugation at 1000 rpm , for 5 min . the hplc analysis was performed using agilent 1200 series system by using a mobile phase composed of water ph 3.5 , adjusted with orthophosphoric acid and acetonitrile ( 55 : 45 v / v ) , and binary eluted at a flow rate of 1.5 ml min . protocols for all the animal studies were approved by institutional ethical committee [ iaec / ccp/12/pr-007 ] . maximum plasma concentration cmax and the time tmax were obtained directly from the individual plasma concentration versus time curves . the terminal half - life , t1/2 , was obtained from log linear regression analysis of the plasma concentration time curves in the terminal phase . the area under plasma concentration time curve ( auc0t , and auc0 ) was determined by linear trapezoidal method . for 90% confidence interval , the auc and cmax values were transformed into their respective logarithms and analysis of variance was calculated using software kinetica 5.0 . the in vivo percentage of the drug absorbed was plotted against the in vitro percentage of the drug released to determine the correlation coefficient . the fraction of the drug absorbed was determined using the wagner nelson method , using the following equation : ( 3)fa=[(ct+keauc0t)keauc0]100 , where fa is the fraction of drug absorbed , ct is the plasma drug concentration at time t , ke is the overall elimination rate constant , and auc0t and auc0 are areas under the curve between time zero and time t and between time zero and infinity , respectively . the optimized orally dissolving formulation of clobazam was subjected to stability studies by packing the individual strip in aluminum foil and loading the formulation on remi stability chamber sc-10 plus ( remi elektrotechnik ltd . , vasai , india ) as per ich guidelines for 180 days at 40c 2c/75% rh 5% . samples were withdrawn at regular intervals and evaluated for in vitro drug release , disintegration time , and tensile strength . ftir spectra of pure drug and physical mixture of pva and clobazam as shown in figures 1(a ) , 1(b ) , and 1(c ) indicate that there was no interaction between drug and film forming polymer used . pure clobazam displays a peak characteristic of c = o stretching vibration at 1694.7 cm , aromatic ch stretching at 3075.4 cm , c c stretching at 1493.5 cm , c n stretching at 11001200 cm , ch bending at 600800 cm , and ch3 bending at 1371 cm ; the spectra of drug with pva showed all characteristics peaks of drug indicating that the drug is compatible with pva . dsc thermograms of clobazam ( pure drug ) , pva ( polymer ) , clobazam and ssg , and clobazam loaded orally dissolving strip as shown in figures 2(a ) , 2(b ) , and 2(c ) illustrated a sharp endothermic peak corresponding to the melting of crystalline clobazam at 180c . the melting endothermic peak of clobazam was not observed in the drug loaded pva orally dissolving strip . this indicates that clobazam was uniformly dispersed and present in an amorphous state in the polymeric matrix . physicochemical characterization of clobazam films as given in table 4 indicated that all the strips prepared with different polymer concentrations were flexible , smooth , transparent , nonsticky , and homogeneous . it was observed that there was no significant difference in the thickness among the strips , which indicated that the orally dissolving strips were uniform . however , the folding endurance of the oral strip f7f9 was found to decrease with increasing content of pva . thus , it appears that the increasing amount of pva decreases the flexibility of the strip . since the surface ph of orally dissolving strips was found to be around neutral ph , there will not be any kind of irritation to the mucosal lining of the oral cavity . all formulations were found to contain uniform quantities of drug ranging between 97.93% and 99.96% , as indicated by content uniformity studies . in vitro disintegration time of the formulated orally dissolving strips increase in the concentration of superdisintegrant decreases the disintegration time of films , which was observed in f1 , f2 , f3 , and f4 ; as the amount of pva increases , the disintegration time increases because of the increasing thickness of the strip but again decreases with increasing amount of ssg and formulation with f6 exhibited minimum disintegration time . mathematical relationship generated using multiple linear regression analysis for the studied variable is expressed as follows : ( 4)disintegration time=28.32222 + 1.56667x1 4.33333x20.075000x1x2 0.33333x12 + 1.66667x22 . the model f - value of 42.62 implies that the model is significant . in vitro drug release was found to decrease with increase in the film forming polymer concentration which may be due to increase in the thickness of the oral strip and due to increase in the time required for wetting and dissolving the drug molecule present in the polymeric matrix but increase with increase in the concentration of the disintegrant . formulations f1 , f2 , and f3 showed drug release up to 79.1% , 85% , and 89% , respectively , as shown in figures 4(a ) , 4(b ) , and 4(c ) at the end of 30 min . film formed with higher quantity of polymer had shown slower dissolution rate ; this might be due to the increase level of pva that results in formation of high viscosity gel layer due to more intimate contact between the particles of polymer resulting in decrease in the mobility of drug particles from the swollen matrix , which leads to a decrease in the release rate . the order of drug release in each set of formulation can be given as ( 5)f1<f2<f3,f4<f5<f6,f7<f8<f9 . the formulation f6 showed a maximum percentage drug release of 96.6% in 30 min as shown in figure 4(b ) . reflection point at 30 min in the curve might be due to the complete solubilization of pva in the medium which improve the wettability and solubilize the drug and provide the fast dissolution rate . further , drop in the percent of drug release after 30 min might be due to the availability of less number of drug molecules in the free void spaces and a decrease in mobility through polymeric matrix . mathematical relationship generated using multiple linear regression analysis for the response variable is expressed as follows : ( 6)% drug release=81.701115.16833x1 + 5.1683x2 + 1.00250x1x2 4.5016x12 + 1.99833x22 . the model f - value of 77.23 implies that the model is significant . tensile strength and % elongation were found to increase with the increasing content of pva which may be due the increase in the elasticity nature of the film forming polymer . mathematical relationship generated using multiple linear regression analysis for the studied variable is expressed as follows : ( 7)tensile strength=2.39111 + 0.56500x1 + 0.31167x20.01500x1x2 0.1916x12 + 0.12833x22 . addition of ssg affects tensile strength and % elongation ; higher concentration of ssg increases thickness and crystallinity of the film , which causes decrease in the tensile strength and % elongation . there was a good linear correlation ( r = 0.9896 0.9999 ) obtained by plotting the percent of clobazam released from all the orally dissolving strip formulations against the square root of time . thus , it was concluded that the release of clobazam from orally dissolving strip formulations followed a diffusion - controlled drug release profile and is in agreement with the higuchi model . the results of multiple linear regression analysis are shown in table 6 , indicating that for all response variables which are disintegration time , tensile strength , and % drug release , the amount of pva ( x1 ) had a negative effect while the concentration of ssg ( x2 ) had a positive effect ; it means that as the amount of ssg increases the tensile strength , disintegration time decreases and there is increase in % drug release , while as the amount of pva is increased , both the tensile strength and disintegration time increase [ 3436 ] . therefore , high level of ssg and medium level of pva should be selected for the rapid disintegration and a faster in vitro drug release of the film . the observed values are in good agreement with the predicted values for the optimized formulation , which demonstrate the feasibility of surface response method in the formulation of oral fast dissolving films . the comparison of observed values and predicted values with % prediction error is shown in table 7 . the data of the response surface plot as shown in figures 5 , 6 , and 7 demonstrated that both x1 and x2 affect the disintegration time , tensile strength , and % release of drug . the surface morphology as shown in figure 8 using scanning electron microscopy of the optimized orally dissolving strip formulation of clobazam depicted smooth surface with some little pores , which is an indication of uniform distribution of drug particles . based on the responses the formulation f6 showing the highest dissolution rate , in vitro disintegration time suitable for fast - dissolving dosage form , and satisfactory tensile strength properties was chosen for subsequent comparative study relative to a marketed clobazam formulation as shown in table 8 . comparative results of in vitro drug release of f6 formulation and marketed formulation ( frisium5 ) as shown in figure 9 indicate that the optimized formulation was comparable to the marketed formulation . the results from the hplc method validation in the rabbit plasma are shown in table 9 . the within day and between day precision as given in terms of % relative standard deviation ranged from 0.5 to 6.2% and from 1.1 to 1.6% , respectively . the mean clobazam plasma concentration time profiles for the prepared orally dissolving strip and the marketed tablets are shown in figure 10 . the statistical comparison of cmax , auc0t , and auc0 indicated no significant difference between the two treatments ( test and reference marketed tablets ) . the 90% confidence intervals for the mean ratio ( test / reference ) of cmax , auc0t , and auc0 were 95.87% , 98.12% , and 99.21% , respectively , and are shown in table 10 , while the acceptable range is 80125% for auc0t and auc0 , and 70143% for cmax , as proposed by the fda . in vitro - in vivo correlation as shown in figure 11 of percentage drug absorbed and percentage drug released showed good in vitro - in vivo correlation for optimized film formulation f6 with r value 0.994 . it was concluded that there was no significant change observed in the weight of orally dissolving strip , tensile strength , and % in vitro drug release . the average clobazam content of formulation f6 after 6-month storage was 95.1 0.17 mg ( ranging from 91.88 to 96.80% ) . therefore , the prepared formulation was stable up to 6 months at 40c ( 75%rh ) . the orally dissolving strips of clobazam prepared using pva as film forming material and ssg as disintegrant by the solvent - casting method showed satisfactory drug dissolution and acceptable physicomechanical characteristics . 3 factorial design was used for the optimization , amongst nine formulations prepared as per the design layout , which indicates that the film prepared using 100 mg of pva and 6% of ssg ( f6 ) showed the highest dissolution rate , suitable in vitro disintegration time , and satisfactory tensile strength and was selected as the optimized formulation . in vivo studies also indicated absence of significant difference between f6 and frisium5 marketed tablets and both exhibited comparable drug plasma level - time profiles . accelerated stability studies results showed that prepared formulation was stable enough for the period of at least 6 months . therefore , the present orodispersible film formulation containing clobazam considered is potentially useful for the treatment of epileptic attack where improved patient compliance and convenience are expected and can be used as an alternative to the fast dissolving tablet formulation .

clobazam orally dissolving strips were prepared by solvent casting method . a full 32 factorial design was applied for optimization using different concentration of film forming polymer and disintegrating agent as independent variable and disintegration time , % cumulative drug release , and tensile strength as dependent variable . in addition the prepared films were also evaluated for surface ph , folding endurance , and content uniformity . the optimized film formulation showing the maximum in vitro drug release , satisfactory in vitro disintegration time , and tensile strength was selected for bioavailability study and compared with a reference marketed product ( frisium5 tablets ) in rabbits . formulation ( f6 ) was selected by the design - expert software which exhibited dt ( 24 sec ) , ts ( 2.85 n / cm2 ) , and in vitro drug release ( 96.6% ) . statistical evaluation revealed no significant difference between the bioavailability parameters of the test film ( f6 ) and the reference product . the mean ratio values ( test / reference ) of cmax ( 95.87% ) , tmax ( 71.42% ) , auc0t ( 98.125% ) , and auc0 ( 99.213% ) indicated that the two formulae exhibited comparable plasma level - time profiles .

pubmed

stroke is a major cause of death and disability world - wide . by 2020 stroke , together with coronary - artery disease , stroke represents an often devastating disruption to life , the majority of survivors experiencing some degree of impairment requiring additional care or support 1 year post - stroke . stroke is an acute event , but may result in significant long - term impact for the individual , such as social isolation , mood disturbance , communication difficulties and reduction in mobility and life roles . recovery following stroke is complex and multidimensional , encompassing bio - medical , psychological and sociological elements [ 911 ] . engagement in self - management practices by individuals with long - term conditions has been suggested as key to promoting recovery and is cited as a means of empowerment and facilitator of improved health outcomes . self - management is a prominent issue in uk health policy [ 1517 ] and has been identified as a key priority for health by organisations independent of the uk government . self - management can be defined as the active management by individuals of their treatment , symptoms , lifestyle , physical and psychological consequences inherent with living with a chronic condition . self - management is an attractive initiative in managing the increasing burden on health and social care resources and reducing associated costs ; the assumption being that effective self - management by an individual reduces their healthcare utilisation [ 2123 ] . self - management interventions ( smis ) are designed to enable people to manage their health more effectively . evaluation therefore must consider two key areas ; firstly whether people develop the skills to manage their own health and secondly , if this consequently results in better health . is complex , not least due to variation in delivery , culture of the sponsoring healthcare organisation and anticipated goals and outcomes . the uk medical research council advocates establishing the theoretical basis of an intervention as a first step in estimating its possible outcomes . currently , evidence suggests that the mediators of change and theoretical premises in smis are unclear [ 2830 ] . firstly , doubt exists regarding the appropriate outcome(s ) to monitor to assist evaluation of the intervention and aid determination of cost - effectiveness and clinical impact . secondly , if the theoretical premises underpinning the intervention are uncertain , intervention fidelity is difficult to monitor and maintain . questions then exist regarding what influences change and how this can be appropriately measured and smis evaluated . smis may be evaluated by examining the effect on health outcomes that potentially change as a consequence of better self - management . using patient reported outcome measures ( proms ) ( e.g. functional status , symptom control , mood and health - related quality of life ) is an important way of to ensuring evaluation considers outcomes important to patients . preliminary investigation of self - management suggests that effective self - management corresponds with positive changes in health behaviour . more recently there has been a focus upon measuring attitudes since these are thought to modify health behaviour [ 2931 ] . proms endeavor to capture information that is not directly observable and unmediated by healthcare professionals ; consequently accurate measurement is contingent on the extent that the prom is an accurate reflection of the variable in question . therefore , it is vital to evaluate whether the measures adopted in smi studies provide legitimate information to evaluate self - management , both the process and obtaining of skills to better manage health and subsequent potential improvements in health . before using an outcome measure in research or clinical practice , it should be assessed and considered to possess adequate psychometric properties . despite the recognised value of reliable and valid outcome measures and the increasing importance of identifying effective smis in stroke , we know of no review that has systematically evaluated international research for the quality of outcome measures used in stroke self - management . the purpose of this article is to systematically review outcome measures used in stroke self - smis , with the aim of informing researchers , healthcare professionals and policy - makers and making recommendations for the design of future outcome measures suitable for use in stroke self - management . this review seeks to systematically examine the outcome measures adopted in stroke smis in terms of the methodology adopted in their development and subsequent strength of their psychometric properties for use with stroke populations . often the methodology adopted in reviews of outcome measures , use differing assessment standards , creating confusion for researchers and clinicians . a recent international delphi study of 57 experts ( 63% response rate ) resulted in a tool to assess the methodological quality of studies on measurement properties , referred to as the consensus - based standards for the selection of health status measurement instruments ( cosmin ) checklist . the cosmin list has good inter - rater agreement and reliability and represents the first critical appraisal tool that is based on the consensus of experts in psychometric theory . cosmin has been used in other systematic reviews examining the measurement properties of outcome measures in a range of health conditions [ 3941 ] . the delphi study consisted of four rounds and sought to reach consensus on the terminology and definitions to be adopted with regard to psychometrics . this consensus offers both researchers and clinicians guidance with regard to some of the complexities of measurement properties . cosmin also addresses modern psychometric theory methodology , such as item response theory , as well as classical test theory . further information on cosmin can be accessed via www.cosmin.nl the properties examined in this systematic review are defined by cosmin and consist of nine items : internal consistency ; reliability ; measurement error ; content validity ; structural validity ; hypothesis testing ; cross - cultural validity ; criterion validity and responsiveness . the purpose of the review is not to make a judgement on the quality of the smi studies , or to synthesize findings to answer questions regarding the effectiveness of the interventions in the review . instead the review focuses upon the value of the outcome measures adopted within stroke smi studies according to their methodological quality , reliability and validity for stroke populations as outlined by cosmin .this is vital since judgments about the results and impact attributed to smis are dependent on valid and reliable measurement . in order to examine the properties of the outcome measures used in stroke smis , it was first necessary to identify which measures were used to evaluate the smis . stroke self - management literature was systematically searched on the following electronic databases by one author ( e.j.b . ) : medline , psychinfo , science direct , web of science and cinahl . the following terms were used to identify existing stroke smi studies : search terms were chosen to represent concepts often linked to self - management ( education , rehabilitation ) , however , studies were excluded unless they specifically stated their purpose was to enhance self - management . article reference lists , website of uk government health department , generic internet search engines , and stroke - specific organisations were also searched . dissertations and conference abstracts were excluded , however , searches for publications by dissertation or conference abstract authors were conducted . selected articles described either ( 1 ) stroke - smi development and/or implementation or ( 2 ) presented outcomes of stroke smis . identified interventions and associated outcome measures were extracted and tabulated . authors screened each abstract to eliminate articles that were not relevant , based on the following inclusion criteria : the study was published in english ; the article addressed self - management specific to stroke ; and was published between january 1990 and june 2011 , to examine the current and most relevant evidence for practice . one reviewer ( e.j.b . ) assessed all relevant full text articles obtained and a second reviewer ( s.d . ) assessed 10% to check reliability . outcome measures included in studies were then identified and recorded ( the version adopted by the smi was selected for review ) . a second literature search then sought to find evidence for the measurement properties ( as outlined by cosmin ) of those outcome measures identified . the following electronic databases were used : medline , psychinfo , science direct , web of science and cinahl . the following search terms were used in conjunction with the title of the identified outcome measure : for example , for a search on the validity of the geriatric depression scale , the search terms stroke and validity and geriatric depression scale was performed . search terms were chosen to represent the properties of outcome measure quality as outlined by the cosmin checklist . no time limitations were set as advocated by cosmin , since older literature on measurement properties is still relevant . this is crucial since a measure s reliability and validity are on - going properties , dependent upon the context and population with which it is used . for example , a measure developed to assess quality of life with a traumatic brain injury population will not necessarily possess acceptable content validity for stroke populations , since the issues faced by both populations may have similarities and differences . article reference lists from the originating studies and generic internet search engines were also searched . discussion between the authors sought to clarify any issues regarding terminology and interpretation of the cosmin checklist and preceded scoring of the identified measurement tools . studies were excluded if they investigated postal or proxy reliability and validity unless this was how they were used in the smis . for outcome measures with more than one result per cosmin criteria , the article stating the most robust results was reviewed . where it was not clear that the study populations were specifically stroke , articles were excluded . one reviewer ( e.j.b . ) assessed all relevant full text articles using a standard data extraction form advocated by cosmin . to ensure consistency of interpretation and scoring , a second reviewer ( s.d . ) independently scored a random 10% of the articles , with discussion between the two reviewers regarding the scores attained . paper authors were contacted for further details where the study reported on early phases , or cited unpublished work . ten items are used to assess whether a study meets the standard for good methodological quality . two items contain general requirements for articles in which item response theory ( irt ) methods and general requirements for the generalisability of the results are applied . where a published paper does not report on a cosmin item for example , if the responsiveness of the measure has yet to be determined , this item is not scored . each item is rated as excellent ( + + + + ) , good ( + + + ) , fair ( + + ) or poor ( + ) . 43 abstracts were identified as potentially relevant studies and were screened ( 46 duplicate records were excluded ) . from these , 19 articles were retrieved and reviewed for inclusion criteria and data extraction ( studies were excluded because they did not meet the detailed criteria or if they reported on an earlier phase of the same study ) . outcome measures within each study were then identified and grouped conceptually into different themes , using content analysis . three studies of stroke - smis originated from the uk ; three from australia ; two from canada ; two from the usa ; two from sweden and one from hong kong . nine studies reported upon interventions aimed at community - dwelling participants ; two upon the acute recovery phase ( <3 months post - stroke ) ; one upon recovery for care home residents and one study did not report details of the setting . four studies delivered individualised interventions ; three utilised workbook interventions ; four tested group smis designed specifically for stroke and two tested existing self - management programs adapted for stroke . interventions were delivered primarily ; by allied health professionals ( six ) ; nurse specialists ( four ) ; researcher ( two ) and lay experts ( one ) . four studies identified primary outcomes ; health - related quality of life ; self - efficacy ; physical functioning and feasibility . although all studies focused upon stroke self - management , none measured stroke self - management as a discrete concept . instead , a range of concepts were measured which presumably were selected to reflect the expected outcomes or process of self - management . evaluation relies upon judgements concerning the process of the smi and the outcome expected following participation in the smi . the majority of measures used sought to measure health outcomes e.g. physical functioning , mood , quality of life ( figure 1 ) . attitudes were also measured which could be considered to more readily reflect the process of self - management e.g. healthcare utilization , medication compliance although the theoretical mechanisms linking self - management to these concepts was not elucidated by the authors . the term unreported is used in this review to describe an outcome measure that has not , at time of writing , been published in peer reviewed publicly available media . unreported measures relate to those developed either by the study authors or through modifications made to existing measures without examination to ensure such assumptions or modifications were valid . therefore , it is not possible to determine if unreported measures meet any of the cosmin criteria . six studies adopted unreported measures of concepts presumably ( although the theoretical links were not explicitly stated by the authors ) , relating to the process of self - management , that had unknown reliability , validity , responsiveness . allen et al . included an unreported measure to assess condition management and patient and carer satisfaction with the intervention . two studies used 10 point visual analogue rating scales ( vass ) to assess usefulness and intelligibility and satisfaction related to the intervention . whilst vass are brief and simple to administer and minimal in terms of respondent burden , without established reliability or validity relating to the underlying construct purported to be measured , they remain of limited value . ljungberg and colleagues designed four questions to assess life satisfaction pre- and post - participation in the smi and sit and colleagues modified an existing stroke knowledge scale ; details of the modifications were absent in the paper . marsden and colleagues used a measure of stroke knowledge test , but stated clearly they were not basing inferences from the data obtained using this measure . forty - three different outcome measures were adopted by studies in this review of measures used in stroke smis . of these , 21 measures ( 49% ) demonstrated some properties in stroke populations , according to the cosmin checklist ( table ii ) . for the remaining measures no evidence could be found for any of the cosmin properties in stroke populations ( n = 16 , 39% ) , or the measures were observer - based assessments ( n = 5 , 12% ) . cosmin property scores in stroke populations of outcome measures used in stroke self - management interventions . a summary of how the measures included in the stroke smi studies scored according to cosmin , when examined for their measurement properties with stroke populations , is shown in figure 2 . none scored in every category of cosmin . where more than one paper addressed a cosmin category , the article which stated the most robust results was scored . the majority of measures scored either excellent rating was content validity . three measures scored excellent in this category as follows ; the stroke adapted sickness impact profile ( sa - sip30 ) ; the stroke self - efficacy questionnaire ( sseq ) and the subjective index of physical and social outcome ( sipso ) . 43 abstracts were identified as potentially relevant studies and were screened ( 46 duplicate records were excluded ) . from these , 19 articles were retrieved and reviewed for inclusion criteria and data extraction ( studies were excluded because they did not meet the detailed criteria or if they reported on an earlier phase of the same study ) . outcome measures within each study were then identified and grouped conceptually into different themes , using content analysis . . three studies of stroke - smis originated from the uk ; three from australia ; two from canada ; two from the usa ; two from sweden and one from hong kong . nine studies reported upon interventions aimed at community - dwelling participants ; two upon the acute recovery phase ( <3 months post - stroke ) ; one upon recovery for care home residents and one study did not report details of the setting . four studies delivered individualised interventions ; three utilised workbook interventions ; four tested group smis designed specifically for stroke and two tested existing self - management programs adapted for stroke . interventions were delivered primarily ; by allied health professionals ( six ) ; nurse specialists ( four ) ; researcher ( two ) and lay experts ( one ) . four studies identified primary outcomes ; health - related quality of life ; self - efficacy ; physical functioning and feasibility . although all studies focused upon stroke self - management , none measured stroke self - management as a discrete concept . instead , a range of concepts were measured which presumably were selected to reflect the expected outcomes or process of self - management . evaluation relies upon judgements concerning the process of the smi and the outcome expected following participation in the smi . the majority of measures used sought to measure health outcomes e.g. physical functioning , mood , quality of life ( figure 1 ) . attitudes were also measured which could be considered to more readily reflect the process of self - management e.g. healthcare utilization , medication compliance although the theoretical mechanisms linking self - management to these concepts was not elucidated by the authors . the term unreported is used in this review to describe an outcome measure that has not , at time of writing , been published in peer reviewed publicly available media . unreported measures relate to those developed either by the study authors or through modifications made to existing measures without examination to ensure such assumptions or modifications were valid . therefore , it is not possible to determine if unreported measures meet any of the cosmin criteria . six studies adopted unreported measures of concepts presumably ( although the theoretical links were not explicitly stated by the authors ) , relating to the process of self - management , that had unknown reliability , validity , responsiveness . allen et al . included an unreported measure to assess condition management and patient and carer satisfaction with the intervention . two studies used 10 point visual analogue rating scales ( vass ) to assess usefulness and intelligibility and satisfaction related to the intervention . whilst vass are brief and simple to administer and minimal in terms of respondent burden , without established reliability or validity relating to the underlying construct purported to be measured , they remain of limited value . ljungberg and colleagues designed four questions to assess life satisfaction pre- and post - participation in the smi and sit and colleagues modified an existing stroke knowledge scale ; details of the modifications were absent in the paper . marsden and colleagues used a measure of stroke knowledge test , but stated clearly they were not basing inferences from the data obtained using this measure . forty - three different outcome measures were adopted by studies in this review of measures used in stroke smis . of these , 21 measures ( 49% ) demonstrated some properties in stroke populations , according to the cosmin checklist ( table ii ) . for the remaining measures no evidence could be found for any of the cosmin properties in stroke populations ( n = 16 , 39% ) , or the measures were observer - based assessments ( n = 5 , 12% ) . cosmin property scores in stroke populations of outcome measures used in stroke self - management interventions . a summary of how the measures included in the stroke smi studies scored according to cosmin , when examined for their measurement properties with stroke populations , is shown in figure 2 . none scored in every category of cosmin . where more than one paper addressed a cosmin category , the article which stated the most robust results was scored . the majority of measures scored either three measures scored excellent in this category as follows ; the stroke adapted sickness impact profile ( sa - sip30 ) ; the stroke self - efficacy questionnaire ( sseq ) and the subjective index of physical and social outcome ( sipso ) . this review examined the methodological quality of studies determining the psychometric properties of outcome measures , used in stroke smis according to criteria outlined by the cosmin checklist . consistent with measurement theory we explored the validity and reliability of these measures for use in people with stroke , not their general use in broader populations . to our knowledge , this is the first review to systematically appraise and summarize the evidence on the quality of outcome measures used in stroke smis . since no study adopted a measure of stroke self - management attitudes or behaviours , the theoretical concepts utilised by studies in the review to measure self - management will first be addressed . the range and number of different published outcome measures adopted by studies in this review may suggest a current lack of consensus regarding the appropriate measures to assist evaluation of stroke smis . alternatively , the use of heterogeneous measures may be reflective of recognition by researchers that self - management embraces a range of differing concepts . the current absence of consensus may in part reflect an underlying lack of consensus about the concept and operation of self - management in stroke . in addition , most smis have been developed for generic audiences , which may partly explain the lack of specific measures developed for stroke self - management . an argument exists for research to investigate the conceptual properties of stroke self - management , to examine which measurement concepts currently being used , if any , are appropriate . some captured health outcomes , such as physical functioning , which the study authors anticipated may be affected by the smi ; others attempted to capture behaviours , such as resource utilisation or attitudes , such as changes in self - efficacy thought to be associated with self - management processes ( figure 2 ) . however , how the concepts measured align with the patient experience of stroke self - management remain unknown . physical function ( pf ) was most often used as an indicator of effective self - management . of the 21 measures possessing at least one property of the cosmin checklist in this review , 11 related to pf ( 52% ) . this is potentially suggestive of an assumption that effective self - management results in improved pf , or that improved pf is a desired outcome . pf appears to remain a dominant concept within stroke rehabilitation , despite increasing evidence of the role of psychosocial factors in recovery [ 5355 ] . for example , the measurement of pf is of limited value in studies that target speech disorder , depression , social participation or cognitive function , debatably all factors in effective stroke self - management . questions regarding the differing priorities of rehabilitation between healthcare professionals and those affected by stroke have been raised before . effective self - management extends beyond the ability to perform certain tasks , encompassing decision making and choices regarding health and behaviour . the role of pf in stroke self - management requires further clarification before it can be adopted as a robust indicator of effective self - management . . however , issues of potential greater importance to patients , for example a change in confidence or increased awareness about how to manage fatigue , may not be captured in measures focused upon management of health behaviours or resource utilisation . there is a need to further conceptualise stroke self - management to ensure that self - management strategies pertinent to people recovering from stroke are captured in existing or new outcome measures . eight of the studies in this review explicitly cited a theoretical basis to the intervention adopted in the study ( table i ) . the most commonly cited theory was psychologist albert bandura s social cognition theory and the concept of self - efficacy ( n = 4 studies ) . self - efficacy can be described as the belief in one s capabilities to organise and execute the course of action required , to produce given achievements . the validity of outcome measures is contingent upon using them for the purpose they were intended for . of the four studies citing self - efficacy as a theoretical premise underpinning the intervention , only two studies utilised outcome measures to reflect change attributed to this theoretical concept in the interventions . the measure adopted by kendall and colleagues , the self - efficacy scale , has unknown psychometric properties in stroke populations , and therefore requires further examination to establish its validity for use in these populations . the stroke self - efficacy scale adopted by jones and colleagues was developed with stroke populations . data were generated with people a relatively short time frame since stroke ( mean duration was 4.2 weeks and 16 days post - stroke for two of the development phases ) . this may not represent sufficient time since stroke for individuals to adequately appraise their situation , especially as some were still in hospital . the relationship to self - management of any of the measures in this review was not explicitly stated by any of the study authors . this suggests that further clarification is required to determine the extent to which they reflect the process or outcomes of self - management . whilst potential theoretical bases for the self - management of long - term conditions , such as self - efficacy , have gained increasing acknowledgement , the role in stroke self - management remains unclear . this is in part due to a lack of robust outcome measures and , in addition , a lack of clarity regarding the purported theoretical foundations of stroke self - management . a paucity of measures scored excellent or good for quality according to the criteria outlined by cosmin ( figure 2 ) . the cosmin checklist does not advocate summarising the quality criteria into one overall quality score , as is often the case in other systematic reviews . since measurement properties are in part affected by the context in which they have been determined , this approach would be misleading . for example in our review , no measure was scored on cross - cultural validity , since the purpose of the review was not to assess how well a measure had been developed and validated in other languages or cultures . outcome measures should be developed with involvement of the target population to identify what is meaningful from their perspective and hence enhance content validity and clinical utility . three measures scored excellent in the content validity category ( sseq ; sipso ; sa - sip30 ) . measures that did not include involvement of users in the development of the measure scored poor on the cosmin checklist , regardless of other aspects of the content validity process which may have been classed fair this is partly as a result of the cosmins scoring method in which the lowest score in any given category counts , but is also indicative of the importance of involving potential users in measurement development . arguably , measures developed without user - involvement have questionable meaning and other types of validity , since without steps in the design to capture the experience of the population to be measured , the context of the measure remains largely that of the measure developers . more recently techniques such as cognitive interviewing , have been used by researchers to ensure the content validity of new measures is optimal . difficulty exists in determining which measures used in the stroke smis in this review reflect self - management with validity since most measures did not score well according to the cosmin criteria . several studies included unreported measures , designed specifically by the authors for the purpose of the smi study [ 44,4852 ] . with the exception of marsden et al . an absence of psychometric data confounds the ability to draw reliable inferences from studies adopting those measures . in addition , a lack of information regarding the development or modification of unreported measures limits the ability to make judgments upon the validity and appropriateness of the measure . a further possible limitation on interpreting data from unreported measures may be a tendency for reporting positive results . without establishment of reliability and validity the outcome measure is little more than a collection of items that have meaning to the developer alone . given the lack of consensus of how stroke self - management operates in the literature , and a lack of consensus upon the theoretical premises grounding stroke smis , the assumptions underpinning unreported measures remain speculative . researchers and clinicians should exercise caution in considering findings from studies adopting unreported measures . of note is that 11 studies ( 85% ) within this review adopted at least one outcome measure without reported validity and reliability with stroke populations . the reporting of minimal , or non - significant , observed changes following stroke smis in those studies including measures without established psychometric properties in stroke populations may be indicative of a lack of relevance and meaningfulness of those measures to stroke populations . problems exist in using unreported measures when determining whether change occurred as a result of an ineffective intervention or due to imprecise measures . measures developed with intended user populations , facilitate the gaining of information about health , illness and the effects of health - care interventions from the perspective of the patient . as well as enhancing content validity , this is of particular relevance to those involved in promoting self - management and increasing patient autonomy , such as nurses and therapists . responsiveness is a necessary property of instruments intended for measuring clinically meaningful change , such as in stroke self - smis . arguments exist that responsiveness should focus upon detecting change that is valued by the person rather than the clinician or researcher . change attributed to an intervention is an important aspect of evaluating clinical effectiveness . in this review , none of these measures scored excellent for this property , and only 15% scored good . aside from inadequate sample sizes , a common finding was that studies often were not clear about what happened to study populations between testing . there is , therefore , a need for future measurement developers to specify these overlooked aspects of development more clearly in subsequent reporting . the majority of smi study populations within this review experienced stroke < 24 months previously , with a number of studies using populations experiencing stroke no more than 6 months previously . this may be a result of sampling to reduce the influence of additional factors upon study outcomes , such as the development of unhelpful coping behaviours , the likelihood of which might increase over time , and out of an assumption that more change may be observed in those early in their recovery . however , in reality the number of people living in the community and recovering from stroke extends beyond those who are 624 months post - stroke . as engagement in self - management activities varies during recovery , particularly following adjustment to stroke as a long - term condition , there is a future need to consider outcome measures sensitive to change(s ) at different durations since stroke . the role of proms , developed using rigorous investigation with the population to be measured extends beyond validating patient experience . proms may improve the quality of interactions between health professionals and patients , assess levels of health and need , and provide evidence of outcomes of services , for the purposes of audit , quality assurance and comparative performance evaluation . this is of particular importance when trying to capture the essence of self - management , since the experience of clients is vital in determining what is valued from their perspective . there is a need to focus upon the development of measures of self - management developed with people recovering from stroke . consensus between reviewers was used to determine eligibility and inclusion of smi articles . whilst we were in agreement however , the use of a standardized critical appraisal tool may assist the selection of articles for future reviews . where interpretation of the cosmin criteria differed , agreement was reached by discussion and consensus . additional reviewers may have further validated this process however the criteria within cosmin are explicitly stated and differences were quickly resolved . data extraction was facilitated by a standardized tool advocated by cosmin , with extraction and scoring checked in a random 10% of articles.we acknowledge that checking of 10% may be viewed as a limitation of this review , however , assert that a systematic process using a standard data extraction tool was followed throughout . that cosmin operates a lowest score counts scoring system may account for the lack of measures scoring well in the measurement property criteria . some studies used otherwise appropriate methodologies , but were rated as poor due to inadequate sample sizes for analyses . for example , for a measure to be rated as good for reliability , measurement error , criterion validity and responsiveness , a sample size of n = 5099 , the sample size required increased to five times the number of items within a measure ( and 100 or 57 * # items but < 100 ) . therefore , if those studies were repeated with larger sample sizes , their ratings according to cosmin could change dramatically . the tendency for measures to score poorly may be reflective of a floor effect of the cosmin checklist . cosmin was developed following consensus of experts in health measurement , therefore if its stringent criteria is to be adopted this is indicative of a need to debate the rigorous methods for measure development required . it is fair to comment that some of the measures in this review were developed before the focus upon involving potential users in measure development . it may be that the measures examined in this review require further development and investigation to establish adequate measurement properties for use with stroke populations . our recommendations for clinicians and researchers seeking to evaluate such interventions would be firstly to clarify the theoretical premise of the intervention in question , as advocated elsewhere . without this step , it is difficult to identify the mechanisms by which the intervention may influence outcomes , and thus difficult to select an outcome measure which appropriately captures the potential outcome . potential outcome measures should be selected on the basis that they appropriately reflect and capture the expected outcome change . this review highlights that the reported theoretical drivers within stroke smis are unclear , not least because they are often not explicitly stated by researchers . the heterogeneity of the outcome measures utilised by smis in this review may indicate a difficulty in determining the expected outcomes of stroke smis . a systematic review demonstrated that interventions with specific aims , such as reduced systolic blood pressure in hypertension or glycosylated haemoglobin levels in diabetes , produced greater effect sizes than those without defined outcomes . further work is therefore warranted to conceptualise stroke self - management and examine the theoretical premises supporting such interventions , and expected outcomes so that appropriate outcome measures which accurately reflect the concept can be selected and/or developed . until such clarification , researchers and clinicians should , where possible , select outcome measures with reliability and validity data in the population to be tested in the intervention . the selection of outcome measures developed with involvement from the target population is also advocated . this ensures that what is meaningful to the patient is more likely to be captured appropriately , thus enhancing content validity . in the meantime , researchers must support clinicians by conducting further work to examine the concept and theoretical premises of self - management and developing appropriate measures if required . the range and number of different published outcome measures adopted by studies in this review may suggest a current lack of consensus regarding the appropriate measures to assist evaluation of stroke smis . alternatively , the use of heterogeneous measures may be reflective of recognition by researchers that self - management embraces a range of differing concepts . the current absence of consensus may in part reflect an underlying lack of consensus about the concept and operation of self - management in stroke . in addition , most smis have been developed for generic audiences , which may partly explain the lack of specific measures developed for stroke self - management . an argument exists for research to investigate the conceptual properties of stroke self - management , to examine which measurement concepts currently being used , if any , are appropriate . some captured health outcomes , such as physical functioning , which the study authors anticipated may be affected by the smi ; others attempted to capture behaviours , such as resource utilisation or attitudes , such as changes in self - efficacy thought to be associated with self - management processes ( figure 2 ) . however , how the concepts measured align with the patient experience of stroke self - management remain unknown . physical function ( pf ) was most often used as an indicator of effective self - management . of the 21 measures possessing at least one property of the cosmin checklist in this review , 11 related to pf ( 52% ) . this is potentially suggestive of an assumption that effective self - management results in improved pf , or that improved pf is a desired outcome . pf appears to remain a dominant concept within stroke rehabilitation , despite increasing evidence of the role of psychosocial factors in recovery [ 5355 ] . for example , the measurement of pf is of limited value in studies that target speech disorder , depression , social participation or cognitive function , debatably all factors in effective stroke self - management . questions regarding the differing priorities of rehabilitation between healthcare professionals and those affected by stroke have been raised before . effective self - management extends beyond the ability to perform certain tasks , encompassing decision making and choices regarding health and behaviour . the role of pf in stroke self - management requires further clarification before it can be adopted as a robust indicator of effective self - management . . however , issues of potential greater importance to patients , for example a change in confidence or increased awareness about how to manage fatigue , may not be captured in measures focused upon management of health behaviours or resource utilisation . there is a need to further conceptualise stroke self - management to ensure that self - management strategies pertinent to people recovering from stroke are captured in existing or new outcome measures . eight of the studies in this review explicitly cited a theoretical basis to the intervention adopted in the study ( table i ) . the most commonly cited theory was psychologist albert bandura s social cognition theory and the concept of self - efficacy ( n = 4 studies ) . self - efficacy can be described as the belief in one s capabilities to organise and execute the course of action required , to produce given achievements . the validity of outcome measures is contingent upon using them for the purpose they were intended for . of the four studies citing self - efficacy as a theoretical premise underpinning the intervention , only two studies utilised outcome measures to reflect change attributed to this theoretical concept in the interventions . the measure adopted by kendall and colleagues , the self - efficacy scale , has unknown psychometric properties in stroke populations , and therefore requires further examination to establish its validity for use in these populations . the stroke self - efficacy scale adopted by jones and colleagues was developed with stroke populations . data were generated with people a relatively short time frame since stroke ( mean duration was 4.2 weeks and 16 days post - stroke for two of the development phases ) . this may not represent sufficient time since stroke for individuals to adequately appraise their situation , especially as some were still in hospital . the relationship to self - management of any of the measures in this review was not explicitly stated by any of the study authors . this suggests that further clarification is required to determine the extent to which they reflect the process or outcomes of self - management . whilst potential theoretical bases for the self - management of long - term conditions , such as self - efficacy , have gained increasing acknowledgement , the role in stroke self - management remains unclear . this is in part due to a lack of robust outcome measures and , in addition , a lack of clarity regarding the purported theoretical foundations of stroke self - management . a paucity of measures scored excellent or good for quality according to the criteria outlined by cosmin ( figure 2 ) . the cosmin checklist does not advocate summarising the quality criteria into one overall quality score , as is often the case in other systematic reviews . since measurement properties are in part affected by the context in which they have been determined , this approach would be misleading . for example in our review , no measure was scored on cross - cultural validity , since the purpose of the review was not to assess how well a measure had been developed and validated in other languages or cultures . outcome measures should be developed with involvement of the target population to identify what is meaningful from their perspective and hence enhance content validity and clinical utility . three measures scored excellent in the content validity category ( sseq ; sipso ; sa - sip30 ) . measures that did not include involvement of users in the development of the measure scored poor on the cosmin checklist , regardless of other aspects of the content validity process which may have been classed fair this is partly as a result of the cosmins scoring method in which the lowest score in any given category counts , but is also indicative of the importance of involving potential users in measurement development . arguably , measures developed without user - involvement have questionable meaning and other types of validity , since without steps in the design to capture the experience of the population to be measured , the context of the measure remains largely that of the measure developers . more recently techniques such as cognitive interviewing , have been used by researchers to ensure the content validity of new measures is optimal . difficulty exists in determining which measures used in the stroke smis in this review reflect self - management with validity since most measures did not score well according to the cosmin criteria . several studies included unreported measures , designed specifically by the authors for the purpose of the smi study [ 44,4852 ] . with the exception of marsden et al . an absence of psychometric data confounds the ability to draw reliable inferences from studies adopting those measures . in addition , a lack of information regarding the development or modification of unreported measures limits the ability to make judgments upon the validity and appropriateness of the measure . a further possible limitation on interpreting data from unreported measures may be a tendency for reporting positive results . without establishment of reliability and validity the outcome measure is little more than a collection of items that have meaning to the developer alone . given the lack of consensus of how stroke self - management operates in the literature , and a lack of consensus upon the theoretical premises grounding stroke smis , the assumptions underpinning unreported measures remain speculative . researchers and clinicians should exercise caution in considering findings from studies adopting unreported measures . of note is that 11 studies ( 85% ) within this review adopted at least one outcome measure without reported validity and reliability with stroke populations . the reporting of minimal , or non - significant , observed changes following stroke smis in those studies including measures without established psychometric properties in stroke populations may be indicative of a lack of relevance and meaningfulness of those measures to stroke populations . problems exist in using unreported measures when determining whether change occurred as a result of an ineffective intervention or due to imprecise measures . measures developed with intended user populations , facilitate the gaining of information about health , illness and the effects of health - care interventions from the perspective of the patient . as well as enhancing content validity , this is of particular relevance to those involved in promoting self - management and increasing patient autonomy , such as nurses and therapists . responsiveness is a necessary property of instruments intended for measuring clinically meaningful change , such as in stroke self - smis . arguments exist that responsiveness should focus upon detecting change that is valued by the person rather than the clinician or researcher . change attributed to an intervention is an important aspect of evaluating clinical effectiveness . in this review , none of these measures scored excellent for this property , and only 15% scored good . aside from inadequate sample sizes , a common finding was that studies often were not clear about what happened to study populations between testing . there is , therefore , a need for future measurement developers to specify these overlooked aspects of development more clearly in subsequent reporting . the majority of smi study populations within this review experienced stroke < 24 months previously , with a number of studies using populations experiencing stroke no more than 6 months previously . this may be a result of sampling to reduce the influence of additional factors upon study outcomes , such as the development of unhelpful coping behaviours , the likelihood of which might increase over time , and out of an assumption that more change may be observed in those early in their recovery . however , in reality the number of people living in the community and recovering from stroke extends beyond those who are 624 months post - stroke . as engagement in self - management activities varies during recovery , particularly following adjustment to stroke as a long - term condition , there is a future need to consider outcome measures sensitive to change(s ) at different durations since stroke . the role of proms , developed using rigorous investigation with the population to be measured extends beyond validating patient experience . proms may improve the quality of interactions between health professionals and patients , assess levels of health and need , and provide evidence of outcomes of services , for the purposes of audit , quality assurance and comparative performance evaluation . this is of particular importance when trying to capture the essence of self - management , since the experience of clients is vital in determining what is valued from their perspective . there is a need to focus upon the development of measures of self - management developed with people recovering from stroke . consensus between reviewers was used to determine eligibility and inclusion of smi articles . whilst we were in agreement however , the use of a standardized critical appraisal tool may assist the selection of articles for future reviews . where interpretation of the cosmin criteria differed , agreement was reached by discussion and consensus . additional reviewers may have further validated this process however the criteria within cosmin are explicitly stated and differences were quickly resolved . data extraction was facilitated by a standardized tool advocated by cosmin , with extraction and scoring checked in a random 10% of articles.we acknowledge that checking of 10% may be viewed as a limitation of this review , however , assert that a systematic process using a standard data extraction tool was followed throughout . that cosmin operates a lowest score counts scoring system may account for the lack of measures scoring well in the measurement property criteria . some studies used otherwise appropriate methodologies , but were rated as poor due to inadequate sample sizes for analyses . for example , for a measure to be rated as good for reliability , measurement error , criterion validity and responsiveness , a sample size of n = 5099 , the sample size required increased to five times the number of items within a measure ( and 100 or 57 * # items but < 100 ) . therefore , if those studies were repeated with larger sample sizes , their ratings according to cosmin could change dramatically . the tendency for measures to score poorly may be reflective of a floor effect of the cosmin checklist . cosmin was developed following consensus of experts in health measurement , therefore if its stringent criteria is to be adopted this is indicative of a need to debate the rigorous methods for measure development required . it is fair to comment that some of the measures in this review were developed before the focus upon involving potential users in measure development . it may be that the measures examined in this review require further development and investigation to establish adequate measurement properties for use with stroke populations . our recommendations for clinicians and researchers seeking to evaluate such interventions would be firstly to clarify the theoretical premise of the intervention in question , as advocated elsewhere . without this step , it is difficult to identify the mechanisms by which the intervention may influence outcomes , and thus difficult to select an outcome measure which appropriately captures the potential outcome . potential outcome measures should be selected on the basis that they appropriately reflect and capture the expected outcome change . this review highlights that the reported theoretical drivers within stroke smis are unclear , not least because they are often not explicitly stated by researchers . the heterogeneity of the outcome measures utilised by smis in this review may indicate a difficulty in determining the expected outcomes of stroke smis . a systematic review demonstrated that interventions with specific aims , such as reduced systolic blood pressure in hypertension or glycosylated haemoglobin levels in diabetes , produced greater effect sizes than those without defined outcomes . further work is therefore warranted to conceptualise stroke self - management and examine the theoretical premises supporting such interventions , and expected outcomes so that appropriate outcome measures which accurately reflect the concept can be selected and/or developed . until such clarification , researchers and clinicians should , where possible , select outcome measures with reliability and validity data in the population to be tested in the intervention . the selection of outcome measures developed with involvement from the target population is also advocated . this ensures that what is meaningful to the patient is more likely to be captured appropriately , thus enhancing content validity . in the meantime , researchers must support clinicians by conducting further work to examine the concept and theoretical premises of self - management and developing appropriate measures if required . this is the first systematic review of international research on outcome measures used and selected in stroke self - smi studies . we have identified important limitations in the measures used to evaluate the effectiveness of stroke self - smis , which has significant implications for the inferences we are currently able to draw about the evidence base . none of the measures used in studies of stroke smis , purported to specifically measure self - management as a discrete concept . this is indicative of the difficulty in conceptualisation and operation of this concept , a view expressed elsewhere . further work is required to determine how the measures identified in this review , align with the concept of self - management . the range of outcomes adopted , the lack of observed changes in outcomes following stroke smis and the lack of consensus surrounding which outcome measures to utilise , indicates that the causal mechanisms of stroke smis remain imprecise . stroke smis have raced ahead of the evidence to support their theoretical basis , operation and effective evaluation . work to conceptualise stroke self - management is required to help identify which outcomes are most appropriate for evaluating interventions , to further inform the theoretical basis for smis and to assist the development of interventions . there is a need for studies to explore the theoretical underpinnings of smi in stroke and for the development of robust outcome measures to enable evaluation of stroke smis .

purposeto systematically review the psychometric properties of outcome measures used in stroke self - management interventions ( smis ) to ( 1 ) inform researchers , clinicians and commissioners about the properties of the measures in use and ( 2 ) make recommendations for the future development of self - management measurement in stroke.methodselectronic databases , government websites , generic internet search engines and hand searches of reference lists . abstracts were selected against inclusion criteria and retrieved for appraisal and systematically scored , using the cosmin checklist.resultsthirteen studies of stroke self - management originating from six countries were identified . forty - three different measures ( mean 5.08/study , sd 2.19 ) were adopted to evaluate self - smis . no studies measured self - management as a discreet concept . six ( 46% ) studies included untested measures . eleven ( 85% ) studies included at least one measure without reported reliability and validity in stroke populations.conclusionsthe use of outcome measures which are related , indirect or proxy indicators of self - management and that have questionable reliability and validity , contributes to an inability to sensitively evaluate the effectiveness of stroke self - smis . further enquiry into how the concept of self - management in stroke operates , would help to clarify the nature and range of specific self - management activities to be targeted and aid the selection of existing appropriate measures or the development of new measures.implications for rehabilitationthe evaluation of complex interventions such as self - management interventions is aided by clear outcome expectations and valid and reliable measurement.this review demonstrates a lack of outcome measures that specifically measure self - management of stroke . a minority of outcome measures that were used as proxy indicators for sm fulfill some of the criteria for quality outlined in the cosmin checklist.clinicians should select measures which appropriately reflect expected outcomes , giving due consideration to the theoretical underpinnings of the intervention . further work is required to establish which measures currently in use , if any , accurately reflect stoke self-management.in the meantime , researchers should seek to develop psychometrically sound measures of stroke self - management to assist effective evaluation of such interventions in stroke .

pubmed

osteoarthritis ( oa ) in knee joint is the most common reason behind functional disability that leads to lowered quality of life in old age people of india . some of the objective factors that may influence oa disease severity include but are not limited to age , height , weight , and bmi . in physiotherapy practice , lowering the pain , other symptoms , improving the activities of daily living ( adl ) , and quality of life ( qol ) are main outcome criteria for oa knee rehabilitation . to achieve this rehabilitative goal , physiotherapists use both self - reported questionnaires as well as performance - based tests to monitor prognosis in the above said parameters , that is , pain , symptoms , adl , and qol . self - reported questionnaires give subjective information about the disease process without examiner bias within short period of time , whereas , performance - based tests objectively measures patient 's ability of perform adl activities . examples for self - reported questionnaire are womac , sf-36 , koos , and so forth . examples for physical performance tests are timed up and go test ( tug ) , six - minute walk test ( 6-mwt ) , stair climbing test , and so forth . physical performance tests are affected by motivation and not affected by psychogenic factors such as beliefs , expectations , cognitive impairments , and cultural , lingual , and educational level [ 3 , 4 ] . possible difference between the two may be performance - based tests measure functional limitation , whereas self - reported questionnaire reveals disability , that is , combination of social and psychological side of the functional limitation . functional limitation is sooner detected by performance - based tests than self - reported questionnaires [ 5 , 6 ] . there is no consensus regarding the use of tests while some prefer to use self - reported questionnaires because they are easy to administer and have high internal consistency features ; others prefer performance - based tests as they consider that these tests are essential for adl evaluation of the patient . ideally , both self - reported and functional tests should be used in knee rehabilitation setting [ 7 , 8 ] . in order to ascertain more valid results , performance - based tests should always be correlated with self - reported measures . in previous studies , womac , sf-36 , and koos are compared with timed up and go ( tug ) test [ 7 , 911 ] . however there is no study that compares self - reported questionnaire with 6-mwt which is more deals with adl as this measures both walking ability as well as endurance . koos is a relatively newer self - reported questionnaire that was developed from womac by roos et al . for oa knee patients . thus , primary objective of present study was to see the correlation of subjectively measured koos questionnaire with objectively measured 6-minute walk test ( 6-mwt ) , age , height , weight , and bmi . present study was a cross - sectional survey study in which data was extracted from individuals with clinical and nonradiographic idiopathic or primary knee acr criteria for clinical diagnosis of idiopathic knee oa were based on knee pain in either knee on most days for at least 1 month in the previous year and at least two of the following signs / symptoms : stiffness , crepitus , bony tenderness , and bony enlargement . exclusion criteria were ( 1 ) self - reports of currently doing lower - extremity exercise 2 times per week ; ( 2 ) self - reports of currently fitness walking 90 minutes per week ; ( 3 ) being unable to read english / urdu questionnaire ( koos ) ; ( 4 ) inflammatory arthritis ; ( 5 ) self - reports of having current knee conditions such as meniscus tears and knee ligament ruptures ; ( 6 ) fracture of spine or lower extremities ; ( 7 ) being scheduled to undergo a major surgical procedure in the next 6 months ; ( 8) those with neurological conditions ; ( 9 ) having contraindications for exercise testing based on american college of sports medicine criteria . total of 251 patients ( 117 males , 134 females ) met the above said criteria and were included for the present study . koos is a self - reported questionnaire consisted of 42 questions in total addressing five patient - related domains including pain ( 9 questions ) , other disease - specific symptoms ( 7 questions ) , activity of daily living ( adl ) ( 17 questions ) , sport and recreation function ( 5 questions ) , and knee - related quality of life ( 4 questions ) . the walking track was a 30 m long empty corridor with marks on every 5 meters . patients were asked to walk back and forth along the 30 m track and to cover the longest possible distance in 6 minutes . at the end of every minute patients two weekly test - retest reliability of walking distance was reported to be 0.87 for patients with knee oa . patients stood on a firm surface in their bare feet to have their height measured . body mass was measured at the end of examination with a calibrated bathroom - type digital scale , on a firm surface . patients were weighed in the standing position , wearing light indoor clothes , but no shoes , jewellery , or heavy clothing . body mass index ( bmi ) was calculated with the formula ( weight/(height ) ) . results are given in mean , standard deviation ( sd ) , and range ( minimum maximum ) . correlation was done using spearman rank correlation since self - reported data was ordinal variable . the spearman correlation coefficient was interpreted as follows : < 0.3 : none ; 0.310.5 : weak ; 0.510.7 : strong ; 0.710.9 : very strong ; and > 0.9 : excellent . table 2 shows mean and standard deviation along with range of all subscales of koos and 6-mwt in primary oa knee patients . overall koos - sports and recreation had a minimum mean score followed by qol subscale . 6-mwt had a weak correlation with koos - adl ( rho 0.461 ) and strong correlation with koos - symptom , koos - pain , and koos - sports ( rho 0.578 , 0.619 , and 0.536 , resp . ) and very strong correlation with koos - qol ( rho 0.733 ) . bmi had a strong correlation with koos - pain , koos - symptom , koos - adl , and koos - sports ( rho 0.683 , 0.641 , 0.523 , and 0.640 , resp . ) and very strong correlation with koos - qol ( rho 0.816 ) . weight had a weak correlation with koos - symptom , koos - adl , and koos - sports ( rho 0.485 , 0.333 , and 0.413 , resp . ) and strong correlation with koos - pain and koos - qol ( rho 0.542 and 0.582 , resp . ) . there was no correlation between age and any of koos subscales except age and koos - qol subscale ( rho 0.159 , p < 0.05 ) . the aim of the present study was to see the correlation between koos five subscales and some of the objective measures , that is , 6-mwt , age , height , weight , and bmi . the results show there is weak ( none ) correlation of koos subscales with height , progressively increase with weight and bmi . mean score of koos all subscales were less than present study results but trend is similar between the two studies meaning sports and recreation subscale is the least followed by qol subscale and symptom subscale was the maximum in both studies . the present study results are in agreement with sabirli et al . who reported strong correlation between timed up and go ( tug ) and all subscales of koos . they reported 0.66 , 0.521 , 0.531 , 0.694 , and 0.561 for pain , symptom , adl , sports , and qol whereas present results for them to 6-mwt was 0.619 , 0.578 , 0.461 , 0.536 , and 0.733 , respectively . . found strong correlation between womac and 5-mwt ( r 0.485 , 0.525 , and 0.577 for pain , stiffness , function subscales , respectively , p < 0.001 for all ) . reported negative correlation between womac pain , function subscales , and 6-mwt ( r 0.205 for pain , p < similarly , maly et al . reported mild but significant correlation between 6-mwt , womac pain , and stiffness subscales ( r 0.39 for pain and r 0.48 for stiffness ) . lin et al . found mild correlation between womac and physical performance tests in oa knee patients ( r 0.330.54 ) . . observed mild but significant correlation between observed and self - reported physical performance ( r 0.200.26 ; p < 0.01 ) . kennedy et al . found mild to moderate correlation ( r 0.210.50 ) between self - report and actual physical performance on 1044 knee arthroplasty patients . stratford et al . reported moderate correlation between self - reported lower extremity function scale ( lefs ) and tug time ( r 0.42 ) on patients waiting for knee arthroplasty . . reported positive correlation between gait cycle and sf-36 pain domain ( r 0.6 - 0.7 , p < 0.001 ) as well as physical function domain ( r 0.5 , p < 0.05 ) . recently , adegoke et al . reported a positive correlation ( r = 0.56 ) between self - reported function and actual physical performance ( stair climbing and tug test ) which is similar to our result ( r = 0.461 ) . reported that weight gain is associated with worsening pain , stiffness , and function in knee pain patients . miller et al . reported that 6 monthly changes in weight is correlated with womac pain ( r 0.346 , p < 0.01 ) , stiffness ( r 0.204 ) , and function ( r 0.310 , p < 0.05 ) , respectively . penserga and tanque reported a positive correlation between womac ( function ) and weight ( r = 0.260 ) . correlation between weight and bmi is similar to that of marks ( 0.764 in present study versus 0.83 and 0.896 in marks ' study ) and their reported r = 0.63 for self - determined walking pace with bmi is similar to our koos - adl with bmi ( r = 0.523 ) . however contradictory to our finding , marks reported lower values for vas pain scale with bmi ( r = 0.352 ) . marks concluded that bmi was significantly correlated with pain using vas ( r 0.270 , p < 0.01 ) and disability using aims ( r 0.357 , p < 0.01 ) . reported bmi significantly correlated with all subscales of womac ( p < 0.001 ) and sf-36 but their correlation was < 0.30 . previous studies also report similar correlation between bmi and womac ( sanghi et al . ; r = 0.592 , 0.634 , 0.749 for pain , stiffness , and adl subscales in klc et al . except cubukcu et al . creamer et al . reported positive correlation ( r = 0.42 ) between womac function subscale and bmi . reported mild positive correlation between bmi and sit to stand performance ( r 0.47 , p < 0.01 ) . marks concluded that age was not significantly correlated with pain using vas ( r 0.122 ) and disability using aims ( r 0.175 ) . penserga and tanque reported lack of correlation between age and womac disability subscales ( r 0.077 ) . lack of correlation between koos pain ( r 0.093 ) , symptom ( r 0.071 ) , and adl ( r 0.036 ) subscales with age and weak correlation between age and koos qol ( r 0.159 ) subscale in oa knee patients have already been published in our previous publication with larger sample size . age found to be insignificant correlation with womac pain ( r 0.01 ) and function ( r 0.06 ) subscales in 257 knee arthroplasty patients . elbaz et al . also reported similar results in symptomatic oa knee patients using womac ( 0.028 , 0.023 , 0.09 for pain , stiffness , and function subscales , resp . ) . reported similar result using womac ( correlations between age and womac pain , stiffness , and function were 0.081 , 0.49 , 0.114 , and resp . ; all were statistically insignificant ) . recently , maly et al . in their study found insignificant correlation between age and pain intensity ( r 0.03 ) and physical performance ( r 0.09 ) . the present study results are in confirmation with previous studies and affirms the usage of koos in indian oa knee patients .

purpose . objective of the present study was to see the correlation of subjectively measured koos questionnaire with objectively measured 6-minute walk test ( 6-mwt ) , age , height , weight , and bmi . participants . 251 subjects with oa knee based on american college of rheumatology criteria . methods . after passing inclusion and exclusion criteria , the following parameters were recorded : age , height , weight , and bmi . then subjects were asked to fill koos questionnaire ; then all subjects were asked to do self - paced walk for 6 minutes . analysis . spearman rank test was done to see the correlation . significant level was set at p < 0.05 . results . 6-mwt had a weak correlation with koos - adl ( rho 0.461 ) and strong correlation with koos - symptom , koos - pain , and koos - sports and very strong correlation with koos - qol . bmi had a strong correlation with koos - pain , koos - symptom , koos - adl , and koos - sports and very strong correlation with koos - qol . weight had a weak correlation with koos - symptom , koos - adl , and koos - sports and strong correlation with koos - pain and koos - qol . all the above values were significant with p < 0.001 . conclusion . koos is strongly positively correlated with 6-mwt and negatively correlated with bmi . its correlation strength has decreased with weight .

pubmed

an increasingly sedentary lifestyle combined with rising consumption of caloric foods has transformed obesity into a global event . obesity is a low - level , chronic inflammatory disease with a multifactorial etiology that includes eating habits , a sedentary lifestyle , and genetic predisposition that promotes the excessive accumulation of body fat [ 2 , 3 ] . in addition to its roles as an energy reservoir and heat insulating material , adipose tissue also secretes cell - signaling molecules such as adipokines . adipose tissue secretes proinflammatory factors like interleukin 6 ( il-6 ) , tumor necrosis factor - alpha ( tnf- ) , and leptin and anti - inflammatory factors as and interleukin 10 ( il-10 ) and adiponectin . studies have demonstrated that the administration of a high - fat diet in rodents promotes metabolic changes , inducing the production of proinflammatory interleukins and a chronic inflammatory process [ 5 , 6 ] . the presence of large concentrations of endotoxins and saturated fatty acids in the diet promotes the activation of toll - like receptor 4 ( tlr-4 ) , which triggers the production of proinflammatory cytokines [ 4 , 5 ] . through receptors associated with transmembrane proteins , these cytokines interfere with the expression of other cytokines , thus affecting metabolic homeostasis [ 7 , 8 ] . among various implications that are closely correlated with obesity , menopause deserves particular attention because of the increase in life expectancy of the population . menopause is defined as the permanent cessation of menstruation due to loss of ovarian function [ 9 , 10 ] . the loss of ovarian function , particularly the decline in estrogen production , promotes adverse changes in the profile of lipoproteins , metabolism of glucose and insulin , distribution of body fat , clotting , and vascular endothelium . in combination with weight gain , these changes place women at a high risk of developing cardiovascular diseases , which are very common in this period of life [ 10 , 11 ] . the physiological actions of estrogens in the body are mediated by two distinct estrogen receptors ( ers ) : er and er ; these receptors are nuclear transcription factors involved in the regulation of several complex physiological processes [ 12 , 13 ] . according to benedusi and colleagues , the inflammatory processes that occur following the menopause result from a reduction in circulating levels of estrogens and their receptors ( ers ) , which have anti - inflammatory properties however , controversy persists as to whether the effects of estrogens are anti- or proinflammatory . studies in premenopausal women suggest a beneficial role for estrogen in the prevention of vascular inflammation and consequent atherosclerosis . estrogen exerts an anti - inflammatory effect on the vasculature , through antioxidant effects , generation of nitric oxide , prevention of apoptosis in vascular cells , suppression of proinflammatory cytokines , and modulation of the renin - angiotensin system . in vitro studies have also demonstrated that estrogen has an anti - inflammatory effect in several cell lineages . the hormonal changes that occur during the menopause are responsible for specific physical and metabolic remodeling that , when associated with weight gain and obesity , have a negative impact on women 's health . therefore , it is essential that both obesity and menopause , in particular the effects of estrogen on the inflammatory state , are investigated to improve women 's quality of life . in this study , we evaluated whether a high - fat diet with or without ovariectomy promoted the inflammatory process in rats by assessing the production of pro- and anti - inflammatory factors and their receptors and tlr-4 protein content . the experimental research committee of the so paulo federal university approved all procedures for the care of the animals used in this study . thirty - day - old female wistar rats were used in this study and were kept under controlled conditions of light ( 12 : 12 h light dark cycle with lights on at 07:00 ) and temperature ( 22 1c ) . during the experimental period the animals were maintained in collective cages and received water and the specific diet ad libitum . thirty - day - old rats were divided into two groups and fed for 4 weeks with one of the following diets : control diet ( c ) or hyperlipidic diet ( h ) . after this period the animals were divided into four groups : sham c : sham group treated with control diet;sham h : sham group treated with hyperlipidic diet;ovx c : ovariectomized group treated with control diet;ovx h : ovariectomized group treated with hyperlipidic diet . sham c : sham group treated with control diet ; sham h : sham group treated with hyperlipidic diet ; ovx c : ovariectomized group treated with control diet ; ovx h : ovariectomized group treated with hyperlipidic diet . the food intake and body weight were measured weekly at 09:00 . the control diet was prepared according to the recommendations of the american institute of nutrition ( ain-93 m ) and the hyperlipidic diet was by ain-93 m modified . the fat content in the control diet ( c ) was 4% containing 0.64 g of saturated fatty acids , and the hyperlipidic diet containing 20% of fat which 7.7 g was saturated fatty acids . the energy content of c diet was 4114.71 kj and of hc diet 6438.58 kj/100 g ( table 1 ) . at 60 days of age , the rats were anesthetized and received intraperitoneal injection of ketamine ( 70 mg / kg ) and xylazine ( 10 mg / kg ) . the ovx group was submitted to bilateral ovariectomy . for this , they were subjected to a muscular incision to open the peritoneal cavity for posterior connection of the uterine tubules and removal of the ovaries . all the animals received antibiotic ( penicillin ) immediately after the surgery and 0.1 ml / kg body weight of ibuprofen 50 mg for 2 days . the animals were euthanized by decapitation on the 90th day of life in the fasting state ( 10 h ) in the early morning to avoid chronobiological variations . the sham groups were in estrus phase ( phase when estrogen secretions exert their biggest influence ) . trunk blood was collected and immediately centrifuged at 4c and serum aliquots were taken and frozen at 80c to measure the concentrations of glucose , triacylglycerols , and total cholesterol using commercials kits from labtest diagnostic sa ( mg , brazil ) . the concentrations of insulin , leptin , and adiponectin were determined by elisa ( linco research inc . the retroperitoneal , parametrial ( par ) , and mesenteric ( mes ) adipose tissues , gastrocnemius muscle ( gast ) , and liver were dissected , weighed , frozen in liquid nitrogen , and stored at 80c until the protein extraction . for determination of carcass lipid and protein content lipid content was measured as described by stansbie et al . and standardized using the method described by oller do nascimento and williamson . briefly , the eviscerated carcass was autoclaved at 120c for 90 min and homogenized with double the mass of water . triplicate aliquots of this homogenate were weighed and digested in 3 ml of 30% koh and 3 ml of ethanol for at least 2 h at 70c in capped tubes . after cooling , 2 ml of 12 n h2so4 was added , and the sample was washed three times with petroleum ether for lipid extraction . results are expressed as grams of lipid/100 g of carcass . for protein measurements , aliquots of the same homogenate ( approximately 1 g ) were heated to 37c for 1 h in 0.6 n koh with constant shaking . after clarification by centrifugation , protein content was measured according to bradford ( bio - rad , hercules , ca ) . adipose tissue depots , gastrocnemius muscle , and liver ( 0.150.3 g ) were homogenized in ice - cold solubilization and total protein extraction buffer ( 100 mm tris , ph 7.5 , 100 mm sodium fluoride , 10 mm sodium orthovanadate , 2 mm phenylmethylsulfonyl fluoride , 10 mm sodium pyrophosphate , and 0.1 mg / ml aprotinin ) . homogenates were centrifuged at 19283 g for 40 min at 4c . the supernatants were saved and the protein concentrations were determined using a bradford assay ( bio - rad , hercules , ca ) with bovine serum albumin as a reference . fifty micrograms of the specific tissue total protein was loaded onto a sodium dodecyl sulfate - polyacrylamide gel ( 5% stacking gel ; 10% running gel ) , separated by electrophoresis , and then electroblotted onto nitrocellulose membranes ( hybond - c extra , amersham ) using a wet electroblotter ( bio - rad , ca , usa ) . after blotting , the membranes were blocked in tris - buffered saline- ( tbs- ) tween buffer , ph 7.5 ( 20 mm tris/500 mm nacl/0.05% tween-20 ) , containing 1% bovine serum albumin and then exposed to specific antibodies diluted in tbs - tween buffer ( ph 7.5 ) containing 1% bsa for 2 h. the membranes then were washed and incubated with anti - rabbit ig or anti - mouse ig conjugated to horseradish peroxidase and diluted to 1/1000 in the same buffer for 1 h. after a series of washes in tbs - tween buffer , the bands were visualized with enhanced chemiluminescence scanned at uvitec ( cambridge ) after adding the ecl reagent ( ge healthcare bio - sciences ab , uk ) . the antibodies against anti - tlr4 ( sc-99183 ) , anti - tnf - r1 ( sc-7895 ) , anti - il-6 r ( sc-660 ) , anti - myd88 ( sc-8197 ) , and anti--tubulin ( sc-58667 ) were obtained from santa cruz biotechnology ( santa cruz , ca , usa ) , and the anti - rabbit ig and anti - mouse ig conjugated to horseradish peroxidase were obtained from sigma ( usa ) . the results were analyzed using two - way analysis of variance followed by tukey 's test . the initial body , liver , gast , and par weights were similar in all groups . in the ovx h group , the ret weight was higher than in the sham c group and the mes weight was higher than in the sham h group . administration of a hyperlipidic diet in the sham group did not modify these parameters compared with the administration of a control diet . however , the association of a hyperlipidic diet with ovariectomy ( ovx h ) caused a significant increase in final body weight . carcass protein content was greater in the ovx c group than both sham groups . however , carcass lipid content in the ovx h group was higher than that in the sham h group ( table 2 ) . the initial 4 weeks of hyperlipidic diet administration did not modify body weight gain and food efficiency ( figures 1(a ) and 1(b ) ) . however , the combination of ovariectomy and administration of a hyperlipidic diet ( ovx h ) increased body weight gain accompanied by high food efficiency ( figures 1(c ) and 1(d ) ) . the serum concentrations of total cholesterol , hdl ( high - density lipoprotein ) cholesterol , glucose , adiponectin , and estradiol were similar in all groups . serum levels of triacylglycerol ( tg ) were decreased in the sham h group compared with the sham c group . serum insulin concentration was lower in the sham h , ovx c , and ovx h groups than in the sham c group . ovariectomy combined with the administration of a hyperlipidic diet caused an increase in serum leptin levels compared with all other groups ( table 3 ) . the cytokines content was similar in the sham c , sham h , ovx c , and ovx h groups and in the liver and gast ( figures 2 and 3 ) . however , administration of a hyperlipidic diet caused a decrease in il-6 and il-10 levels in mes adipose tissues . furthermore , administration of a hyperlipidic diet associated with ovariectomy decreased the tnf- content of mes ( figure 4 ) . the il-6 content was decreased by the hyperlipidic diet ; however , in combination with ovariectomy , this effect was reversed in ret adipose tissues ( figure 5 ) . the hyperlipidic diet caused a decrease in il-10 levels , but ovariectomy had the opposite effect , and the tnf- content of ret adipose tissues was high following ovariectomy ( figure 5 ) . the il-6 receptor , tnf receptor , tlr-4 , and myd88 protein content did not differ between groups in the liver ( figure 6 ) , gast ( figure 7 ) , and mes adipose tissues ( figure 8) . in this study , we demonstrated that administration of a hyperlipidic diet for 8 weeks combined with short - term ovariectomy promoted an increase in body weight gain and a decrease in metabolic efficiency , accompanied by hyperleptinemia . the cytokines content of the liver and gast was unchanged ; however , levels of all cytokines analyzed were decreased in mes adipose tissues . also , we demonstrated that 4 weeks of ovariectomy ( ovx c group ) per se did not modify these parameters compared with the sham c group . however , ryou et al . demonstrated that 8 weeks of ovariectomy in rats caused an increase in body weight and food efficiency with elevated plasma leptin . these results suggest that the time period after ovariectomy may influence body weight gain and food efficiency . previously , jen et al . showed that high - fat diet fed male rats were heavier than chow - fed male rats by the sixth week of a high - fat diet , whereas female rats were heavier by the ninth week . consistent with this , we demonstrated that the administration of a hyperlipidic diet for 8 weeks compared with a control diet did not cause an increase in body weight gain in female rats . . showed that ingestion of a high - fat diet for 8 weeks did not modify body or tissue weight . however , the combination of a high - fat diet and ovariectomy caused an increase in body weight gain accompanied by an increase in ret and mes weight , similar to our findings . this excessive weight gain is primarily due to fat deposition , reflected by increased serum leptin levels , which rise in direct proportion to fat mass . carcass lipid content in the ovx h group was higher than in the sham h and sham c groups . diets rich in saturated fatty acids decrease uncoupling protein-1 ( ucp-1 ) activity in brown adipose tissue liu et al . showed that low estrogens levels , characteristic of ovariectomy , increase ucp-1 expression in subcutaneous adipose tissue from 4 weeks until 12 weeks of ovariectomy . in contrast , ko et al . indicated that a high - fat diet combined with ovariectomy decreased ucp-1 levels relative to controls . also , it has been reported that ovariectomy causes a decrease in the lipolytic responsiveness to norepinephrine of fat cells and an increase in adipose tissue lipoprotein lipase ( lpl ) . lpl hydrolyzes tg from chylomicrons and very - low - density lipoproteins to free fatty acids and monoacylglycerols . a hyperlipidic diet was shown to cause an increase in lpl activity in white adipose tissue . by these mechanisms , ovariectomy promotes increased adiposity , particularly in rats receiving a hyperlipidic diet . taken together , these findings imply that a high - fat diet combined with ovariectomy may cause a decrease in energy expenditure and an increase in adipose tissue deposits , probably via enhanced uptake of tg from chylomicrons . also , in the present study , it was observed that serum tg was lower in sham h group compared with sham c group . studies have reported that low - fat , high - carbohydrate diets increase the liver de novo lipogenesis and cause hypertriglyceridemia compared to high - fat diets [ 29 , 30 ] . . showed that the administration of estrogen promoted a slight decrease in body weight without entailing a significant reduction in adiposity index or in ret weight in obese rats . these data suggest that this treatment is insufficient to restore these parameters , although there is a 77% reduction in the expression of lpl , which would reduce fat deposition by decreasing fatty acid uptake from the circulation . however , this is inconsistent with studies in which estrogen replacement managed to recover both body weight and adiposity index in female control rats . this discrepancy may be related to the structural form of estrogen used , in addition to the dosage and duration of estrogen replacement and the age at which the female rats were ovariectomized . increase in serum leptin concentration was observed only in ovx h group , suggesting that the decrease in estrogen could potentiate the effect of high - fat diet on leptin secretion . hyperleptinemia may arise as a result of the increased body weight of rats in the ovx h group . showed that animals fed a high - fat diet were hyperleptinemic and overweight compared with animals receiving a control diet . it is established that serum leptin levels rise with increasing adiposity and are directly proportional to fat mass . loss of efficacy of endogenous or exogenous leptin in the therapeutic maintenance of body weight is attributable to the development of resistance to leptin , a common occurrence in obese individuals . possible explanations for this include a lack of leptin at central sites resulting from defective transport , for instance , through the blood and the brain barrier , decreased production of leptin by the hypothalamus , or interruption of signal transduction between leptin and receptors in the hypothalamus . saravanan et al . showed that animals fed a high - fat diet had a state of hyperleptinemia accompanied by overweight when compared to animals that received control diet . it is understood that ovariectomy promotes hyperphagia and weight gain , and although this hyperphagia is transitional , the gain in body weight is more durable . estrogen replacement has been shown to decrease food intake and restore weight in ovariectomized rats . these facts suggest that estrogen is involved in the modulation of energy homeostasis by direct action on the appetite and central pathways regulating energy and by controlling the secretion of hormonal signals peripherals such as leptin , which in turn regulates effector pathways central and essential to the integration of the hypothalamus in energy homeostasis . content , administration of a hyperlipidic diet in this study caused a decrease in il-6 and il-10 content in both ret and mes adipose tissues . previously , our group showed that administration of a lard diet for 8 weeks promoted a decrease in il-10 in ret and mes adipose tissues of male mice , with no effect on il-6 or tnf-. cani et al . observed an increase in lps , accompanied by elevated mrna concentrations of pai-1 , il-1 , tnf- , and f4/80 in subcutaneous adipose depots and pai-1 , il-1 , and f4/80 in mes adipose tissues , of high - fat diet fed male mice relative to control diet fed mice . these results suggest that the presence of female hormones could modify the response to a high - fat diet in terms of il-6 adipose tissue content , especially in ret adipose tissues . previously , lafontan and berlan reported that the physiology , metabolism , and function of white adipose tissue vary in a depot - specific manner . several factors could contribute to this , such as difference in the amount of hormones receptors , blood flow , hormone , cytokines , and polypeptides production . for instance , it has been demonstrated by yamashita et al . that aerobic training was effective in reducing adipokines levels related to inflammation in mesenteric adipose tissue but not in retroperitoneal adipose tissue . in this study , the rats were euthanized , between 9:00 and 12:00 h , in the estrus phase , a period when estrogen secretions exert their biggest influence . in fact , in vitro studies demonstrated an inhibitory effect of estradiol on il-6 production in several cell types in humans and rodents , including macrophages , monocytes , osteoblasts , and bone stromal cells [ 3941 ] . conversely , ovariectomy combined with a hyperlipidic diet promoted a decrease in the tnf- , il-6 , and il-10 content of mes adipose tissues . riant et al . demonstrated that estrogens , particularly chronic e2 administration to ovariectomized mice , enhance the expression of inflammatory factors , such as il-6 and tnf- , in mice fed with a high - fat diet for 4 or 12 weeks , indicative of a proinflammatory effect of e2 , at least in visceral adipose tissues . accordingly , in this study , we observed a decrease in tnf- in mes adipose tissues in the ovx h group compared with the sham h group , supporting the idea of a possible in vivo proinflammatory effect of estrogen on white adipose tissue , in special visceral one . both hyperlipidic diet and estrogen have been reported to influence the circadian rhythm modifying the expression of clock genes involved in the regulation of metabolism and protein expression [ 43 , 44 ] . in this sense , cano et al . demonstrated that high - fat diet disrupts the daily variations of circulating of several adipocytokines , particularly by a reduction in plasma concentration of tnf- from 9:00 to 13:00 h , compared to control diet treated rats . it could be speculated that the results found in the present study , related to the effect of diet and ovariectomy on adipose tissues cytokines content , could be related to the disruption on the clock genes , since the presence of circadian clock genes has been reported in fat . content in white adipose tissue by the administration of a hyperlipidic diet and/or ovariectomy , protein levels of il-6r , tnfr1 , tlr-4 , and myd88 were similar between groups in all tissues studied ( figures 6 , 7 , and 8) . myd88 knockout mice showed no response to the tlr-4 ligand lps in terms of inflammatory mediator production by macrophages , b cell proliferation , or endotoxin shock [ 47 , 48 ] . however , in the case of tlr-4 stimulation , lps - induced activation of nf-b and c - jun n - terminal kinases ( jnk ) was observed with delayed kinetics , even in myd88 knockout cells , although these cells did not produce any inflammatory cytokines in response to lps . taken together , it is possible to speculate that the modification in cytokines content in white adipose tissue observed in this study may have been stimulated by another mechanism , such as mapk dependent pathway . in conclusion , our results demonstrate that administration of a hyperlipidic diet for 8 weeks combined with short - term ovariectomy did not alter the cytokine content of the liver and gastrocnemius muscle but caused a decrease in all analyzed cytokines in mesenteric adipose tissue . however , ovariectomy in rats fed a hyperlipidic diet caused hyperleptinemia and increased body weight gain , food efficiency , and carcass lipid content , which , in combination with long - term ovariectomy , may contribute to the induction of proinflammatory processes , particularly those associated with a high - fat diet . these results emphasize that , after the menopause , women must decrease their consumption of fat .

four - week - old female wistar rats were divided into two groups and fed a control diet ( c ) or a hyperlipidic diet ( h ) for 4 weeks . rats from each group underwent ovariectomy ( ovx ) or sham surgery ( sham ) . they received c or h for the next four weeks . the body weight gain ( bw ) , food efficiency ( fe ) , and carcass lipid content were higher in the ovx h than in the sham h. the ovx h exhibited a higher serum leptin level than other groups . il-6 , tnf- , and il-10 content of mesenteric ( mes ) adipose tissue was lower in the ovx h than in the ovx c. il-6 , tnf- , and il-10 content of retroperitoneal ( ret ) adipose tissue was lower in the sham h than in the sham c. the sham h showed decreased tg relative to the sham c. similar results were obtained in relation to il-6r , tnfr1 , tlr-4 , and myd88 contents in the mes and ret white adipose tissue among the groups . a hyperlipidic diet for 8 weeks combined with short - term ovariectomy decreases the cytokine content of mes adipose tissues but increases bw , enhancing fe and elevating serum leptin levels . these suggest that the absence of estrogens promotes metabolic changes that may contribute to installation of a proinflammatory process induced by a hyperlipidic diet .

pubmed

hemoptysis is one of the most common symptoms in patients with respiratory diseases , when massive , has a mortality rate of more than 50% . therefore , significant or life - threatening hemoptysis calls for quick treatment to stop bleeding . in the past , the control of hemoptysis had been obtained using surgical methods if conservative management failed to be effective . but patients with massive hemoptysis may be associated with high morbidity and mortality using conservative or surgical management . it is a safe and effective treatment , not only for massive hemoptysis , but also for chronic or recurrent hemoptysis that impairs the patient 's quality of life . the first procedure for embolization therapy is a complete angiography of the pulmonary vascular tree when bleeding bronchial arteries are seen , embolization therapy is carried out . this retrospective study was undertaken to determine the demographic characteristics , clinical features , results of imaging techniques , complications related to bae , and follow - up of 344 patients underwent bae . this study was a retrospective analysis of 344 cases , who underwent bronchial arteriography at tianjin haihe hospital between 2006 and 2013 . the patients were hospitalized due to hemoptysis , with the volume of blood ranging 502000 ml/24 h. each patient underwent standard medical management , but the hemoptysis still occurred , so they underwent bronchial arteriography at first and underwent bae when necessary . there were 186 men and 158 women , with a mean age of 57 years old ( ranging 1783 years old ) . two hundred and thirty - four patients had acute and massive hemoptysis , and 110 patients had chronic and recurrent hemoptysis ( recurrent rate at least 3 times in 1 year ) . the data for outcome analysis were obtained from the clinical records , all of the radiological images were reviewed as possible . we collected such data for analysis : clinical features ; chest computed tomography ( ct ) scan ; bronchial ateriography , bae , and results ; embolization material ; complications related to bae ; and follow - up . about 317 patients ( 92.2% ) were suggestive of causes of hemoptysis , and 27 patients ( 7.8% ) showed focal hemorrhage . blood lobe could be identified by chest ct scan in 105 cases ( 30.5% ) . bleeding source could be localized in unilateral lung but could not be defined in an exact lobe in 90 cases ( 36.2% ) . both of the lungs had unusual film but not focal hemorrhage in 149 patients ( 43.3% ) . the findings of angiography suggested that few direct sign found during arteriography , most of the indirect appearances that support a site of bleeding include tortuosity , hypertrophy , hypervascularity , aneurysms , extravasation , and bronchial artery to pulmonary artery or vein shunting . in 131 patients ( 38.1% ) , only left - side bronchial arteries were abnormal ; in 69 patients , only right - side bronchial arteries were abnormal ; in the other 144 patients , bilateral bronchial arteries were abnormal . as the statistics , 944 responsible bronchial arteries involves in according to the angiography : 275 right bronchial arteries ; 200 left bronchial arteries ; 170 combined right and left bronchial trunk ; 123 right intercostal arteries ; other 176 arteries [ figure 1 ] . we analyzed the responsible arteries for tuberculosis , bronchiectasis , bronchogenic carcinomas and undefined reasons as follow [ table 1 ] . a spinal artery was identified in three patients , one spinal artery originated from the left bronchial artery , one from the right intercostal artery , and one from the right subclavian artery . the numbers of responsible arteries for different diseases despite of which kind of causations , the main responsible arteries were right bronchial artery , left bronchial artery and combined right and left bronchial trunk ; more right intercostal arteries were involved than left . bronchial artery embolization was performed in 336 of 344 patients ( 97.7% ) , artery spasm and closure occurred in six patients , arterial subintimal dissection occurred in one patient , and the responsible vessel could nt be super - selective in one patient because of combining with the spinal artery . overall , there were 1530 embolization coils for the 336 patients with a total of 920 arteries embolized ( including bronchial and nonbronchial systemic arteries ) . according to the chest ct scanning and bronchial arteriography , we concluded the etiologies of the hemoptysis in 344 cases : tuberculosis in 190 patients ( 55.2% ) , bronchiectasis in 99 patients ( 28.8% ) , malignancy in 20 patients ( 5.8% ) , undefined in 18 patients ( 5.2% ) , pneumonia in six patients ( 1.8% ) , deformity of bronchial artery in six patients ( 1.8% ) , bronchial artery aneurysm in one patients ( 0.2% ) , other pulmonary disease in four patients ( 1.2% ) . bronchial artery embolization was successful in controlling hemoptysis immediately in 330 of 344 patients ( 96% ) . 61 patients ( 17.7% ) had recurrent hemoptysis within 1 month after undergoing bae , only three of them were performed a second bae , two patients had rebleeding in the previous site , one was found new bleeding area . three of these patients died of massive hemoptysis in 1 month after undergoing embolization ( two patients with bronchogenic carcinoma and one with tuberculosis ) , one patient with severe chronic obstructive pulmonary disease died of hypercapnia in 3 days after undergoing embolization . the two patients in whom an embolization procedure could not be performed ( one because of spinal artery combined with the hemorrhage vessel and one was under subintimal dissection ) , taken medical treatment for 1 week , the hemoptysis were gradually controlled . each patient performed bae had a repeated chest ct scanning after 1 month , which showed that there was no coil dropping or shifting . seventy four patients ( 21.5% ) had recurrent hemoptysis over 1 month after undergoing bae . forty - four patients were performed a repeated embolization , all of the bronchial arteriograms bronchial arteriograms showed there was no dropping of the coil , in 17 patients developed recanalization of the previously embilzed vessels , the remaining 27 patients developed new blood vessels responsible for hemorrhage . one hundred and seven patients ( 31.1% ) had a fever ( < 38.5c ) after bae . chest pain postprocedure occurred in 65 patients ( 18.9% ) , back pain in 51 patients ( 14.8% ) , shoulder pain postprocedure in 29 patients ( 8.4% ) , and transient dysphagia or lower limb numbness in three patients ( 0.9% ) . all of the patients had been followed up over 1 month after bae except the four patients had been died within 1 month . two hundred and forty - eight patients had a telephone call follow - up recently ( follow - up periods ranging from 6 months to 8 years ) . it revealed that 28 patients had been dead , six of them died of massive hemorrhage , seven died of deterioration of lung disease but not bleeding , the remainder died of other diseases or accident . four patients died within 1 year after due to advanced lung cancer ( one had a massive bleeding out of controlling ) , 24 patients died during 24 years after bae . twenty - one patients have recurrent hemoptysis after repeated bae , the volume of bleeding is < 5 ml / d . one hundred and ninety - nine patients had follow - up periods ranging from 6 months to 8 years without evidence of recurrent hemoptysis . about 317 patients ( 92.2% ) were suggestive of causes of hemoptysis , and 27 patients ( 7.8% ) showed focal hemorrhage . blood lobe could be identified by chest ct scan in 105 cases ( 30.5% ) . bleeding source could be localized in unilateral lung but could not be defined in an exact lobe in 90 cases ( 36.2% ) . both of the lungs had unusual film but not focal hemorrhage in 149 patients ( 43.3% ) . the findings of angiography suggested that few direct sign found during arteriography , most of the indirect appearances that support a site of bleeding include tortuosity , hypertrophy , hypervascularity , aneurysms , extravasation , and bronchial artery to pulmonary artery or vein shunting . in 131 patients ( 38.1% ) , only left - side bronchial arteries were abnormal ; in 69 patients , only right - side bronchial arteries were abnormal ; in the other 144 patients , bilateral bronchial arteries were abnormal . as the statistics , 944 responsible bronchial arteries involves in according to the angiography : 275 right bronchial arteries ; 200 left bronchial arteries ; 170 combined right and left bronchial trunk ; 123 right intercostal arteries ; other 176 arteries [ figure 1 ] . we analyzed the responsible arteries for tuberculosis , bronchiectasis , bronchogenic carcinomas and undefined reasons as follow [ table 1 ] . a spinal artery was identified in three patients , one spinal artery originated from the left bronchial artery , one from the right intercostal artery , and one from the right subclavian artery . the numbers of responsible arteries for different diseases despite of which kind of causations , the main responsible arteries were right bronchial artery , left bronchial artery and combined right and left bronchial trunk ; more right intercostal arteries were involved than left . bronchial artery embolization was performed in 336 of 344 patients ( 97.7% ) , artery spasm and closure occurred in six patients , arterial subintimal dissection occurred in one patient , and the responsible vessel could nt be super - selective in one patient because of combining with the spinal artery . overall , there were 1530 embolization coils for the 336 patients with a total of 920 arteries embolized ( including bronchial and nonbronchial systemic arteries ) . according to the chest ct scanning and bronchial arteriography , we concluded the etiologies of the hemoptysis in 344 cases : tuberculosis in 190 patients ( 55.2% ) , bronchiectasis in 99 patients ( 28.8% ) , malignancy in 20 patients ( 5.8% ) , undefined in 18 patients ( 5.2% ) , pneumonia in six patients ( 1.8% ) , deformity of bronchial artery in six patients ( 1.8% ) , bronchial artery aneurysm in one patients ( 0.2% ) , other pulmonary disease in four patients ( 1.2% ) . bronchial artery embolization was successful in controlling hemoptysis immediately in 330 of 344 patients ( 96% ) . 61 patients ( 17.7% ) had recurrent hemoptysis within 1 month after undergoing bae , only three of them were performed a second bae , two patients had rebleeding in the previous site , one was found new bleeding area . three of these patients died of massive hemoptysis in 1 month after undergoing embolization ( two patients with bronchogenic carcinoma and one with tuberculosis ) , one patient with severe chronic obstructive pulmonary disease died of hypercapnia in 3 days after undergoing embolization . the two patients in whom an embolization procedure could not be performed ( one because of spinal artery combined with the hemorrhage vessel and one was under subintimal dissection ) , taken medical treatment for 1 week , the hemoptysis were gradually controlled . each patient performed bae had a repeated chest ct scanning after 1 month , which showed that there was no coil dropping or shifting . seventy four patients ( 21.5% ) had recurrent hemoptysis over 1 month after undergoing bae . forty - four patients were performed a repeated embolization , all of the bronchial arteriograms bronchial arteriograms showed there was no dropping of the coil , in 17 patients developed recanalization of the previously embilzed vessels , the remaining 27 patients developed new blood vessels responsible for hemorrhage . one hundred and seven patients ( 31.1% ) had a fever ( < 38.5c ) after bae . chest pain postprocedure occurred in 65 patients ( 18.9% ) , back pain in 51 patients ( 14.8% ) , shoulder pain postprocedure in 29 patients ( 8.4% ) , and transient dysphagia or lower limb numbness in three patients ( 0.9% ) . all of the patients had been followed up over 1 month after bae except the four patients had been died within 1 month . two hundred and forty - eight patients had a telephone call follow - up recently ( follow - up periods ranging from 6 months to 8 years ) . it revealed that 28 patients had been dead , six of them died of massive hemorrhage , seven died of deterioration of lung disease but not bleeding , the remainder died of other diseases or accident . four patients died within 1 year after due to advanced lung cancer ( one had a massive bleeding out of controlling ) , 24 patients died during 24 years after bae . twenty - one patients have recurrent hemoptysis after repeated bae , the volume of bleeding is < 5 ml / d . one hundred and ninety - nine patients had follow - up periods ranging from 6 months to 8 years without evidence of recurrent hemoptysis . earlier , besides medical treatment , surgery was the fast and best method to control the bleeding . but it required patients many preparations , such as chest ct , pulmonary function , even bronchoscopy to evaluate the patient physical condition and determine the range of surgery . some preparations were impossible for the patients in emergency conditions , the impairment of surgery was so great for a lot of people that gravely lowered the quality of life . bae is a well - established procedure used to control massive hemoptysis since it first performed in 1974 . subsequently , bae was widely used , because non - operable patients could be treated and other patients could be stabilized prior to surgery . in clinical , about 1 hour is needed to prepare before bae , and the physical requirements of patient are lower than for surgery . the technology is mostly described the outcomes in control of hemoptysis due to varies causations . in some conditions , interventional embolization can be used before surgery . in our study , of the 344 cases , an immediate control of bleeding was achieved with embolization in 330 patients ( 96% ) , similar to the immediate success rate was reported 7598% earlier . recurrent bleeding despite apparently adequate embolotherapy remains a considerable problem , some reported with bleeding occurring in 942% of patients after embolization . in our series , rebleeding occurred within 30 days in 61 of 344 patients ( 17.7% ) . a second embolization procedure was performed in three patients , two patients were found rebleeding in the previous points , and one was found new bleeding arteries . overall the patients , 54 of whom rebleeding over 1 month in the follow - up , 44 of whom were performed a second bronchial arteriography . in 27 patients , an additional bronchial or nonbronchial collateral artery was embolized resulting in the successful control of hemoptysis , 17 patients developed recanalization of the previously embilzed vessels and had been embolizied again . in the recurrent hemoptysis patients , the etiologies were mainly due to cancer , tuberculosis , and severe bronchietasis ( at least two lobes were suffered ) . the disease development contributed to the rebleeding . in our series , a patient with cancer was performed in bae for three times , and a patient with tuberculosis was performed for five times , both of whom were not suitable for surgery . so it has shown that bae is an effective procedure with which to definitively treat some patients with hemoptysis and can be repeatedly utilized . as we know , the source of massive hemoptysis in 90% of cases due to the brochial circulation , as well as the remaining 10% divided equally between the pulmonary circulation ( 5% ) and other systemic arterial supply to the lungs . it is well - documented that the anatomy of bronchial arteries is variable form person to person , typically origin arises from the descending aorta between the levels of the t5 and t6 vertebrae . four classic patterns of origin were described : type i ( 40% ) : two on the left and one on the right , presenting as an intercostobronchial trunk ( icbt ) ; type ii ( 21% ) : one on the left and one icbt on the right ; type iii ( 20% ) : two on the left and one on the right ( one icbt and one bronchial artery ) ; type iv ( 9.7% ) : one on the left and two on the right ( one icbt and one bronchial artery ) . according to the statistics , the main responsible vessels for bleeding are right bronchial arteries , left bronchial arteries , combined right and left bronchial trunk , right intercostal arteries . in different etiologies , the rate of abnormal arteries is coincidence with the result . the bronchial arteriography showed that more right intercostal arteries combining with bronchial artery than left . the distribution and frequency of embolization of the arteries were agreed with kwon et al . usually , a spinal artery can originate from a bronchial artery in up to 5% of patients , with right side being more common than the left side . as our observation , a spinal artery was identified in three of our patients , with two occurring on the right side and one occurring on the left side . our result showed that tuberculosis ( 55.2% ) , bronchiectasis ( 28.8% ) , malignancy ( 5.8% ) , undefined ( 5.2% ) , pneumonia ( 1.8% ) , deformity of bronchial artery ( 1.8% ) , bronchial artery aneurysm ( 0.2% ) , other pulmonary disease ( 1.2% ) . , the etiologies of hemoptysis included tuberculosis ( 34% ) , bronchiectasis ( 26% ) , aspergilloma ( 18% ) , neumoconiosis in coal miners ( 13% ) , and bronchogenic carcinoma ( 3% ) . knott - craig et al . retrospectively studied 120 patients with hemoptysis and found that the right lung was the source in 62% and the left lung in 38% , with the right upper lobe being the area that was most commonly affected . our results were that the right lung was the source of bleeding in 131 patients ( 38.1% ) , the left lung was the source in 69 patients ( 20.0% ) , and both lungs were the source in 144 patients ( 41.8% ) . the common complications of bae included subintimal dissection , arterial perforation by a guidewire , and reflux of embolic material into the artery without adverse sequelaed during the procedure , and fever , chest pain , dyspnea , transient dysphagia , groin hematoma , even headache or blindness etc . several patients developed transverse myelitis as a result of inadvertent embolization of the spinal arteries . during the procedure of bae , when a spinal artery was founding , but in contrast , mal et al . observed the brown - sequard 's syndrome , following three episodes of spinal cord complications , which regressed after 4 months without sequelae ; paraparesis with spontaneous regression after 2 weeks ; and complete paraplegia without regression . if a spinal artery arises from a bronchial artery , we will only embolize the bronchial artery if we can achieve a stable distal position well beyond the spinal artery origin . our patients showed transistent complications such as fever , chest pain , shoulder pain , groin hematoma , all the symptoms were disappeared in 48 hours , and fewer of our patients experienced any neurologic sequelae . observed the outcome of bae : the hemoptysis control rate was 93.1% at the end of 2 weeks , 85.7% at the end of 1 month , 79.5% at 90 days , 63.2% at 180 days , 51% at 1 year and 38.7% at the end of 2 years . the follow - up of our patients revealed that 28 patients had died , and six from massive hemorrhage and the remainder as a result of their disease process . twenty - one patients have recurrent hemoptysis after repeated bae , the volume of bleeding is < 5 ml / d , taking medicine can control the bleeding . one patient suffered recurrent hemoptysis wanted to take a surgical management , but the diffuse disease in lung limited the management . there was no follow - up information available on 92 patients , because they did not return after treatment and the telephone numbers were null . the remaining 199 patients had follow - up periods ranging from 6 months to 8 years without evidence of recurrent hemoptysis . in summary , hemoptysis represents a significant clinical entity with high morbidity and potential mortality . medical management ( in terms of resuscitation and bronchoscopic interventions ) and surgery bae procedures represent the first - line treatment for hemoptysis arising from bronchial arterial source . in our study , it was very useful in obtaining immediate bleeding control which was slightly better than that in other report . the technique was a relatively safe and effective method for control the hemoptysis , importantly , it can be used repeatedly . in our study , more than 50% patients had massive hemoptysis , about 70% patients ca nt be identified bleeding point by chest ct , 41% patients hemorrhage source from two lungs according the bronchial arteriography . although the long - term outcome in some patients is not good , bae may be the only life - saving treatment option in patients who are poor surgical candidates .

background : hemoptysis is a significant clinical entity with high morbidity and potential mortality . both medical management ( in terms of resuscitation and bronchoscopic interventions ) and surgery have severe limitations in these patients population . bronchial artery embolization ( bae ) represents the first - line treatment for hemoptysis . this article discusses clinical analysis , embolization approach , outcomes and complications of bae for the treatment of hemoptysis.methods:a retrospective analysis of 344 cases , who underwent bronchial arteriography at tianjin haihe hospital between 2006 and 2013 . several aspects of outcome were analyzed : demographics , clinical presentation , radiographic studies , results , complications and follow - up of bae.results:three hundred and forty - four consecutive patients underwent bronchial arteriography , 336 of 344 patients ( 97.7% ) performed bae ; there were 1530 coils for 920 arteries embolized ; the main responsible sources for bleeding were right bronchial artery ( 29.7% ) , left bronchial artery ( 21.6% ) , combined right and left bronchial trunk ( 18.4% ) , right intercostal arteries ( 13.3% ) ; 61 patients ( 17.7% ) had recurrent hemoptysis within 1 month after undergoing bae , 74 patients ( 21.5% ) had recurrent hemoptysis over 1 month after undergoing bae ; the common complications of bae included subintimal dissection , arterial perforation by a guide wire , fever , chest pain , dyspnea , etc . the follow - up was completed in 248 patients , 28 patients had been dead , 21 patients still bleed , 92 patients had lost to follow-up.conclusions:the technique of bae is a relatively safe and effective method for controlling hemoptysis . the complications of bae are rare . although the long - term outcome in some patients is not good , bae may be the only life - saving treatment option in patients who are poor surgical candidates .

pubmed

helicobacter pylori ( hp ) infection may cause several clinical manifestations , ranging from asymptomatic to significant gastroduodenal disease including ulcer , bleeding , perforation , and adenocarcinoma . no pathogenic mechanism is known to explain the above diversity . celiac disease ( cd ) is a t - cell - mediated disorder of the small bowel triggered by gluten in susceptible subjects . the damage is not confined only to the small bowel but may affect the gastric mucosal structure and function . in both conditions , hp and cd , a systemic humoral immune response is detected , but much interest has been focused on the local immune inflammatory reaction . it is defined by the presence of > 25 intraepithelial lymphocytes ( iels ) ( surface and upper foveolar epithelium ) per 100 epithelial cells , without taking into account the mononuclear inflammatory cell infiltration of the lp . this condition may be recognized endoscopically as varioliform gastritis , nodularity , hypertrophic gastropathy , and aphtous erosions [ 5 , 6 ] . a similar histological entity has been reported in association with a variety of gastric infections , inflammatory diseases , and in autoimmune disorders affecting the gastric mucosa including cd , in adults [ 79 ] , as well as in pediatric patients [ 1012 ] . additionally , hp is a major etiology for lg , extensively documented in adults [ 13 , 14 ] and children [ 15 , 16 ] . furthermore , investigations of the relationship between hp infection and cd have yielded conflicting results [ 9 , 17 ] , probably because of the different prevalence of hp in the populations studied . others have focused on the hp - related lg in cd [ 6 , 8 ] and recently on the link between anemia , hp , and cd . the pathogenetic relationship between cd , hp and lg is even more complicated since gastric hp existence attenuates duodenal lesions in cd patients . it is generally accepted that the major cytokine response to hp and cd has a t - helper 1 ( th1)-type profile . the exact nature of the cellular response contributing to this inflammatory profile has not been determined , and less so in children . conflicting results exist in the literature on the lymphocytic subpopulations , quantity and quality , infiltrating the gastric mucosa in cd and hp . as lg is a morphologic endpoint of numerous etiologies , the literature is ripe with controversy and since extensive characterization of the lymphocytic populations infiltrating the gastric mucosa in cd and hp infected children is lacking , the present study was undertaken . the aims were to look for additional quantitative and qualitative histological features , to define the immunophenotype of the gastric mucosa of the two most prevalent etiologies of pediatric lg ( cd and hp infection ) . our secondary aim was to demonstrate a unique histopathology profile of lg by immunohistocemistry staining , trying to clarify the interrelationship between hp and cd in the pediatric population , thus shedding new light on the two entity 's enigma . forty patients referred for endoscopy due to upper gastrointestinal symptoms ( mostly recurrent abdominal pain , or iron deficiency anemia ) were included in the study . patients were divided into 4 groups group1 is eight normal controls ( without cd nor hp infection ) ; group 2 is ten patients with new active cd without hp infection ; group 3 is twelve celiac negative patients infected by hp ; and group 4 is ten new active cd patients infected by hp ( table 1 ) . none of these patients had any other gastrointestinal underlying disease including evidence for food allergy , giardiasis , or inflammatory bowel disease . the diagnosis of cd was based on the accepted histological findings supported by positive serology for cd ( antiendomysial and antitissue transglutaminase antibodies ) . all cd patients were under normal gluten containing diet at the time of diagnosis . hp status was assessed according to conventional biopsy - based criteria plus positive urease test . all patients underwent esophago - gastro - duodenoscopy using gif - xp 20 endoscope ; pentax , tokyo , japan . at least 6 biopsies were obtained : 3 from the second part of the duodenum for diagnosing or exclusion of cd , and 3 from the antrum ; one for quick urease test and two for histologic examination . sections obtained from gastric biopsies were immediately fixed in buffered formalin and embedded on edge in paraffin . the diagnosis of lg was established if 25 lymphocytes per 100 gastric epithelial cells infiltrated the surface epithelium . furthermore , we estimated the extent to which the inflammatory infiltrate involved the lp and the mucosal glands . in addition , the number of mucosal and submucosal lymphoid aggregates were counted . to clarify the nature of these inflammatory cells three m sections were prepared from formalin - fixed , paraffin - embedded tissue blocks , air dried , and subjected to deparaffinization with xylene and absolute alcohol . the immunoperoxidase stains were performed using a panel of antibodies that included cd20 , cd3 , cd4 , cd8 , cd57 , cna42 , and ki67 ( table 2 ) , with appropriate dilutions as recommended by the manufactures , using the ventana es autoimmunostainer and the iviewdab detection kit from ventana . the gastric iels were counted on the hematoxylin - eosin stains per 100 consecutive gastric surface mucosal cells with a x400 magnification ( objectivex10 ) . the density of subepithelial lymphocytes was determined semiquantitatively using a 4 tier grading system , according to the percentage of the area in the lp infiltrated by the inflammatory cells . grade 0 is if less than 5% of the area was infiltrated by inflammatory cells , grade 1 is between 530% , grade 2 is between 3060% , and grade 3 is if > 60% of the area was infiltrated . in order to evaluate the inflammatory glandular involvement , glands containing inflammatory cells were counted out of a fixed total number of superficial glands ; calculated as the percent of involved glands . continuous variables were compared across groups using analysis of variance ( anova ) with bonferroni 's correction for multiple comparisons . discrete ( semi quantitative ) variables were compared using kruskal - wallis nonparametric one - way analysis of variance , with multiple comparisons . a p - value less or equal than 0.05 was considered significant . demographic data of the four examined groups of patients are summarized in table 1 . the mean age of all groups and gender 's ratio were not statistically significant different . in cd as well as in hp gastritis patients , increased numbers of iel compatible with the diagnosis of lg ( 45.518.5 per 100 gastric epithelial cells ) compared to patients without lg ( 10.84.5 per 100 gastric epithelial cells ) , were observed . the highest proportion of lg ( 50% ) was found in cd patients without hp infection , followed by patients with cd infected by hp ( 20% ) , hp positive patients without cd ( 8.3% ) and none in the control group . the distribution and total number of lymphoid aggregates in the antrum among the four different groups are shown in table 3 . patients infected with hp had the highest number of lymphoid aggregates irrespective of the cd status . three cd patients with hp and nine hp infected patients without cd had more than 2 lymphoid aggregates . the mean number of intraepithelial lymphocytes ( iels ) positive for cd3 was increased significantly in cd patients with or without hp infection compared to controls [ p0.01,p0.05 respectively ] , ( table 4 ) . similarly the mean number of cd8 + iels was increased significantly in cd patients with or without hp infection compared to controls ( p<0.05 ) ( figure 1 ) . hp infection did not contribute to the number of cd8 + iels . in the hp infected group without cd , the number of cd8 + iels was not significantly different from the normal controls [ table 4 ] . a slight increase in cd8 + lymphocytes was noted in the lp and intra mucosal glands in the cd group patients compared with all the other groups , although these results did not reach statistical significance . cd4 + lymphocytes were slightly increased in the lp and in the mucosal glands of the hp+ patients compared with controls and cd cases . no statistical significant differences were found for the number and distribution of b lymphocytes ( cd20 + ) , natural killer ( nk ) lymphocytes ( cd57 ) and follicular dendritic cells ( cna42 ) within all different examined compartments ( iels , lp and glands ) . in order to investigate the differential contribution of hp infection to lg in an inflammatory condition like cd , the gastric tissue inflammatory subpopulation profile was investigated , by immunohistochemistry , and compared between the 4 groups of patients . past studies focused mainly on the surface epithelial infiltrate as well as on the superficial pit gastric epithelium invasion by lymphocytes , and to a lesser extent on the infiltrate in the lp and in the gastric glands . the main finding of the present study was the highest prevalence lg in pediatric cdhp followed by cdhp . forty five percent of cd patients are estimated to have lg like in our study [ 8 , 12 ] . however , lg appears to be similarly frequent in hp positive children with and without cd . previous studies demonstrated that lg was found to be more common in hp positive children without cd than in hp negative children without cd . there are still controversial results on the contribution of cd and hp infection to lg . moreover , the association between these pathologies to lg is not well established [ 1315 ] . lymphocytic gastritis was reported in 3645% of children with cd [ 6 , 10 , 13 ] , and disappears after a gluten free diet . hp infection is less frequently found in patients with lg [ 13% ] than with the usual chronic antral gastritis [ 6590% ] . cd is considered to be a population at greatest risk for lg compared to hp affected patients . our study confirmed the published results of an increased number of iels in both cd and hp infected patients . surprisingly , cd patients with associated hp infection showed a lower rate of lg . this evidence might be explained by the well known limited roll of bacterial infection in cytotoxicity . hp convergent the immune response towards th2 response and suppress the th1 immune response . previous investigators have published several sets of data concerning the composition of the lymphocytic infiltrates in the different diseases [ 1620 ] . drut et al found that lg in pediatric cd patients contains a peculiar cd3 , cd7 and cd8 intraepithelial lymphocyte population , that is not associated with the presence of cd4 , cd20 , cd56 and cd57 iels . in hp gastritis there is an increased number of mononuclear cells in the gastric lp , including b and t lymphocytes , plasma cells , macrophages and mast cells . it has been shown that hp stimulates b lymphocytes and causes an increase in their numbers predominantly in the lp . although immunity against hp infection appears not to be dependent on b cells , the role of t cells still remains to be clarified . it appears however that the b cell proliferation might be driven by activated lymphocytes ( cd4 + cells ) that might recall and activate mononuclear phagocytes . demonstrated that the cellular response includes an innate nonspecific response represented mainly by polymorphonuclear cells and macrophages , as well as a t cell response with abundant positive staining with anti - cd8 antibodies , was observed indicative of a predominance of suppressor / cytotoxic t lymphocytes both in the lp and in the epithelium . similarly we also showed the presence of cd3 + iels in both diseases ( cd ; hp infection ) . despite a potential additive effect between these two pathological processes , it is impossible to differentiate between them , based only on the number of the cd3 + iels in the antral mucosa . cd3 staining is a pan t lymphocyte marker and does not differentiate between cd4 helper and cd8 suppressor / cytotoxic cells , therefore we aimed to characterize the t cell subsets immunophenotype ( cd4 + , cd8 + , cd57 ) . in our study the cd8 + iels were significantly higher in cd patients ( 20/100 epithelial cells ) , compared to controls ( 1/100 epithelial cells ) or hp infected patients without cd ( 2/100 epithelial cells ) . we assume that in undiagnosed patients with histological features compatible with lg , higher counts of cd8 + iels may imply that the diagnosis is cd rather than hp gastritis . our findings support the published data that cd8+/cd4 iels are involved significantly in the pathogenesis of cd . in our study , the low expression of cd57 in the lymphoid cells , both in the epithelium , in the lp as well as in the mucosal glands indicate that nk cells may play a negligible role in these two pathologies . we also tried to differentiate between these two causative agents of lg by looking at the proliferation marker , ki67 . although both conditions were associated with a prominent adaptive immune activity , no increase in the proliferation index of the surface epithelium was demonstrated in these pathologies . dendritic cells ( cna42 + ) which serve as professional antigen presenting cells did not show an increased expression in both pathologies . this may indicate that the process of antigen presentation occurred in the lymph nodes . this notion is supported by the absence of cd4 ( t helper ) cells in the immune cell infiltrates found in the affected tissues . in addition to all the above , we confirmed the published results that the highest number of lymphoid follicles was observed in a similar proportion of children with or without cd , who were hp positive independent of the presence of lg . the inflammatory infiltrate in the lp and in the mucosal glands does not contribute to the differentiation between these diseases . in summary , our study aimed to explore the contribution of cd and hp infection to lg and to characterize the different immunoprofiles of the gastric inflammatory cells involved in these diseases . we were looking for an applicable histological tool that might differentiate between cases of cd and hp infection with overlapping clinical and histological features . we suggest that in very young patients infected by hp and suspicion for having atypical cd ( negative serology with increased iels with normal villous architecture - marsh i classification ) with a debatable diagnosis of cd versus hp infection , the number of cd8 + iels in the antrum might hint toward the diagnosis of cd rather than hp infection , and the number of lymphoid follicles directs toward the diagnosis of hp infection . thus , it is important to include immunohistochemical analysis of cd8 lymphocytes in the antrum in undefined cases of cd .

lymphocytic gastritis ( lg ) is associated with helicobacter pylori ( hp ) and celiac disease ( cd ) . we aimed to clarify the relationship between hp infection and cd by defining a unique histopathology profile of lg in these two diseases . forty patients who underwent upper endoscopy were divided into four groups : eight controls , ten active cd patients without hp , twelve cd negative with hp , and ten active cd with hp infection . antral samples were assessed by immunohistochemical staining for cd20 , cd3 , cd4 , cd8 , cd57 , cna42 , and ki67 for lymphoid aggregates , intraepithelial lymphocytes ( iels ) number , density of lamina propria ( lp ) lymphocytes , and inflammatory glandular involvement . only iels positive for cd3 and cd8 were increased significantly in cd patients with or without hp infection . hp did not contribute to the number of cd8 iels . in complicated cases with hp and suspicious for cd , the number of cd8 + iels hints toward a cd rather than hp infection .

pubmed

angiogenesis , the process by which the existing vascular network expands to form new blood vessels , is required for the growth of solid tumors . for this reason , vascular endothelial growth factor ( vegf ) , a primary stimulant of angiogenesis , binds and activates vegf receptor 1 ( vegfr1 ) and vegfr2 . vegf is an important and powerful factor increasing vascular permeability and promoting metastasis . without blood vessels , the tumors can not be larger than a few millimeters , so the inhibition of angiogenesis with the use of several drugs could represent an important tool in cancer treatment for several reasons . ( 1 ) angiogenesis occurs at high levels during fetal development , the menstrual cycle , and in wound healing . therefore , the treatments should have low toxicity ; in fact , they might be expected to interfere with this process and should not harm most normal dividing cells . ( 2 ) the antiangiogenic treatments should not be designed to attack directly the cancer cells . the targets of several of these treatments are normal processes controlled by normal cells and not by the tumor cells themselves . the high mutation rates of cancer cells that often render chemotherapy ineffective will not interfere with these drugs . in this paper , we underline the importance of inhibition of vegf as attractive therapeutic target in the treatment of cancer . vegf is a primary stimulant for tumor angiogenesis , making it a critical target for cancer therapy [ 3 , 4 ] . in breast cancer , elevated levels of vegf actually , bevacizumab , a humanized monoclonal antibody that binds human vegf and prevents vegf from binding vegfr1 and vegfr2 , is approved for the treatment of metastatic her2/neu - negative breast cancer . anti - vegf therapy with bevacizumab , the phenethylamine of the 2c family 2c3 or the fully human antibody that inhibits vegf binding to vegfr2 r84 inhibits the growth of established orthotopic mda - mb-231 breast cancer cell line in severe combined immunodeficiency ( scid ) mice , reduces tumor microvessel density , and limits the infiltration of tumor - associated macrophages , but it is associated with elevated numbers of tumor - associated neutrophils [ 9 , 10 ] . selective inhibition of vegfr2 with an anti - vegf antibody is sufficient for effective blockade of the protumorigenic activity of vegf in breast cancer xenografts . these findings further define the complex molecular interactions in the tumor microenvironment and provide a translational tool that may be relevant to the treatment of breast cancer . inhibition of vegf binding to vegfr2 by 2c3 has been shown to reduce tumor size both in pancreatic [ 1113 ] and breast tumors . also the effect on tumor growth after the treatment with r84 in an orthotopic breast cancer model , similar to 2c3 , has been evaluated . in fact , mda - mb-231 cells ( 5 10 ) were injected into the mammary fat pad of nonobese diabetic nod / scid mice , and the therapy was initiated on day 26 after tumoral cell injection , when tumor volume reached 150 mm . in this orthotopic human breast cancer xenograft model , the chronic treatment with r84 , 2c3 , or bevacizumab significantly reduced ( p < .001 ; days 44 and 48 versus control ) the tumoral growth , such that there was a 55% , 62% , and 58% decrease , respectively , in tumor volume compared with control - treated animals . thus , these data show that inhibition of the vegf factor is sufficient to reduce the mass volume of mda - mb-231-derived tumors . to determine if the effect of r84 , 2c3 , and bevacizumab on mda - mb-231 tumor growth in vivo could be due directly to the block of vegf activation of tumor cells , the tumor cell proliferation and migration were also evaluated in vitro , demonstrating that mda - mb-231 cells migrated strongly toward vegf and , consequently , this migration was blocked by the addition of 2c3 , r84 , or bevacizumab . vegf is an abundant cytokine in the tumor microenvironment and is known to stimulate immune cell chemotaxis ; however , few studies have looked directly at the effect of inhibitors of vegf on immune cell infiltration into tumors . it has been shown previously that 2c3 inhibits macrophage infiltration in an orthotopic pancreatic cancer model [ 16 , 17 ] . in a mouse model of thyroid cancer , in nonobese diabetic ( nod)/scid mice , inoculated with mda - mb-231 cells , anti - vegf therapy with 2c3 , r84 , or bevacizumab reduced tumor microvessel density by 50% , 45% , and 58% , respectively . cancer and inflammation are connected by two pathways : the intrinsic and the extrinsic pathways . these two pathways converge , resulting in the activation of transcription factors , such as nuclear factor-b ( nf-b ) , signal transducer and activator of transcription 3 ( stat3 ) , and hypoxia - inducible factor 1 ( hif1 ) , in tumour cells . these transcription factors modulate the production of inflammatory mediators , including cytokines and chemokines . these factors recruit and activate various leukocytes . the cytokines activate the same key transcription factors in inflammatory cells , stromal cells , and tumor cells , resulting in more inflammatory mediators being produced and a cancer - related inflammatory microenvironment being generated . the mediators and cellular effectors of inflammation are important constituents of the local environment of tumours ; in some cancers , inflammatory conditions are present before a malignant change occurs . to examine the importance of inflammatory microenvironment on tumor site , where a massive macrophage infiltration is associated , breast tumors from bevacizumab , 2c3 , or r84 treatment groups were analyzed using three different macrophage markers : the mouse f4/80 antigen , a 160 kd glycoprotein expressed by murine macrophages , and cd16 + cells , a cluster of differentiation found on the surface of natural killer cells , neutrophil polymorphonuclear leukocytes , monocytes , and macrophages . immunofluorescence staining of tumor sections revealed that anti - vegf therapy with 2c3 , r84 , and bevacizumab reduced cluster of differentiation the infiltration of f4/80 + and of cd16 + natural killer cells , neutrophil polymorphonuclear leukocytes , monocytes and macrophages . the reduction of macrophage infiltration in the phase of treatment with 2c3 and r84 is presumably due to expression of vegfr2 on tumor - associated macrophages . the major findings of this study were that 2c3 , r84 , and bevacizumab effectively decrease tumor size , microvessel density , and macrophage infiltration in an orthotopic model of breast cancer . these data are consistent with previous findings [ 1618 ] and support the concept that reducing macrophage infiltration is an important aspect of anti - vegf therapy . in addition , these studies showed changes in other immune cell infiltrates : myeloid - derived suppressor cells ( mdscs ) , dendritic cells ( dcs ) , and neutrophils , following anti - vegf therapy . in fact , infiltrating inflammatory cells constitute a large component of the overall tumor mass . vegf is a major chemoattractant for inflammatory cells , including macrophages , neutrophils , dendritic cells , myeloid - derived suppressor cells , and t - cells [ 20 , 21 ] . to evaluate if inhibition of vegf reduces angiogenesis and modulates immune cell infiltration of orthotopic breast cancer xenografts , roland et al . use preclinical models of breast cancer to compare the effect of different anti - vegf therapies on breast cancer growth , vascular parameters , immune cell infiltration , and intratumoral cytokine levels . they confirmed that the inhibition of vegf receptor activation resulted in changes in intratumoral levels of il-1 and cxcl1 that correlated with changes in immune cell infiltration . in fact , ma et al . constructed a plasmid encoding vegf shrna to knock down vegf both in vitro and in vivo . in vitro , specificity and potency of the targeting sequence were first validated in a549 lung adenocarcinoma cells by rtpcr and elisa assays . in vivo the vegf shrna expressing plasmids were administered systemically in combination with low dose of cis - diclorodiamminoplatino ( ddp ) that is an antineoplastic chemotherapy agent that interferes with all phases of the cell cycle by binding to dna through the formation of crosslinks between complementary strands . the combinated treatment of the two agents had a significantly enhanced antitumoral effect compared with the treatment with pshvegf or ddp alone , resulting in reduction of the tumor weight by 83.13% . to demonstrate that the therapeutic effects were related to downregulation of vegf expression instead of other nonspecific reactions , it was analyzed the distribution of immunoreactive vegf in the tumors and observed a general decrease of vegf staining in the tumors belonging to the mice treated with pshvegf , whereas the tumors belonging to the mice treated with pshhk exhibited significantly more vegf staining . consistently , the elisa assay showed that pshvegf caused significant reduction in intratumoral vegf expression compared with pshhk . these data reinforce the hypothesis that the effect of anti - vegf agents extends beyond the inhibition of angiogenesis because many immune cells express vegfrs , including macrophages , neutrophils , mdscs , dcs , and t - cells [ 23 , 24 ] . in fact , it was shown a reduction in macrophage number in tumors from animals treated with anti - vegf therapy with respect to the control mice . vegf is a potent stimulator of endothelial cell survival , mitogenesis , migration , and differentiation [ 1 , 2 ] . several vegf inhibitors have been approved by the us food and drug administration for the treatment of tumors or age - related macular degeneration [ 3 , 4 ] ( figure 1 ) . recently , in many clinical trails , angioinhibitors were also being used in combination with conventional chemotherapy . clinical trials generally combine very low dose of chemotherapy followed by angioinhibitor therapy . combination of angioinhibitors will need to be tested vigorously in the future , as single angioinhibitors are approved for use of cancer . preventive angioinhibitory therapy may also be possible in the future , because angioinhibitory therapy is generally less toxic and less susceptible to induction of acquired drug resistance .

cancer is the second leading cause of death in the world after cardiovascular diseases . some types of cancer cells often travel to other parts of the body through blood circulation or lymph vessels , where they begin to grow . this process is recognized as metastasis . angiogenesis is the formation of new blood vessels from existing vessel . normally angiogenesis is a healthy process , that helps the body to heal wounds and repair damaged body tissues , whereas in cancerous condition this process supports new blood vessels formation that provide a tumor with its own blood supply , nutrients and allow it to grow . the most important proximal factor for angiogenesis is the vascular endothelial growth factor vegf . angioinhibition is a form of targeted therapy that uses drugs to stop tumors from making new blood vessels . therefore , in this paper we analyse the importance of vegf as target of cancer therapy , analysing murine models .

pubmed

vulvar and vaginal atrophy ( vva ) , resulting from the loss of estrogen stimulation on vaginal and vulvar tissue , is a common medical condition in postmenopausal women one that will occur in most postmenopausal women at some point in their lives.1,2 there are an estimated 64 million postmenopausal women in the united states ( us ) , and as many as 32 million women may suffer from vva symptoms including dyspareunia ( pain during sexual intercourse ) , vaginal dryness , and vaginal irritation.1,3,4 vva is chronic , progressive , and , unlike vasomotor symptoms , will not resolve with time and without treatment . left untreated , vva symptoms can not only cause discomfort but can also negatively impact women s quality of life , including sexual relationships and emotional well - being.1,5 severe vva may affect other quality - of - life aspects , including clothing choices , exercise options , and general pelvic floor comfort . the recently published revive ( real women s views of treatment options for menopausal vaginal changes ) survey , administered to a large cohort of postmenopausal women in the us , offers many insights into the impact of vva symptoms on women s lives . findings of generally poor understanding of vva among women , coupled with concerns about efficacy , convenience , and safety of vaginal over - the - counter ( otc ) products and prescription therapies for vva , emphasize the need for better communication between women and their healthcare providers ( hcps ) about vva and its treatment options.6 this article reviews findings from the revive survey , discusses implications of these findings for hcps who care for postmenopausal women , and provides practical treatment strategies for the care of women with vva . the revive survey was an online evaluation of postmenopausal women in the us , conducted from may 31 , 2012 through june 14 , 2012 , and published online on may 16 , 2013.6 a total of 15,576 women aged 4575 years were contacted through knowledgepanel ( gfk custom research , princeton , nj ) , a demographically representative panel of us citizens , making the revive survey the largest study cohort of postmenopausal women in recent years . of 10,486 women who responded , 8081 ( 77% ) identified themselves as postmenopausal ( ie , having no menstrual period for the previous 12 months for natural or surgical reasons ) . among 8081 postmenopausal women , 3046 ( 38% ) reported 1 symptom consistent with vva ( dyspareunia , vaginal dryness , local irritation , tenderness , bleeding with sexual activity , or pain with exercise ) . the common medical terms vva and vulvar and vaginal atrophy were unfamiliar to most women reporting vva symptoms . most women were unaware that their vaginal symptoms could caused by menopause or hormonal changes ; only approximately one - quarter of women specifically identified menopause as the cause of their symptoms.6 thus , the revive survey demonstrates that many postmenopausal women have low awareness and poor understanding of vva and its associated symptoms . these findings show the contrast between perceptions of vva symptoms and other symptoms that are more readily associated with menopause ( eg , hot flushes ) . women who associate vva with menopause may assume that it will abate over time , similar to vasomotor symptoms . almost half of the study population had never discussed their vva symptoms with an hcp . forty percent of women with vva symptoms said they expected hcps to initiate this conversation ; however , among those who had discussed vva symptoms , the hcp was the initiator only 13% of the time . similarly , among participants who had an hcp for gynecologic needs , only 19% reported being asked about sexual health during routine examination . the most common reasons for not mentioning symptoms to hcps were the assumption that their symptoms were a natural part of aging or were not bothersome enough at that time.6 among women who initiated discussions about vva , 73% waited until a scheduled physical examination and ~50% waited greater than 7 months to do so.6 the most common symptoms prompting a visit specifically to discuss vva were vaginal irritation ( 50% ) , dyspareunia ( 27% ) , and vaginal dryness ( 24% ) . among women who discussed vva symptoms with an hcp , ~50% felt neutral or negative about the information and recommended treatment options they received.6 this inadequate understanding of vva , along with poor communication between women and their hcps regarding vva , may contribute to delayed diagnosis and treatment . data from the revive survey demonstrate the considerable impact of vva on women s lives . the most commonly reported vva symptoms among postmenopausal women were vaginal dryness ( 55% ) , dyspareunia ( 44% ) , and local irritation ( 37% ) ( table 1).6 more than half of participants reported that vva had the greatest impact on enjoyment of sex ( table 2 ) . in all , 12% of women without a sexual partner noted they were not seeking one because of discomfort caused by their vva symptoms . approximately one in four women reported that other areas of their life were negatively affected by vva , including sleep , general enjoyment of life , and temperament . although some participants in the revive survey stated that the earliest onset of vva symptoms was premenopause ( 13% ) , or during the first year after their last menstrual period ( 20% ) , most ( 67% ) reported that their earliest symptoms began during postmenopause ( greater than one year after their last menstrual period).6 the onset of individual symptoms occurred at variable times , with irritation being the most likely symptom occurring premenopause , and dryness or tenderness more likely to begin during the first year after cessation of menstrual periods . use of vva - specific treatments ( vaginal otc products [ eg , astroglide and replens ] or vaginal prescription therapies [ eg , estrace , vagifem , and estring ] ) was reported by 40% of participants.6 otc products were used by 67% of those who ever used a treatment , and vaginal estrogen therapies were used by 27% . even among women who had discussed symptoms with an hcp , the use of otc products as monotherapy was common ( 62% ) ; 23% of women who had spoken with an hcp were on prescription therapy and 15% of women were using vaginal prescription therapy + otc vaginal therapy . reported limitations of participants current otc or prescription treatments included inadequate symptom relief , inconvenience , and dislike of the accommodations needed for vaginal administration ( ie , privacy and nighttime administration ) . previous population - based surveys710 have identified that many women who are using vaginal therapies to treat their vva symptoms are dissatisfied and discontinue treatment because they had concerns about side effects , or had found the treatment messy and inconvenient or not to have an effect on their symptoms . an additional analysis of data collected in the revive survey6 looked at women s perspectives on their satisfaction / dissatisfaction with their current treatments for vva including otc lubricants and moisturizers as well as prescription vaginal estrogen therapies . of the 3046 postmenopausal women in the revive survey who reported vva symptoms , 41% were currently using some form of treatment for vva . however , of those women who actually had been given a clinical diagnosis of vva ( 9% recalled being given a diagnosis ) , 27% were not using any treatment for their symptoms neither a prescription nor otc treatment . only 35% of postmenopausal women currently on any treatment for vva ( otc , prescription vaginal estrogens , or both ) were satisfied with their current vva treatment , with women using vaginal estrogen generally more satisfied than women using otc lubricants or moisturizers ( 42% vs. 32% , respectively ) . in the assessment of dislikes among current treatment options , long - term safety ( 41% ) messiness ( 43% ) was the major concern identified by users of otc lubricants or moisturizers ( table 3 ) . many women recognized that otc lubricants or moisturizers were able to neither restore the vagina to its natural state ( 39% ) nor provide adequate relief of vva symptoms ( 28% ) . for the women who remained nave to treatment despite a diagnosis of vva , major concerns for not initiating treatment included safety concerns ( 28% ) and concerns about hormone exposure ( 16% ) . however , many of these women ( 44% ) felt their symptoms were not bothersome enough to warrant treatment . with regard to satisfaction with current treatment(s ) , 40% of those using vaginal estrogen therapy felt they had no other treatment option , with an additional 22% feeling inconvenienced with the mode of administration . in addition , 38% of all women who had ever used prescription products to treat vva chose not to refill their vaginal estrogen therapy because of a variety of concerns related to safety or side effects ( 45% ) including long - term safety concerns ( 28% ) , administration ( 8% ) , messiness ( 15% ) , or overall treatment efficacy ( 47% ) , with 16% citing not enough relief from vva symptoms . similar concerns were raised by those women who discontinued the use of otc lubricants and moisturizers , including issues related to administration ( 10% ) , messiness ( 14% ) , and overall treatment efficacy ( 38% ) . some women also stated the symptoms not being bothersome enough as a reason for discontinuing their current therapy ( otc , 9% ; vaginal estrogen , 14% ) . when asked about preferences regarding the method of treatment for their vva symptoms an oral treatment was highly preferred among younger patients as well as those patients who had never used any vva treatment , especially among those who indicated a direct preference . for those women currently using any type of treatment for their vva symptoms , there was no difference in preference between an oral treatment ( 35% ) or a vaginal treatment ( 41% ) ( table 4 ) . the common medical terms vva and vulvar and vaginal atrophy were unfamiliar to most women reporting vva symptoms . most women were unaware that their vaginal symptoms could caused by menopause or hormonal changes ; only approximately one - quarter of women specifically identified menopause as the cause of their symptoms.6 thus , the revive survey demonstrates that many postmenopausal women have low awareness and poor understanding of vva and its associated symptoms . these findings show the contrast between perceptions of vva symptoms and other symptoms that are more readily associated with menopause ( eg , hot flushes ) . women who associate vva with menopause may assume that it will abate over time , similar to vasomotor symptoms . almost half of the study population had never discussed their vva symptoms with an hcp . forty percent of women with vva symptoms said they expected hcps to initiate this conversation ; however , among those who had discussed vva symptoms , the hcp was the initiator only 13% of the time . similarly , among participants who had an hcp for gynecologic needs , only 19% reported being asked about sexual health during routine examination . the most common reasons for not mentioning symptoms to hcps were the assumption that their symptoms were a natural part of aging or were not bothersome enough at that time.6 among women who initiated discussions about vva , 73% waited until a scheduled physical examination and ~50% waited greater than 7 months to do so.6 the most common symptoms prompting a visit specifically to discuss vva were vaginal irritation ( 50% ) , dyspareunia ( 27% ) , and vaginal dryness ( 24% ) . among women who discussed vva symptoms with an hcp , ~50% felt neutral or negative about the information and recommended treatment options they received.6 this inadequate understanding of vva , along with poor communication between women and their hcps regarding vva , may contribute to delayed diagnosis and treatment . data from the revive survey demonstrate the considerable impact of vva on women s lives . the most commonly reported vva symptoms among postmenopausal women were vaginal dryness ( 55% ) , dyspareunia ( 44% ) , and local irritation ( 37% ) ( table 1).6 more than half of participants reported that vva had the greatest impact on enjoyment of sex ( table 2 ) . in all , 12% of women without a sexual partner noted they were not seeking one because of discomfort caused by their vva symptoms . approximately one in four women reported that other areas of their life were negatively affected by vva , including sleep , general enjoyment of life , and temperament . although some participants in the revive survey stated that the earliest onset of vva symptoms was premenopause ( 13% ) , or during the first year after their last menstrual period ( 20% ) , most ( 67% ) reported that their earliest symptoms began during postmenopause ( greater than one year after their last menstrual period).6 the onset of individual symptoms occurred at variable times , with irritation being the most likely symptom occurring premenopause , and dryness or tenderness more likely to begin during the first year after cessation of menstrual periods . use of vva - specific treatments ( vaginal otc products [ eg , astroglide and replens ] or vaginal prescription therapies [ eg , estrace , vagifem , and estring ] ) was reported by 40% of participants.6 otc products were used by 67% of those who ever used a treatment , and vaginal estrogen therapies were used by 27% . even among women who had discussed symptoms with an hcp , the use of otc products as monotherapy was common ( 62% ) ; 23% of women who had spoken with an hcp were on prescription therapy and 15% of women were using vaginal prescription therapy + otc vaginal therapy . reported limitations of participants current otc or prescription treatments included inadequate symptom relief , inconvenience , and dislike of the accommodations needed for vaginal administration ( ie , privacy and nighttime administration ) . previous population - based surveys710 have identified that many women who are using vaginal therapies to treat their vva symptoms are dissatisfied and discontinue treatment because they had concerns about side effects , or had found the treatment messy and inconvenient or not to have an effect on their symptoms . an additional analysis of data collected in the revive survey6 looked at women s perspectives on their satisfaction / dissatisfaction with their current treatments for vva including otc lubricants and moisturizers as well as prescription vaginal estrogen therapies . of the 3046 postmenopausal women in the revive survey who reported vva symptoms , 41% were currently using some form of treatment for vva . however , of those women who actually had been given a clinical diagnosis of vva ( 9% recalled being given a diagnosis ) , 27% were not using any treatment for their symptoms neither a prescription nor otc treatment . only 35% of postmenopausal women currently on any treatment for vva ( otc , prescription vaginal estrogens , or both ) were satisfied with their current vva treatment , with women using vaginal estrogen generally more satisfied than women using otc lubricants or moisturizers ( 42% vs. 32% , respectively ) . in the assessment of dislikes among current treatment options , long - term safety ( 41% ) messiness ( 43% ) was the major concern identified by users of otc lubricants or moisturizers ( table 3 ) . many women recognized that otc lubricants or moisturizers were able to neither restore the vagina to its natural state ( 39% ) nor provide adequate relief of vva symptoms ( 28% ) . for the women who remained nave to treatment despite a diagnosis of vva , major concerns for not initiating treatment included safety concerns ( 28% ) and concerns about hormone exposure ( 16% ) . however , many of these women ( 44% ) felt their symptoms were not bothersome enough to warrant treatment . with regard to satisfaction with current treatment(s ) , 40% of those using vaginal estrogen therapy felt they had no other treatment option , with an additional 22% feeling inconvenienced with the mode of administration . in addition , 38% of all women who had ever used prescription products to treat vva chose not to refill their vaginal estrogen therapy because of a variety of concerns related to safety or side effects ( 45% ) including long - term safety concerns ( 28% ) , administration ( 8% ) , messiness ( 15% ) , or overall treatment efficacy ( 47% ) , with 16% citing not enough relief from vva symptoms . similar concerns were raised by those women who discontinued the use of otc lubricants and moisturizers , including issues related to administration ( 10% ) , messiness ( 14% ) , and overall treatment efficacy ( 38% ) . some women also stated the symptoms not being bothersome enough as a reason for discontinuing their current therapy ( otc , 9% ; vaginal estrogen , 14% ) . when asked about preferences regarding the method of treatment for their vva symptoms an oral treatment was highly preferred among younger patients as well as those patients who had never used any vva treatment , especially among those who indicated a direct preference . for those women currently using any type of treatment for their vva symptoms , there was no difference in preference between an oral treatment ( 35% ) or a vaginal treatment ( 41% ) ( table 4 ) . results of the revive survey verify that many women suffering from vva symptoms do not discuss them with their hcp . they may find it easier to discuss hot flushes rather than vaginal symptoms such as painful sex , because of the sensitivity of the latter topic.11 however , even women who have discussed vva with their hcp may not have had an optimal experience , as illustrated by the high number of women who continue using otc products despite inadequate symptom relief . bridging communication gaps in the difficult topics of sexual health and menopause can result in increased patient adherence and satisfaction with vva treatment . this can be accomplished not only by physicians but also by hcps in gyn and primary care practices such as nurse practitioners and physician assistants who may have more opportunity to engage in in - depth discussions and provide individualized care.12,13 when a potentially uncomfortable topic arises , a key to fostering open communication is to first put the woman at ease by normalizing the conversation , commenting that vva is a common medical condition that most postmenopausal women experience . this can often be accomplished by prefacing questions about vva with general broad statements such as many women have vaginal changes after menopause , so i ask all of my patients about vaginal and sexual health , or tell me if you have experienced symptoms of vaginal changes , such as dryness . opening statements such as these can be followed by more specific questions about bothersome vaginal symptoms.11,14,15 willingness to discuss these issues can even be facilitated in the waiting room by providing a comfortable environment and pamphlets or other materials addressing the topic . if signs of vva are present , although the patients did not complain of any symptoms , a statement can be made such as i notice some changes that i ve seen in many postmenopausal women that may cause symptoms such as vaginal dryness or pain with intercourse , followed by tell me about any symptoms you may have experienced . simple screening questions can be incorporated into routine visits for women in their mid-40s to facilitate an open and honest dialogue about vva and the potential negative impact of vva symptoms . the goal is to identify women who are experiencing vva symptoms but have not mentioned them because of assumptions and misconceptions such as those revealed in the revive survey . before the publication of the women s health initiative study , hormone therapy was prevalent and however , the utilization and acceptance of systemic hormone replacement therapy is currently low,16 resulting in more women presenting with vva symptoms . with the trend toward using the lowest possible hormone dose for the shortest possible time , vva can even occur among postmenopausal women who are taking systemic hormone therapy.17 furthermore , as shown by the revive survey , women often do not understand that vva is related to menopause . effective patient education might begin with the explanation that vva is a common medical condition associated with reduced estrogen levels after menopause , though less well known than vasomotor symptoms ( hot flushes ) . at the same time , hcps should point out that , unlike hot flushes , vva will worsen over time if left untreated.1 findings from the revive survey indicate that , for some women , symptom onset may occur at early stages of menopause and postmenopause ; these women may experience vva symptoms for a prolonged period if treatment is not initiated as soon as symptoms appear . additionally , it may be helpful to describe the physical changes occurring in the vaginal tissues that result in vva and how these changes lead to the symptoms that women experience . changes in the number of superficial and parabasal cells in the vagina lead to reduced moisture and elasticity , which contribute to symptoms such as vaginal dryness and dyspareunia . increases in vaginal ph lead to greater susceptibility to infection , which in turn can produce symptoms such as itching and irritation . visual examination findings , such as vaginal dryness , petechiae , pallor , friability , and redness of the mucosa , may also prompt discussion of menopausal vaginal changes ( fig . 1 ) . an effective way to describe these changes to patients may be as follows : before menopause , due to the presence of estrogen , the vagina is moist and has ridges and folds , like pleats , that allow the vagina to be more flexible and adaptable for intercourse and childbirth . without estrogen , physical changes occur in the vagina , leading to symptoms such as painful sex and dryness , and the chemistry of the vagina changes , resulting in greater likelihood of infection , itching , and irritation . a suggested approach to screening and managing vva and associated symptoms is presented in figure 2 . another important point to emphasize during patient education is that otc products do not effectively treat the underlying pathological causes of vva and therefore do not halt or reverse the progression of this condition . however , certain prescription therapies ( ie , estrogens and estrogen agonist / antagonist therapies ) directly improve the physical changes underlying vva by increasing superficial cells and reducing parabasal cells and vaginal ph . although otc products may be the reasonable first - line option for women with mild symptoms thus , if the otc product is tried and found to be ineffective , as was the case for ~40% of the revive survey participants who used them , the patient would already have a prescription in hand , and thus treatment would not have to be delayed . women should be counseled on the many treatment options available for vva - related symptoms,18 and they can make choices based on their personal preferences and needs . treatment adherence is improved when women participate in the decision - making process . table 5 provides an overview of currently available options for vva , including otc products ( lubricants and moisturizers ) , vaginal estrogen therapies ( conjugated equine estrogen and estradiol creams , estradiol tablet and ring ) , and a new non - estrogen oral therapy , ospemifene , which is an estrogen agonist / antagonist recently approved by the us food and drug administration for the treatment of moderate to severe dyspareunia associated with postmenopausal vva . the revive survey offers important insights on vva from postmenopausal women experiencing undesirable vaginal symptoms . this survey illustrates that women s awareness / understanding of vva is low , and almost half of the revive survey participants had not discussed their vaginal symptoms with their hcp . moreover , 40% of women expected their hcp to initiate a conversation about menopausal symptoms . these findings underscore the need for increased vigilance by hcps caring for postmenopausal women . for women approaching menopause , simple screening questions about vaginal symptoms can be asked during routine visits , and the responses can help identify those in need of treatment . in addition , proactive patient education can be provided on the physical changes underlying vva and how the condition can progress if left untreated . only 41% of respondents were current treatment users , whereas 27% had never been treated and 33% had stopped using treatment . the use of otc products that do not treat the physiological changes underlying vva was prevalent , even among women who had discussed their symptoms with an hcp . nurse practitioners ( nps ) should engage their patients in a candid discussion of symptom severity , preferences , and concerns regarding vva treatments . this approach can help guide treatment selection and patient education , which ultimately may improve patient adherence and outcomes . hcps are in a position to proactively identify women suffering from vva , provide essential patient education regarding the condition , and guide their patients to appropriate treatment .

vulvar and vaginal atrophy ( vva ) is a chronic and progressive medical condition common in postmenopausal women . symptoms of vva such as dyspareunia , vaginal dryness , irritation , and itching can negatively impact sexual function and quality of life . the revive ( real women s views of treatment options for menopausal vaginal changes ) survey assessed knowledge about vva and recorded attitudes about interactions with healthcare providers ( hcps ) and available treatment options for vva . the revive survey identified unmet needs of women with vva symptoms such as poor understanding of the condition , poor communication with hcps despite the presence of vaginal symptoms , and concerns about the safety , convenience , and efficacy of available vva treatments . hcps can address these unmet needs by proactively identifying patients with vva and educating them about the condition as well as discussing treatment preferences and available therapies for vva .

pubmed

in denmark , women planning to conceive are advised to take a daily supplement of 400 g folic acid ( fa ) until the 12th week of gestation to reduce the risk of neural tube defects ( ntds).1 starting fa supplementation before conception is an important component of effective prevention of ntds , since the neural tube closes at gestational week 6 , only a few weeks after the pregnancy may be recognized.2 fa is primarily taken as single supplements or as a component of multivitamins ( mvs ) and other vitamin preparations made for pregnancy planners or pregnant women . despite campaigns promoting the fa recommendation in denmark in 1999 and 2001 , women in the preconceptional period appear to have neither adequate dietary intake of folate nor adequate supplementation from fa tablets or mv supplements . according to the national survey of dietary habits from 2003 to 2008 , only 510% of danish women of fertile age have a sufficient intake of dietary folate.3 the most recent data on periconceptional use of fa supplements in denmark are from the danish national birth cohort ( dnbc ) . between 2000 and 2002 , the proportion of fa compliers among dnbc participants who planned their pregnancy increased from 14% to only 22% , and thus remained low even after the campaigns.4,5 several retrospective studies of pregnant women in other countries also reported poor compliance , with 12% of women in the uk,6 17% in norway,7 2330% in australia8,9 and 44% in the us10 reporting fa supplement use in the preconception period . in addition , a prospective cohort study reported that only 6% of the women who became pregnant within 3 months of being interviewed in a general woman s survey in the uk followed the fa recommendation.11 in previous studies , young age , low education and income , smoking , overweight , and parity were reported to be predictors of noncompliance.58,10 to date , no study has examined other health - related predictors of compliance during the preconceptional period , and we lack recent estimates of compliance with the fa recommendation reported from pregnancy planners during the preconceptional period . in the current study , we estimated the prevalence of fa and mv supplement use and identified sociodemographic , lifestyle , reproductive , and medical predictors of such use among danish pregnancy planners . the present study was a cross - sectional analysis of baseline data from an internet - based prospective cohort study of danish pregnancy planners snart - gravid ( soon pregnant).the study design has been described in detail elsewhere.12,13 briefly , the study was initiated in june 2007 . participants were enrolled via the study website , and data were collected by email and self - administered questionnaires . recruitment was achieved by a pop - up advertisement placed on a well - known health - related website ( http://www.netdoktor.dk ) and two press releases . before enrolment , potential participants entering the study website were required to read a consent form and fill in a screening questionnaire in order to confirm eligibility . further , participants were required to provide a valid email address and their personal civil registry ( cpr ) number , which is a unique ten - digit personal identification number allowing linkage to a number of nationwide registries . eligible women were invited to complete a baseline questionnaire and bimonthly follow - up questionnaires for 12 months or until conception occurred , after which active follow - up ended . participants were initially randomized to receive either a short- or a long - form baseline questionnaire . completion rates and missing data were similar for both questionnaire versions.13 eligible women were danish residents aged 1840 years , living in a stable relationship with a male partner , not using birth control , not receiving any type of fertility treatment , and attempting to conceive for no more than 12 months . from june 1 , 2007 to august 3 , 2011 of these , one woman did not provide a valid cpr number in the screener , one woman was excluded because she was already pregnant ( 14 weeks ) , and 533 women were excluded because they had been attempting to conceive for more than 12 months . in total , 5383 eligible women enrolled in the study during the 4-year study period . data on preconceptional use of fa and mv supplements and sociodemographic , lifestyle , reproductive , and medical variables were assessed at baseline . in the baseline questionnaire , women were asked do you take vitamins on a regular basis daily or almost every day ? , how long have you been taking vitamins on a regular basis less than one year , 15 years , more than 5 years , or do nt know ? , and which of the following vitamins or minerals do you take on a regular basis mvs , vitamin a , beta - carotene , vitamin b , vitamin c , vitamin d , vitamin e , fa , calcium , magnesium , selenium , or other ? participants who reported multivitamin or wrote the name of an mv product were defined similarly , participants who reported folic acid or wrote folate were defined users were defined as women who used either fa supplements , mv supplements , or both . women who used single - vitamin or mineral supplements other than fa and women who did not take any dietary supplements were defined nonusers . a total of 197 ( 3.7% ) women did not answer the initial vitamin question and were also defined as nonusers . participants reported weight , height , physical activity , and smoking history in the baseline questionnaire , allowing the calculation of body mass index ( bmi ) , total metabolic equivalents ( mets ) , and pack - years of smoking . bmi was calculated by dividing weight ( kg ) with height squared ( m ) . mets were estimated by summing the mets from moderate and vigorous physical activity ( hours per week multiplied by 3.5 and hours per week multiplied by 7.0 , respectively).14 data on smoking were categorized as pack - years of ever - smoking where 1 pack - year was defined as smoking 20 cigarettes per day in 1 year . to assess sociodemographic predictors of compliance among pregnancy planners , we examined the association between preconceptional fa or mv supplement use and the following variables : age ( categorized as < 25 , 2529 , 3034 , and 35 years ) , schooling ( primary and lower secondary school , high school , and other ) , vocational training ( none , short [ <3 years ] , medium [ 34 years ] , and long [ > 4 years ] ) and total monthly household income ( < 12,500 , 12,50024,999 , 25,00039,999 , 40,00064,999 , and 65,000 dkk / month ) . we also examined lifestyle factors that could indicate intense efforts to conceive , such as intercourse frequency ( categorized as < 1 , 13 , and 4 times per week ) and months attempting pregnancy at study entrance ( 01 , 24 , 56 , and 712 months ) . furthermore , compliance with other health recommendations , such as attending the national screening program for cervical cancer ( pap smear ) during the last 3 years ( none vs one or more ) , smoking history ( never smoked , < 5 , 59 , and 10 pack - years ) , current alcohol intake ( none , 13 , 47 , 814 , and 15 drinks / week ) , bmi ( < 18.5 , 18.524.9 , 2529.9 , 3034.9 , and 35 kg / m ) , and engagement in physical activity ( < 10 , 1019 , 2039 , and 40 mets / week ) , were also of interest . finally , we studied the associations with previous spontaneous abortion ( yes vs no ) , parity ( nulliparous vs parous ) , and medical conditions that may increase health awareness in relation to pregnancy , which included hypertension ( yes vs no ) , diabetes ( yes vs no ) , thyroid disease ( yes vs no ) , pelvic inflammatory disease ( yes vs no ) , and infection with chlamydia ( yes vs no ) . we estimated the prevalence proportion ( pp ) of fa or mv users in each level of study predictors . multivariate binomial regression was used to obtain prevalence proportion differences ( ppds ) with 95% confidence intervals ( cis ) for each predictor level in relation to the reference group . the multivariate analyses for the pp and ppd for each study predictor were adjusted for all other covariates . stata statistical software ( version 11.2 ; college station , texas ) was used for all analyses . missing values ranged between 0.1% ( schooling ) and 8.7% ( total monthly household income ) . because 1540 ( 28.6% ) participants were initially randomized to receive the short - form baseline questionnaire , they did not receive the questions about hypertension , diabetes , thyroid disease , and pelvic inflammatory disease . the present study was a cross - sectional analysis of baseline data from an internet - based prospective cohort study of danish pregnancy planners snart - gravid ( soon pregnant).the study design has been described in detail elsewhere.12,13 briefly , the study was initiated in june 2007 . participants were enrolled via the study website , and data were collected by email and self - administered questionnaires . recruitment was achieved by a pop - up advertisement placed on a well - known health - related website ( http://www.netdoktor.dk ) and two press releases . before enrolment , potential participants entering the study website were required to read a consent form and fill in a screening questionnaire in order to confirm eligibility . further , participants were required to provide a valid email address and their personal civil registry ( cpr ) number , which is a unique ten - digit personal identification number allowing linkage to a number of nationwide registries . eligible women were invited to complete a baseline questionnaire and bimonthly follow - up questionnaires for 12 months or until conception occurred , after which active follow - up ended . participants were initially randomized to receive either a short- or a long - form baseline questionnaire . eligible women were danish residents aged 1840 years , living in a stable relationship with a male partner , not using birth control , not receiving any type of fertility treatment , and attempting to conceive for no more than 12 months . from june 1 , 2007 to august 3 , 2011 of these , one woman did not provide a valid cpr number in the screener , one woman was excluded because she was already pregnant ( 14 weeks ) , and 533 women were excluded because they had been attempting to conceive for more than 12 months . in total , 5383 eligible women enrolled in the study during the 4-year study period . data on preconceptional use of fa and mv supplements and sociodemographic , lifestyle , reproductive , and medical variables were assessed at baseline . in the baseline questionnaire , women were asked do you take vitamins on a regular basis daily or almost every day ? , how long have you been taking vitamins on a regular basis less than one year , 15 years , more than 5 years , or do nt know ? , and which of the following vitamins or minerals do you take on a regular basis mvs , vitamin a , beta - carotene , vitamin b , vitamin c , vitamin d , vitamin e , fa , calcium , magnesium , selenium , or other ? participants who reported multivitamin or wrote the name of an mv product were defined similarly , participants who reported folic acid or wrote folate were defined users were defined as women who used either fa supplements , mv supplements , or both . women who used single - vitamin or mineral supplements other than fa and women who did not take any dietary supplements were defined nonusers . a total of 197 ( 3.7% ) women did not answer the initial vitamin question and were also defined as nonusers . participants reported weight , height , physical activity , and smoking history in the baseline questionnaire , allowing the calculation of body mass index ( bmi ) , total metabolic equivalents ( mets ) , and pack - years of smoking . bmi was calculated by dividing weight ( kg ) with height squared ( m ) . mets were estimated by summing the mets from moderate and vigorous physical activity ( hours per week multiplied by 3.5 and hours per week multiplied by 7.0 , respectively).14 data on smoking were categorized as pack - years of ever - smoking where 1 pack - year was defined as smoking 20 cigarettes per day in 1 year . to assess sociodemographic predictors of compliance among pregnancy planners , we examined the association between preconceptional fa or mv supplement use and the following variables : age ( categorized as < 25 , 2529 , 3034 , and 35 years ) , schooling ( primary and lower secondary school , high school , and other ) , vocational training ( none , short [ <3 years ] , medium [ 34 years ] , and long [ > 4 years ] ) and total monthly household income ( < 12,500 , 12,50024,999 , 25,00039,999 , 40,00064,999 , and 65,000 dkk / month ) . we also examined lifestyle factors that could indicate intense efforts to conceive , such as intercourse frequency ( categorized as < 1 , 13 , and 4 times per week ) and months attempting pregnancy at study entrance ( 01 , 24 , 56 , and 712 months ) . furthermore , compliance with other health recommendations , such as attending the national screening program for cervical cancer ( pap smear ) during the last 3 years ( none vs one or more ) , smoking history ( never smoked , < 5 , 59 , and 10 pack - years ) , current alcohol intake ( none , 13 , 47 , 814 , and 15 drinks / week ) , bmi ( < 18.5 , 18.524.9 , 2529.9 , 3034.9 , and 35 kg / m ) , and engagement in physical activity ( < 10 , 1019 , 2039 , and 40 mets / week ) , were also of interest . finally , we studied the associations with previous spontaneous abortion ( yes vs no ) , parity ( nulliparous vs parous ) , and medical conditions that may increase health awareness in relation to pregnancy , which included hypertension ( yes vs no ) , diabetes ( yes vs no ) , thyroid disease ( yes vs no ) , pelvic inflammatory disease ( yes vs no ) , and infection with chlamydia ( yes vs no ) . we estimated the prevalence proportion ( pp ) of fa or mv users in each level of study predictors . multivariate binomial regression was used to obtain prevalence proportion differences ( ppds ) with 95% confidence intervals ( cis ) for each predictor level in relation to the reference group . the multivariate analyses for the pp and ppd for each study predictor were adjusted for all other covariates . stata statistical software ( version 11.2 ; college station , texas ) was used for all analyses . missing values ranged between 0.1% ( schooling ) and 8.7% ( total monthly household income ) . because 1540 ( 28.6% ) participants were initially randomized to receive the short - form baseline questionnaire , they did not receive the questions about hypertension , diabetes , thyroid disease , and pelvic inflammatory disease . overall , 412 ( 7.7% ) women used fa supplements exclusively , 1100 ( 20.4% ) used mv supplements exclusively , and 1831 ( 34.0% ) used both , yielding 62.1% users . a total of 82 ( 1.5% ) used single vitamins or minerals other than fa , and 1958 ( 36.4% ) did not use any dietary supplements , yielding 2040 ( 37.9% ) nonusers of fa or mv supplements ( table 1 ) . among users , after mutual adjustment for covariates , higher age was associated with increased prevalence of fa or mv use ( table 2 ) . our data also indicate that women who finished high school were more likely to use fa or mvs compared with women with less schooling . similarly , compared with women with a long vocational training ( > 4 years ) , ppds for having no vocational training and a short ( <3 years ) vocational training were 5.7% and 7.1% , respectively , and compared with total monthly household of 65,000 dkk / month , ppd for < 12,500 dkk / month was 8.7% . while intercourse frequency 1 times / week was associated with an increased prevalence of fa or mv use , there was no clear association between months attempting to conceive at study entrance and fa or mv use . furthermore , women who had pap smears once or more during the last 3 years were more likely to use fa or mv supplements compared with women who did not have pap smears ( ppd was 9.7% for none versus one or more ) . kg / m ) were associated with a decreased prevalence of fa or mv use . compared with women who never smoked , ppd for 10 pack - years of smoking was 11.3% . compared with no alcohol intake ppd for 15 drinks / week was 18.4% . compared with normal weight ( bmi = 18.524.9 kg / m ) , ppds of fa or mv use for bmis of 3034.9 and 35 kg / m were 5.1% and 7.1% , respectively . furthermore , women who engaged in physical activity were more likely to use fa or mvs than sedentary women ( < 10 mets / week ) . among women who had a previous spontaneous abortion , the ppd of fa or mv use was 7.7% higher than women who did not report having a previous abortion . finally , there was no clear association between being diagnosed with either hypertension , diabetes , thyroid disease , pelvic inflammatory disease , or infections with chlamydia and preconceptional fa or mv supplement use . our data indicate that 62% of the 5383 pregnancy planners in this study adhere to the danish fa recommendation . by interpreting users as compliers , we may overestimate the proportion of compliers following the fa recommendation . the questionnaire provided information on whether the women took an fa or mv supplement or not , but there was no information on the exact dose of fa ingested or the brand name of the fa or mv product . in addition , mv supplements have no standard definitions,1720 it can not be assumed that all mvs contained fa . however , most mvs on the danish market contain 400 g fa , especially those made for use during pregnancy . the prevalence among pregnancy planners in our study is higher than the prevalence of preconception fa ( 12%44%)510 and mv use ( 11%)8 reported by pregnant women in previous studies . however , these studies collected data during the second and third trimesters , which may decrease the recall of fa and mv use during the preconceptional period . in addition , the studies were relatively small , with 588 women attending an antenatal clinic in melbourne in gestational weeks 3638,8 1541 women attending an antenatal ultrasound screening in gestational weeks 1719 in oslo,7 and 327 pregnant women calling the california teratogen information service in gestational week 13 in the us.10 consequently , prevalence estimates reported in these studies may have been imprecise . the definition of compliance with the fa recommendation differs among studies . in the study by forster et al,8 29% took prepregnancy fa supplements , but only 23% reported taking fa for at least 4 weeks before pregnancy . in the study by braekke and staff,7 17% of this proportion may be more comparable with our findings , since some users in our study may have been in gestational weeks 04 at baseline . women with unplanned pregnancies are less likely to use fa supplements before pregnancy . in the studies by forster et al8 and goldberg et al,10 the prevalence of unplanned pregnancy was 19% and 36% , respectively . these studies estimated fa use among all pregnancies ( planned and unplanned ) , making their results difficult to compare with ours . in the study by braekke and staff,7 there was no information about unplanned pregnancies or contributions of fa from mv supplements , both increasing the possibility of underestimation of the proportions of fa users in this study . in denmark , the largest and most recent study of lifestyle factors in relation to compliance with the fa recommendation was conducted among 18,294 pregnant women ( gestational weeks 1012 ) enrolled in the dnbc during 20002002.5 in this study , fa supplements were derived from single fa tablets , mv tablets , and vitamins made for pregnant or lactating women . compliance was defined as taking 320 g fa per day , from 4 weeks before the date of the last menstrual period until gestation week 6 . a total of 76% of the pregnancies were planned , and the overall proportion of women who complied with the recommendation was only 16% among planners . during the 10-year time period to the present study , there may have been an increase in the awareness as well as more available information about prepregnant recommendations , which may partly explain the observed increase in the prevalence of compliers with the fa recommendation found in this study . in contrast to the dnbc , our data on supplement use and potential predictors were reported during the preconceptional period , and information was recorded prospectively , which increases the validity of the study . on the other hand , snart - gravid study participants may be more health - conscious than the general population of pregnancy planners , possibly overestimating the prevalence of fa or mv use . even so , it is unlikely that an overestimation of supplement use would meaningfully distort the associations between predictors and fa or mv use . in addition , differences in sociodemographic factors among snart - gravid study participants compared with the general population may also contribute to an overestimation of fa or mv use . our study agrees with previous studies that found that maternal age,5,7,10 education,5,6,10 income,6,8 smoking,5,8 and bmi10 were associated with preconception fa use . although a history of childbirth may increase awareness of preconceptional fa use , previous studies found that a previous childbirth either decreased the likelihood of fa use in subsequent pregnancies or was not related to fa use.5,7,8 we found only little association between parity and fa use in our study . the positive associations between previous spontaneous abortion and intercourse frequency and fa or mv use found in our study have not been reported previously . these findings may indicate a relation between a high desire to conceive and increased knowledge about the preconceptional fa guidelines . while lifestyle recommendations during pregnancy are widely available , less advice is available for women trying to conceive . in addition to the fa recommendation , danish women planning a pregnancy are advised to avoid alcohol altogether.1 a total of 32% reported no alcohol intake in our study , which was slightly more than the 12% reported from the dnbc previously.21 in addition , we found the lowest proportion of fa or mv users among women with the highest alcohol intake . together with the concern that excessive alcohol intake impedes the normal bioavailability and metabolism of folate,22 these results underscore the need to target women at high risk of not following the fa recommendation , as well as those not following other preconceptional guidelines . being diagnosed with a chronic disease has previously been associated with increased dietary supplement use in general.23 pregnancy planners with a chronic illness might be more prone to use fa or mvs because of increased health awareness , but we found little association between a diagnosis of hypertension , diabetes , thyroid disease , pelvic inflammatory disease or infections with chlamydia and preconceptional use of fa or mvs . our data indicate that 62% of the 5383 pregnancy planners in this study adhere to the danish fa recommendation . by interpreting users as compliers , we may overestimate the proportion of compliers following the fa recommendation . the questionnaire provided information on whether the women took an fa or mv supplement or not , but there was no information on the exact dose of fa ingested or the brand name of the fa or mv product . in addition , mv supplements have no standard definitions,1720 it can not be assumed that all mvs contained fa . however , most mvs on the danish market contain 400 g fa , especially those made for use during pregnancy . the prevalence among pregnancy planners in our study is higher than the prevalence of preconception fa ( 12%44%)510 and mv use ( 11%)8 reported by pregnant women in previous studies . however , these studies collected data during the second and third trimesters , which may decrease the recall of fa and mv use during the preconceptional period . in addition , the studies were relatively small , with 588 women attending an antenatal clinic in melbourne in gestational weeks 3638,8 1541 women attending an antenatal ultrasound screening in gestational weeks 1719 in oslo,7 and 327 pregnant women calling the california teratogen information service in gestational week 13 in the us.10 consequently , prevalence estimates reported in these studies may have been imprecise . the definition of compliance with the fa recommendation differs among studies . in the study by forster et al,8 29% took prepregnancy fa supplements , but only 23% reported taking fa for at least 4 weeks before pregnancy . in the study by braekke and staff,7 17% of this proportion may be more comparable with our findings , since some users in our study may have been in gestational weeks 04 at baseline . women with unplanned pregnancies are less likely to use fa supplements before pregnancy . in the studies by forster et al8 and goldberg et al,10 the prevalence of unplanned pregnancy was 19% and 36% , respectively . these studies estimated fa use among all pregnancies ( planned and unplanned ) , making their results difficult to compare with ours . in the study by braekke and staff,7 there was no information about unplanned pregnancies or contributions of fa from mv supplements , both increasing the possibility of underestimation of the proportions of fa users in this study . in denmark , the largest and most recent study of lifestyle factors in relation to compliance with the fa recommendation was conducted among 18,294 pregnant women ( gestational weeks 1012 ) enrolled in the dnbc during 20002002.5 in this study , fa supplements were derived from single fa tablets , mv tablets , and vitamins made for pregnant or lactating women . compliance was defined as taking 320 g fa per day , from 4 weeks before the date of the last menstrual period until gestation week 6 . a total of 76% of the pregnancies were planned , and the overall proportion of women who complied with the recommendation was only 16% among planners . during the 10-year time period to the present study , there may have been an increase in the awareness as well as more available information about prepregnant recommendations , which may partly explain the observed increase in the prevalence of compliers with the fa recommendation found in this study . in contrast to the dnbc , our data on supplement use and potential predictors were reported during the preconceptional period , and information was recorded prospectively , which increases the validity of the study . on the other hand , snart - gravid study participants may be more health - conscious than the general population of pregnancy planners , possibly overestimating the prevalence of fa or mv use . even so , it is unlikely that an overestimation of supplement use would meaningfully distort the associations between predictors and fa or mv use . in addition , differences in sociodemographic factors among snart - gravid study participants compared with the general population may also contribute to an overestimation of fa or mv use . our study agrees with previous studies that found that maternal age,5,7,10 education,5,6,10 income,6,8 smoking,5,8 and bmi10 were associated with preconception fa use . although a history of childbirth may increase awareness of preconceptional fa use , previous studies found that a previous childbirth either decreased the likelihood of fa use in subsequent pregnancies or was not related to fa use.5,7,8 we found only little association between parity and fa use in our study . the positive associations between previous spontaneous abortion and intercourse frequency and fa or mv use found in our study have not been reported previously . these findings may indicate a relation between a high desire to conceive and increased knowledge about the preconceptional fa guidelines . while lifestyle recommendations during pregnancy are widely available , less advice is available for women trying to conceive . in addition to the fa recommendation , danish women planning a pregnancy are advised to avoid alcohol altogether.1 a total of 32% reported no alcohol intake in our study , which was slightly more than the 12% reported from the dnbc previously.21 in addition , we found the lowest proportion of fa or mv users among women with the highest alcohol intake . together with the concern that excessive alcohol intake impedes the normal bioavailability and metabolism of folate,22 these results underscore the need to target women at high risk of not following the fa recommendation , as well as those not following other preconceptional guidelines . being diagnosed with a chronic disease has previously been associated with increased dietary supplement use in general.23 pregnancy planners with a chronic illness might be more prone to use fa or mvs because of increased health awareness , but we found little association between a diagnosis of hypertension , diabetes , thyroid disease , pelvic inflammatory disease or infections with chlamydia and preconceptional use of fa or mvs . our findings indicate that more than one - third of the pregnancy planners in this study do not follow the preconceptional fa recommendation . we found that pregnancy planners with other risky behaviors , such as smoking and alcohol use , obesity , and being physically inactive , are less likely to comply with the fa recommendation or use mvs .

purposecompliance with the danish preconceptional folic acid ( fa ) recommendation a daily supplement of 400 g is reported to be poor . uncertainty remains , however , about the prevalence of compliers and health - related predictors of compliance in the preconceptional period.methodswe used self - reported baseline data from 5383 women , aged 1840 years , enrolled in an internet - based prospective cohort study of danish pregnancy planners during 20072011 . we estimated the prevalence proportions of fa or multivitamin ( mv ) use in relation to selected sociodemographic , lifestyle , reproductive , and medical characteristics . multivariate binomial regression was used to obtain prevalence proportion differences with 95% confidence intervals for each level of study predictors , adjusted for all other predictors.resultsoverall , 7.7% of women used fa supplements , 20.4% used mv supplements , 34.0% used both , 1.5% used other single vitamins or minerals , and 36.4% did not use any dietary supplements . the prevalence of fa or mv supplement use was higher among older women , women with higher education and income , and women with healthy lifestyle factors such as being a nonsmoker , nondrinker , physically active , maintaining a normal body mass index and having regular pap smears . greater intercourse frequency and a history of spontaneous abortion were also positively associated with fa or mv supplement use . we found no clear association between use of fa or mv supplements and a diagnosis of hypertension , diabetes , thyroid disease , pelvic inflammatory disease , or chlamydia.conclusiona large proportion of pregnancy planners do not use fa or mv supplements . pregnancy planners with generally risky lifestyle behaviors are less likely to comply with the fa recommendation .

pubmed

peripheral arterial disease ( pad ) commonly results from progressive narrowing of arteries in the lower extremities due to atherosclerosis . it has become evident that pad is an important predictor of substantial coronary and cerebral vascular risk [ 2 , 3 ] . the most common symptomatic manifestation of mild to moderate atherosclerotic pad is intermittent claudication , observed in ~5% of individuals older than 60 years [ 5 , 6 ] . several studies , using the ankle - brachial index , have revealed that the presence of pad , even when being asymptomatic and in patients with no history of other cardiovascular diseases , is a marker of greatly increased cardiovascular morbidity and mortality [ 7 , 8 ] . oxidative stress , through the extra generation of reactive oxygen species ( ros ) , is also involved in the development and progression of pad . moreover , risk factors associated with pad , similar to those for coronary heart disease , include high blood concentrations of total cholesterol and ldl - cholesterol . inhibitors of 3-hydroxy-3-methylglutaryl coenzyme a ( hmg - coa ) reductase , called statins , are well - known lipid - lowering drugs . statins not only reduce lipid levels , but they also have important pleiotropic effects , improving endothelial function , reducing oxidative stress , affecting the lipid metabolism enzyme activities , and so forth [ 1214 ] . three main reasons have been suggested for initiating statin therapy for patients with pad of the lower limbs : ( 1 ) prevention of coronary heart disease ; ( 2 ) treatment of lower extremity peripheral arterial disease ; and ( 3 ) perioperative treatment of patients with peripheral arterial disease . although several trials have shown that treatment with statins is associated with a reduction in coronary heart disease events , such as stroke [ 18 , 19 ] and mortality [ 2023 ] , most of these trials did not specifically address the treatment of patients with pad . the aim of the present study , therefore , was to evaluate lipid peroxidation parameters and total antioxidant capacity in the serum of patients with pad before surgery as well as 35 days and 710 days after surgery . we also compared these parameters with those in a group of patients receiving simvastatin therapy . the study group consisted of pad patients , treated either surgically ( group i , n = 35 males , aged 61 8 years ) or pharmacologically ( group ii , n = 18 males , aged 62 7 years ) in the department of general and vascular surgery at the poznan university of medical sciences . patients included in group i were admitted for surgical treatment ( implantation of an aortobifemoral bypass graft ) . they were diagnosed by measurement of the ankle brachial index ( abi ) and arteriography of the lower limbs , and were clinically stable without any accompanying diseases . the patients treated surgically had shown critical ischemia ( abi less than 0.90 , rest pain , ulcer or necrosis of the lower limbs , and ankle pressure 50 mmhg ) . blood samples were collected without anticoagulant , in the fasting state , before surgery as well as 35 days and 710 days after surgical treatment . group ii had been receiving statin therapy , with 20 mg of simvastatin daily , for at least 3 months . the study protocol was approved by the ethics committee of poznan university of medical sciences , and the subjects were fully informed and gave their written consent . all the reagents were of analytical grade of purity ( sigma - aldrich , st . spectrophotometric measurements were carried out on sp-8001 uv / visible spectrophotometer ( metertech inc . , taiwan ) . the subjects ' lipid profile ( total cholesterol , hdl cholesterol , ldl cholesterol , and triglycerides ) , glucose , uric acid , apolipoproteins ( apo a - i , apo b ) and fibrinogen concentrations in serum were measured by standard laboratory procedures , using the roche cobas 6000 analyzer system , dade behring bn ii system , and the sysmex ca-500 series system . the total antioxidant potential ( capacity ) of serum was determined by ferric reducing antioxidant power ( frap ) assay according to the benzie and strain method , based on the reduction of ferric ion fe(iii ) to ferrous ion fe(ii ) at a low ph . this method measures the change in absorbance at 593 nm owing to the formation of a blue - colored fe(ii)-tripyridyltriazine complex from the colorless oxidized fe(iii ) form , by the action of electron - donating antioxidants . lipid peroxidation was measured by estimation of both lipid hydroperoxides ( loohs ) and malondialdehyde ( mda ) concentrations by the sodergren method and ohkawa et al . the concentration of thiol ( -sh ) groups was determined according to the hu method . ceruloplasmin , in the presence of o - dianisidine hydrochloride in an acidic solution ( ph 5.0 ) , forms a stable yellow - brownish product . absorbance was measured at 540 nm and ceruloplasmin activity was expressed in u / l ( mol of oxidized substrate in 1 ml of serum in 1 min ) . the paired student 's t - test ( to compare the values before and after surgery ) , the two - tailed unpaired t - test ( to compare group i and group ii ) , and the f - test were used for statistical analysis of the data . the mean changes ( ) in lipid peroxidation parameters and antioxidant capacity , that is , 35 days ( 1 ) and 710 days ( 2 ) after surgery , in relation to the values obtained before surgery , were calculated . pearson 's correlation coefficients r1 and r2 were computed for the results found before surgery and the changes observed during treatment ( 1 , 2 ) . as shown in table 1 , generally , the clinical parameters of the two groups of patients included in this study did not differ significantly . however , the concentrations of total cholesterol and ldl cholesterol were found to be significantly higher in group i before surgery than those in group ii on simvastatin therapy . group i patients before surgery had enhanced oxidative stress , as demonstrated by elevated serum levels of mda , in comparison to those treated pharmacologically . we did not observe any significant decrease in looh and mda concentrations in group i patients after surgery ( figures 1 and 2 ) . the total antioxidant capacity ( expressed as frap ) and -sh group concentrations in the serum of patients on statin therapy and in those before surgery were similar ( figures 3 and 4 ) . however , a decrease was observed in frap level 35 days after surgery , and 710 days after surgery , it was significantly lower in comparison to the values before surgery and on simvastatin therapy . moreover , thiol group concentrations 35 days postoperatively were lower than those before surgery and 710 days after surgery and were lower than those in patients treated with simvastatin ( figure 4 ) . a significant difference in the oxidase activity of ceruloplasmin between group ii and group i before surgery was observed . its oxidase activity in serum was higher in group ii and 710 days after surgery in group i , in comparison to the values before and 35 days after surgery ( figure 5 ) . in addition , all the studied parameters ( looh , mda , frap , -sh groups , and ceruloplasmin ) in group i patients before surgery were significantly correlated with mean changes at 35 days ( 1 ) and/or 710 days ( 2 ) after surgery ( table 2 ) . oxidized ldl is a biochemical marker of oxidative damage and has atherogenic effects , including the stimulation of foam cell formation and activation of the inflammatory process in the vascular wall . the findings of the present study are broadly consistent with the hypothesis that lipid peroxidation may be one mechanism through which several risk factors may promote pad . , we observed an increased mda level in the patients before surgery , which suggests that they were in a state of high oxidative stress . at neutral ph , mda exists as a low reactive enolate anion , but it is still toxic and may damage many biologically important molecules . determination of the lipid peroxidation product mda in pad is important because , as was shown earlier , mda damages collagen by forming transverse intramolecular bonds that cause rigidity of blood vessels . in addition , modification of native ldl by mda causes the accumulation of cholesteryl esters within the cell in which the atherosclerotic reaction takes place . several trials have shown that statin therapy relieves symptoms and improves function in patients with lower extremity pad [ 32 , 33 ] . mda concentrations in the serum of the patients treated with simvastatin were lower than those in group i patients before surgery . since pad patients are usually undertreated with regard to the use of lipid - lowering agents , compared to patients with coronary heart disease , our results provide strong support for the efficacy of statin therapy in pad . human cells and tissues contain many antioxidant enzyme systems and nonenzymatic antioxidants , which protect against the action of toxic free radicals . the frap assay used here is a useful indicator of the body 's antioxidant status , which determines the level of non - enzymatic antioxidants , such as uric acid , ascorbic acid , -tocopherol , and some protein - containing -sh groups . an increase in frap level in the patients before surgical treatment is consistent with similar results obtained by gawron et al . in a group of patients with coronary heart disease and by lantos et al . in patients with hypertension . an increased antioxidant capacity of serum seems to be response to an increase in the concentration of lipid peroxidation products . the reduction in frap level observed in our group i patients 35 and 710 days after surgery can be partly explained by a decrease in their serum antioxidant status and an increase in serum oxidant levels following surgery , as indicated by the negative correlations found ( table 2 ) . in their study , girona et al . they showed that simvastatin therapy increases the resistance of both ldl and hdl to oxidation . in addition , simvastatin inhibits the ability of activated macrophages to oxidize ldl in vitro in a dose - dependent manner . a similar effect has been shown for other statins , such as atorvastatin , which reduces ldl levels and hdl oxidation and protects paraoxonase an enzyme bound to hdl . with regard to frap in our study , simvastatin therapy ( 20 mg daily ) did not influence the total antioxidant potential . however , shin et al . found a significant increase in antioxidant ability in their group of hypercholesterolemic patients treated with 40 mg simvastatin daily . they suggested the possibility of a dose - dependent effect of simvastatin on plasma antioxidant status . this could be the reason why we did not detect any effect of simvastatin on frap in our study . glutathione and other proteins , such as albumin , are an important part of serum antioxidant activity against free radicals . the thiol groups react with peroxide radicals in the first stage of the oxidation reaction and also act in the lag phase , which is an accepted marker of serum oxidizability . in our group of patients with pad , the decrease in thiol group concentrations , observed 35 days after surgery , followed by an increase 710 days after surgery , may be explained by oxidative stress intensification in reperfusion and by the actions of -sh groups , leading to their reduction . furthermore , a decrease in both lag phase and albumin level has also been reported in the serum of patients with aneurysmal or arterial occlusive disease . some studies have confirmed that ceruloplasmin is an independent risk factor for cardiovascular disease [ 43 , 44 ] . authors of that study suggest that increased ceruloplasmin activity is related to the acute phase response to inflammation and necrosis in atherosclerosis obliterans and results from increased ros production , mostly of superoxide radicals . therefore , the preferential oxidative destruction of thiol groups can explain the greater susceptibility of ceruloplasmin to oxidative stress . recent data show that statins may reduce oxidative stress by decreasing the generation of ros , thereby synergizing with the biological effects of antioxidants . the results of our study show that ceruloplasmin activity was influenced by both simvastatin therapy and surgical treatment , but only 710 days postoperatively . in our opinion , simvastatin may be implicated in antioxidant properties of ceruloplasmin , as reflected by the increase in its activity in men on simvastatin therapy . several in vitro studies have indicated that ceruloplasmin is a potent catalyst of ldl oxidation and that has been attributed to the presence of a reduced copper atom on its surface . ros dissociate copper from ceruloplasmin and therefore can markedly alter its structure and function , which could explain the initial lack of differences in its activity 35 days after surgery . in view of the escalating prevalence of pad in our aging population , further studies are needed to clarify precisely not only the mechanisms involved but the relative importance of the lipid - related and nonlipid - related effects of statins as well . results of this study show diverse oxidative state responses to surgical treatment and confirm the beneficial effects of statin therapy in pad .

the various risk factors for peripheral arterial disease ( pad ) are almost identical to those for atherosclerosis and include abnormal levels of lipids or lipoproteins . lipid peroxidation parameters and total antioxidant capacity in the serum of male patients with pad before surgery as well as 35 days and 710 days after surgery were measured . we also compared these parameters with those in a group of patients receiving simvastatin therapy . concentrations of lipid hydroperoxides ( loohs ) and malondialdehyde , the total antioxidant capacity ( assessed by ferric reducing antioxidant power assay ) , concentration of thiol ( -sh ) groups , and ceruloplasmin activity were determined spectrophotometrically in pad patients treated surgically ( group i ) or pharmacologically ( group ii ) . the patients before surgical treatment had significantly higher concentrations of malondialdehyde but lower ceruloplasmin activity than those observed in group ii , treated with simvastatin . no significant differences before surgery in ferric reducing antioxidant power or thiol concentrations were found between the two groups . however , in group i , both ferric reducing antioxidant power and thiol group concentrations decreased 35 days postoperatively , and ceruloplasmin activity increased 710 days after surgical treatment . the presented results demonstrate diverse oxidative stress responses to surgical treatment and confirm the beneficial effects of statin therapy in pad .

pubmed

in recent years , the demand for herbal remedies in nigeria has been on the rise . yet there are little or no scientific data to support medicinal claims of these herbal formulations . herbals are usually sought after for their health benefits and these have become common medicines in many nigerian homes . it is recognized that herbal remedies prepared from medicinal plants may have several traditional applications especially in developing nations like nigeria where access to formal healthcare is limited . however , the increasing commercial promotion of herbals necessitate the need for assessment of safety and validity of medicinal claims . in recent past , several studies have demonstrated the toxic effects of indiscriminate use of packaged herbals [ 1 , 4 , 5 ] . furthermore , the general belief that herbal medicines are natural , effective , and without adverse effects has immensely contributed to the upsurge in the patronage of herbal formulations [ 68 ] . the popular belief that herbal remedies are without toxic or undesirable effects these and other factors form part of several concerns associated with the use of herbal remedies in developing nations . in nigeria , studies have revealed that the number of nigerian medicinal plants screened for the validation of biological activity far outweighs the assessment for potential toxic compounds and contaminants [ 2 , 10 , 11 ] . also , many herbal formulations being promoted and marketed in nigeria lack scientific data to support acclaimed medicinal benefits and may pose great health risk to the numerous unsuspecting consumers . according to the fijk leaflet , the herbal is prepared from a mixture of 13 medicinal plants including cassia alata , citrus medica var acida , aloe vera , cassia augustifolia , cassia siamea , kyaha sengalensis , xylopia aethiopica , gongronema latifolium , khaya grandifoliola , morinda lucida , anthocelesta djalonensis , citrullus lanatus , and aloe barbaris . this must have contributed to the wide patronage and consumption among the nigerian populace . according to the fijk label , the herbal remedy is indicated for treatments of several ailments which include obesity , diabetes , sexual improvement , menstruation pains , infertility , high blood pressure , crawling sensation , and malaria , amongst others . there are scientific reports on the medicinal relevance of the individual plant constituents of the fijk mixture . however , there are no empirical data to support claims of medicinal benefits or otherwise of fijk . a search through online research libraries including the cochrane , pubmed , and google - scholar revealed the absence of scientific data on fijk herbal remedy . the scenario makes likely toxic potential from repeated exposure to the fijk mixture largely unknown . for the safety of public health , it is essential that herbal remedies have available scientific data on safety / toxicity profiles in order to aid informed choices by consumers . this study aimed to determine the effect of oral and repeated exposure to fijk on rat plasma biochemical indices and liver morphology . commercial reagent kits for the assay of alkaline phosphatase ( alp ) , aspartate aminotransferase ( ast ) , alanine aminotransferases ( alt ) , total cholesterol ( tc ) , total protein ( tp ) , triglyceride ( tag ) , and high density lipoprotein cholesterol ( hdl - c ) were as supplied by randox diagnostics , crumlin , uk . twenty - four male wistar rats with weight between 190 and 200 g were obtained from the experimental animal farm at the university of ilorin , ilorin , nigeria . the wistar rats were housed in animal cages in a well - ventilated experimental room . the rats were allowed to acclimatize for a period of 14 days before the commencement of treatments . handling of animals was in accordance with relevant institutional and ethical guidelines as approved for scientific study . mg / kg : received fijk at a dosage of 15 mg / kg body weight . fijk 30 mg / kg : received fijk at a dosage of 30 mg / kg body weight . fijk 45 mg / kg : received fijk at a dosage of 45 mg / kg body weight . control : received distilled water . 15 mg / kg : received fijk at a dosage of 15 mg / kg body weight . fijk 30 mg / kg : received fijk at a dosage of 30 mg / kg body weight . fijk 45 mg / kg : received fijk at a dosage of 45 mg / kg body weight . the dosages were selected based on corresponding therapeutic doses for human consumption as recommended by the herbal producer . the treatment was daily and lasted for 21 days . at the end of treatments , the blood samples were collected into edta bottles , spun at 5000 g for 10 minutes using a refrigerated centrifuge ( anke tdl-5000b , shanghai , china ) to obtain the plasma , which was subsequently used for the biochemical analysis . the liver tissues were excised , weighed , and fixed in buffered neutral formalin and used for histopathological examinations . the biochemical indices were determined in rat plasma using a uv / vis spectrophotometer ( jenway , staffordshire , united kingdom ) where applicable . the levels of rat plasma total protein ( tp ) , aspartate aminotransferase ( ast - ec : 2.6.1.1 ) , alanine aminotransferase ( alt - ec : 2.6.1.2 ) , alkaline phosphatase ( alp - ec : 3.1.3.1 ) , and lipid profile including total cholesterol ( tc ) , triglyceride ( tag ) , high - density lipoprotein - cholesterol ( hdl - c ) , and low - density lipoprotein cholesterol ( ldl - c ) were determined using randox assay kits . reduced glutathione level ( gsh ) was determined by the procedure described by ellman with slight modification . lipid peroxidation was ascertained by monitoring the degrees of lipid peroxides in the supernatant fraction of plasma using the method described by niehaus jr . and samuelsson . the rat liver was fixed in 10% buffered neutral formalin ( bnf ) immediately following excision from animals . capture and scoring for morphological changes were done by a pathologist blind to the treatments , at the pathology unit , university of ilorin teaching hospital , ilorin , nigeria . data were subjected to statistical analysis using the one - way anova ( graphpad software inc . , san diego , ca ) . the oral administration of fijk to rats did not produce significant change to average body weights of animals , even though gradual decreases in rat body weights were observed for groups that received fijk herbal mixture ( figure 1 ) . likewise , the relative organ to body weight ratio showed no significant difference among the various treatment groups ( figure 2 ) . however , the oral administration of fijk herbal mixture at double and triple doses caused significant reduction in rat plasma protein level compared to the control group . the determination of lipid profile showed that the oral exposure of rats to fijk at the various doses did not produce significant change in the plasma level of total cholesterol when compared to the control . however , the exposure to fijk at the highest dosage caused significant elevation in rat plasma level of tryglyceride . further assessment of lipid profile revealed that the daily administration of fijk mixture depleted rat plasma level of hdl - c ( figure 3 ) . meanwhile , the level of rat plasma ldl - c rose with increasing doses of fijk herbal . furthermore , the atherogenic index was elevated significantly in the groups exposed to fijk mixture ( figure 4 ) . on the other hand , rat plasma alp level was lowered ( p < 0.05 ) following oral exposure to fijk herbal mixture but the reduction was only significant in the group that received the highest dose of fijk . in contrast , treatment of rats with fijk herbal mixture at all doses significantly increased the levels of plasma alt and ast relative to the control group ( figure 5 ) . to assess whether oral exposure of fijk in rats would predispose to lipid peroxidation , the plasma level of mda was determined . there was significant elevation in rat plasma level of mda in the fijk treated groups relative to the control ( figure 6 ) . conversely , oral administration of fijk to rats significantly depleted the plasma levels of gsh compared to the control . the light microscopic examination of rat liver sections for morphological changes revealed lesions caused by fijk herbal mixture ( figure 7 ) . the fijk herbal mixture caused hepatic alterations including hypochromic , haemorrahage , ruptured vascular channels , and hyperemia . however , the rat liver in the control group had no incidence of this cellular lession . unfortunately the use of herbal products is not strictly regulated in nigeria thus making them freely available , a scenario which predisposes to possible abuse by consumers . although herbal mixtures enjoy wide patronage in nigeria , little is known about likely toxicity that may be associated with repeated consumption . herbal remedies may have recognizable therapeutic effects ; they also may have toxic side effect . more so , many herbal preparations lack scientific facts to back up acclaimed medicinal benefits . currently , there are no available empirical data on fijk herbal mixture . the present study sought to determine the influence of oral and repeated adminstration of fijk herbal mixture on rat biochemical and morphological parameters . this is coming against the background that previous studies have demonstrated toxic potentials of packaged herbals [ 4 , 10 , 11 ] . in the present study , although rat liver weights were reduced in the treatment groups , the relative organ weights were not significantly affected by exposure fijk mixture . however , the evaluation of rat lipid profile revealed that oral administration of fijk herbal mixture did not significantly alter the total cholesterol in all the treatment groups . however , there were dose - dependent increases ( p < 0.05 ) to the plasma levels of tag and ldl - c in rats orally exposed to fijk herbal mixture . conversely , levels of plasma hdl - c were significantly lowered by repeated administration of fijk mixture and the effect was dose dependent . the lipid profile indices are useful in monitoring health status of the cardiovascular system . although the rat total cholesterol level was not affected by fijk herbal mixture administration , elevated levels of tag and ldl - c may predispose to cardiovascular related disorders . increased level of ldl - c has been associated with higher risk of atherosclerosis while elevated level of hdl - c is linked to reduced occurences of cardiovascular disorder [ 19 , 20 ] . the level of rat plasma hdl - c decreased with increasing doses of fijk leading to elevated atherogenic index . the atherogenic index can be used to predict the risk for development of cardiovascular disorders . therefore , the low ratio of hdl - c to ldl - c , caused by repeated oral exposure to fijk , may implicate increased tendency for the development of atherosclerosis . the rat plasma levels of alt and ast were elevated by exposure to repeated administration of fijk mixture relative to control . the alt and ast are normally found in the red blood cells , liver , heart , and kidney tissues . the levels of alt and ast have long been used to assess the functions of liver . increased plasma levels of both alt and ast have been linked to tissue toxicity [ 21 , 22 ] . normally , a basal level of the enzymes is found in the plasma ; however , when there is cellular damage , the enzymes extrude into the extracellular fluid thus raising the concentrations in the plasma . in the present study , the significant alterations to levels of rat plasma alt and ast this is further supported by the fact that the ratio of ast to alt in plasma was less than one ( < 1 ) , which may indicate ensuing acute or chronic liver injury . on the other hand , the rat plasma alp levels were significantly reduced in the group which received the highest dosage of fijk herbal mixture . the decreased level may be as a result of inactivation or decreased protein synthesis . previous studies have shown that herbal mixtures have capability to alter the levels of alt , ast , and alp in rats [ 1 , 9 ] . furthermore , in order to evaluate whether the oral and repeated exposure to fijk mixture was capable of predisposing to oxidative stress , we determined the levels of rat plasma mda and gsh . the mda is a by - product of lipid peroxidation while the gsh level could be used to assess the antioxidant status of a cellular system . the increased level of rat plasma mda suggested the presence of lipid peroxidation caused by oral exposure to fijk herbal mixture . the increased mda level in rats may be due to the generation of free radical species potentiated by exposure to fijk mixture . on the other hand , ensuing oxidative stress caused by the oral administration of fijk was further demonstrated by the depleted level of rat plasma gsh . the gsh molecule is a nonenzymatic antioxidant capable of scavenging free radicals and it could also serve as substrate to other antioxidant enzymes like the glutathione peroxidase . the low level of gsh may be attributed to increased usage in order to scavenge free radical species or consumption as substrate by antioxidant enzymes which function to protect against oxidative damage . recent studies have associated oral and repeated exposure to herbal mixtures with lipid peroxidation and oxidative stress [ 1 , 5 ] . the light microscopic examinations of rat liver sections showed intact and normal cellular architecture in the control group . however , the examination of rat liver sections in groups exposed to various dosages of fijk mixture revealed incidences of morphological lesions . the rat hepatic alterations caused by exposure to fijk herbal mixture included haemorrahage , ruptured vascular channels , hyperemia , and inflammation . the histopathological findings support the biochemical alterations caused by oral and repeated exposure to fijk herbal mixture and are reminiscent of rat hepatic injury . this is consistent with a previous report which demonstrated organ toxicity caused by herbal formulations . the study revealed elevated atherogenic index caused by oral and repeated exposure to fijk mixture in rats . furthermore , oral and repeated exposure to fijk herbal mixture in rats caused lipid peroxidation and potentiated hepatic lesion .

this study determined the effect of the oral and repeated administration of fijk herbal mixture on rat biochemical and morphological parameters . twenty - four wistar rats were distributed into four groups of 6 . group a served as control and received oral administration of distilled water daily . the experimental groups b , c , and d were daily and orally exposed to fijk herbal mixture at 15 , 30 , and 45 mg / kg , respectively . treatments lasted for 21 days . the rats were sacrificed under mild diethyl ether anesthesia 24 hr after cessation of treatment . the blood and liver samples were collected and used for the biochemical and morphological analyses . oral exposure to fijk caused elevated levels of rat plasma alt , ast , triglycerides , ldl , and mda . in contrast , rat plasma hdl , gsh , and alp levels were lowered by fijk oral exposure . also , the herbal remedy caused a dose - dependent elevation in the plasma atherogenic index . the histopathology examinations of rat liver sections revealed inimical cellular alterations caused by repeated exposure to fijk . study provides evidence that oral and repeated exposure to fijk in rats raised the atherogenic index and potentiated oxidative stress as well as hepatic injury .

pubmed

identifying carriers of autosomal recessive or x - linked disorders before pregnancy has the potential to benefit prospective parents . couples can become aware of the possible genetic risks to future offspring and of the reproductive options available . these options include not only prenatal diagnosis followed ( or not ) by termination of the pregnancy in case of an affected fetus or by coming to terms with the risk , but also the choices of using preimplantation genetic diagnosis , using donor sperm or oocytes , seeking adoption or refraining from having children . in some culturally related marriage practices there are two approaches to the identification of carriers : carrier screening and carrier testing . carrier screening is defined as the detection of carrier status in persons who do not have an a priori increased risk for having a child with a certain disease , whereas with carrier testing , the persons do have a higher a priori risk based on their or their partners personal or family history ( castellani et al . , 2010 ) . carrier screening can be offered on an individual basis , but also as an organized screening programme , either during or before pregnancy . while most screening programmes in health care aim to prevent , treat and alleviate disease , above and beyond these aims , the particular goal of preconceptional carrier screening is to strengthen reproductive autonomy and informed decision - making . if offered preconceptionally , this can mean less time constraints , less pressure and less emotional stress than when a test is performed during pregnancy ( bombard et al . , 2010 ) internationally , the potential of preconceptional carrier screening has been studied extensively for cystic fibrosis ( cf ) ( bekker et al . , 1993 ; tambor et al . , 1994 ; axworthy et al . these studies have repeatedly revealed positive attitudes towards cf carrier screening from health care providers ( poppelaars et al . , 2004a ; mcclaren et al . , 2008 ) , patients and their relatives ( poppelaars et al . , 2003 ) and from the general public ( poppelaars et al , 2004c ) . furthermore , preconceptional carrier screening for cf for couples with no family history of cf was recommended a decade ago by the national institutes of health ( nih ) ( national institutes of health , 1999 ) , the american college of medical genetics ( acmg ) and the american college of obstetricians and gynaecologists ( acog ) ( grody et al . , 2001 ) . despite this , screening is not currently offered in most countries . carrier testing for disorders such as cf is still usually restricted to families and partners of cf patients and carriers . apart from some specific regions ( e.g. thalassemia carrier screening in cyprus and sardinia ) and specific communities ( e.g. carrier screening for ashkenazi jews ) , traditional healthcare systems have hesitated to implement carrier screening programmes for the entire population of couples planning a pregnancy . various concerns underlie this stance , including the psychological impact ( axworthy et al . , 1996 ) and risk of stigmatization / discrimination of being a carrier ( mcqueen , 2002 ) , impairment of the freedom of choice because of the undue pressure on individual choice ( godard et al . , 2003 ) , medicalization ( poppelaars et al . , 2004c ) , the fact that couples are difficult to reach before conception ( lakeman et al . , 2009 ) , and provider - related barriers such as a lack of familiarity with genetics and selection criteria for offering screening ( morgan et al . , 2004 ) . recently , commercial companies have been offering preconceptional carrier tests directly - to - consumers . these direct - to - consumer ( dtc ) tests provide information about monogenic diseases and the risk of having affected offspring . dtc genetic testing is often defined as the offer and/or marketing of genetic tests directly to the public without the intermediary of a health care professional from the traditional health care system . as has been suggested by the human genetics commission , however , we also include in this discussion tests that are commissioned by the consumer from a commercial company outside the traditional health care system but where a medical practitioner or a health professional is involved in the provision of the service ' ( human genetics commission , 2010 ) . the availability of dtc genetic testing services in general has created a huge debate about the desirability of these activities ( howard and borry , 2008 ; hunter et al . , 2008 ; kaye , 2008 ) . however , until now , these discussions have mainly focused on the commercial dtc offer of genome - wide - testing ; a service which offers individual risk assessments for common disorders ( janssens et al . , 2008 ; melzer et al . , 2008 ) with limited or no medical or health benefits to the individual user . various concerns were raised with regard to this dtc offer of genetic tests , including , among others , concerns related to the validity and utility of tests offered , the absence of individualized medical supervision , the lack of an adequate consent procedure , the absence and/or quality of pre- and post - test genetic counselling , the inappropriate testing of minors , the respect for privacy and the potential burden on public health resources . because of the great heterogeneity in types of dtc genetic tests , and the fact that the consequences of their results vary widely , a one - size - fits - all approach is not appropriate for all dtc genetic tests . since preconception carrier screening has been accepted by various medical associations , dtc testing for these tests may be regarded differently when compared with the dtc tests for common disorders that have been criticized because of , for example , the lack of clinical utility ( i.e. how likely the test is to significantly improve patient outcomes ) and analytical validity ( i.e. how accurately and reliably the test measures the genotype of interest ) . therefore , the aim of this article is to discuss various ethical issues raised by the dtc offer of preconceptional carrier tests for autosomal recessive disorders from commercial companies that are not embedded in regular health care . in the following order , this article will present and discuss : ( i ) the current offer of preconceptional carrier tests through online providers ; ( ii ) the implications for the informed consent procedure and the possible consequences for the provision of information ; ( iii ) the need for medical supervision and follow - up and ( iv ) the current offer in relation to the appropriate use of existing resources . the details of the offer from each company , however , vary greatly . in may 2010 , the company dna direct ( www.dnadirect.com ) advertised one individual carrier test ( for cf ) and one carrier testing panel ( for ashkenazi jews ) . on its website professional medical groups including the nih ( ) and acog recommend that cf carrier screening be offered to all couples who are planning a pregnancy or are currently pregnant ( https://www.dnadirect.com/web/article/testing-for-genetic-disorders/cystic-fibrosis/30/who-should-consider-testing accessed 19/01/2011 ) . the company dnatraits says that they are committed to making all medically validated tests available to consumers rapidly , inexpensively and understandably ( www.dnatraits.com/compare accessed 03/05/2010 ) and is selling a limited number of individual tests ( e.g. for alpha-1-antitrypsin deficiency ) as well as panels of disorders ( e.g. an ashkenazi jewish genetic disease panel or a sickle cell / beta - thalassaemia panel ) . along with risk assessment information for other disorders , pathway genomics ( www.pathway.com ) and 23andme ( www.23andme.com ) include in their full genome testing report the carrier status for 37 and 24 different single - gene conditions , respectively . finally , in february 2010 the company counsyl ( www.counsyl.com ) launched their offer of a prepregnancy universal carrier test which tests an individual or couple for over 100 , mostly autosomal recessive , genetic diseases . counsyl considers these activities as a cause , a campaign to finally end the needless suffering of preventable genetic disease ( https://www.counsyl.com/about/counsyl/ accessed 19/01/2011 ) . these abovementioned dtc genetic tests providers do not make a distinction between screening individuals with an a priori low population risk or testing individuals with a high a priori probability of having an affected child . because of this increasing possibility for determining an individuals risk of transmitting a condition to his or her offspring and the potential opportunities and threats generated by these results to individual citizens and society as a whole , the health council of the netherlands ( 2008 ) , for example , emphasized the need for criteria for a responsible offer of genetic dtc screening as well . in this specific report , the original genetic screening criteria , as outlined by the who ( wilson and jungner , 1968 ) , which relate to the prerequisites for starting community - wide screening , are extended with a much larger view on screening . specific individuals being invited to undergo a medical test included in the definition of a genetic carrier screening programme , but also a wider population is included by highlighting the opportunity to be tested in brochures or in the press , via advertising or promotion by commercial providers. screening can take the form of large - scale programmes for which all pregnant women , all newborn babies or all men or women in a particular age group are eligible . but it can also entail people responding to an advertisement or a website offer for a health check with a clinic or a health organization ( health council of the netherlands , 2008 ) . the large number of monogenic disorders included in the dtc genetic testing companies services is in clear contrast with the selected number of disorders that is usually considered for inclusion in preconception carrier screening programmes in the traditional health care system . on the basis of generally accepted genetic screening guidelines ( health council of the netherlands , 2008 ; european society of human genetics , 2003 ; uk national screening committee , 2003 ; nuffield council on bioethics , 2006 ) , carrier testing for a larger number of disorders challenges at least two established specifications that have been followed in this field until now : ( i ) the condition should be an important health problem , and ( ii ) there should be a suitable test with known predictive value . moreover , testing should lead to a favourable ratio of advantages to disadvantages , in which the voluntary character of consent , the information provision , the embedding in medical supervision and the appropriate use of existing resources are guaranteed ( see below ) . first , an important criterion that should be fulfilled before a screening programme is initiated is that the condition should be an important health problem ( european society of human genetics , 2003 ; uk national screening committee , 2003 ) or a significant health problem ( health council of the netherlands , 2008 ) . significance does not necessarily relate to the number of people affected ; it also addresses the severity of a health problem even if a condition is rare ( european society of human genetics , 2003 ; health council of the netherlands , 2008 ) . the universal carrier test offered by counsyl , therefore , is whether dtc carrier testing should be seen as an activity for which these screening criteria are applicable . a disorder such as gaucher disease , for example , was already a matter of debate with regard to its integration in a panel for ashkenazi jews , since the most common gaucher disease mutation in this population leads to a highly variable but usually milder phenotype ( zimran et al . , 1997 ; zuckerman et al . , 2007 ) . this then raises the question of whether it is acceptable to systematically identify carrier couples for disorders that are less severe ( borry et al . , 2008 ) . severity judgments are complex and a particularly challenging base on which to make reproductive choices . providing severe and possibly less severe disorders within the same panel undermines the consistency of that panel . secondly , it has been emphasized that screening should be based on a simple , safe , precise and validated test ( uk national screening committee , 2003 ) with known predictive value ( european society of human genetics , 2003 ) . although the analytical validity of the test for each mutation included in panels offered by dtc genetic testing companies has likely been validated , the clinical validity ( i.e. how consistently and accurately the test detects or predicts the intermediate or final outcomes of interest ) of the panel of mutations may be far from 100% . moreover , the clinical utility of testing for some disorders is debatable as some homozygotes , because of low penetrance , may never develop overt disease and/or the expression may be variable ( levenson , 2010 ) . for example , this is the case for hereditary haemochromatosis , which is also included in the counsyl test panel . in addition , companies offering large panels of disorders may base their inclusion criteria on technical and economic aspects rather than on policy considerations , which take into account , among other things , carrier frequency , severity of the disorder and feasibility of testing in a particular population . it also raises doubts about the individual disorders included in these large testing panels , and questions whether more is really better ( leib et al . , 2005 ) . moreover , the question is raised of whether good information and informed decision - making is still possible when the test panel contains such a heterogeneous group of disorders , for which test sensitivity and specificity are variable . to ensure informed decision - making , it is crucial that individuals receive the necessary information about the purpose of the test , the reproductive choices resulting from the test , the reliability and limitations of the test , the possible psychological impact and the potential consequences of the test for the individual and his / her family members ( mcqueen , 2002 ) . privacy and confidentiality of the results , as well as possible consequences related to their disclosure to third parties , such as insurance companies and employers , should also be discussed . moreover , the nature of the reproductive options makes it imperative to adopt a non - directive approach with potential users . although some dtc genetic testing companies do include a lot of this information on their websites , the question is whether , while being commercially driven , the information presented is balanced enough to enable informed choice . moreover , according to a recent study ( lachance et al . , 2010 ) , many users would struggle to find and understand the important information on companies websites needed to make an informed decision . finally , although all companies selling dtc genetic tests require some form of consent from the consumer when ordering a test , the process of informed decision - making can not be reduced to clicking a box to accept the terms of service or signing a document . educating people about carrier testing 2009 ) , and the fact that carrier tests have a test sensitivity of < 100% ( causing a residual risk to people who are not found to be carriers ) make the goal of transmitting information about these tests a non - trivial matter . studies have shown that a significant proportion of participants who tested negative when undergoing preconceptional screening for cf or hbps wrongly believed that they were definitively not carriers , while some carriers falsely believed that they were only likely to be carriers ( axworthy et al . , 1996 ; honnor et al . , 2000 ; an offer of dtc testing is likely to face the same problems in educating people as has been demonstrated in these screening programmes . multiple studies have also demonstrated that both carriers and non - carriers may experience negative feelings , such as anxiety and worry , when participating in genetic screening , although anxiety levels often decrease after a few months ( bekker et al . , 1994 ; henneman et al . , the dtc offer of genetic tests is worrisome in the light of these findings , where the practices of existing dtc genetic testing companies mainly only offer written information found on their website . as dna direct puts it : pretest education is provided at no additional charge through in - depth , online materials for all tests ( www.dnadirect.com/web/consumers accessed 4/05/2010 ) . pre- or post - test counselling is often only possible at an additional cost ( $ 250/hour for dna direct ; $ 99/hour for pathway genomics ) , which may be a barrier for consumers to ask for it , although some insurance companies may reimburse it . in contrast , dnatraits offers a free optional consultation with their genetic counsellors before testing and a free mandatory consultation after to discuss the results ( http://www.dnatraits.com/services accessed 04/05/2010 ) . 23andme does not offer pre- or post - test counselling and urges consumers to seek the advice of health professionals if questions or concerns arise ( https://www.23andme.com/about/consent/?version=1.3 accessed 19/01/2011 ) . similarly , counsyl initially considered their test as an at - home carrier test , as a successor to the at - home pregnancy test and did not consider offering any pre- or post - test counselling ( http://precedings.nature.com/documents/4192/version/1 accessed 19/01/2011 ) . it has been suggested that general practitioners or midwives may be the most appropriate health care providers to offer carrier screening , since they can provide preconception care as part of ongoing primary care ( poppelaars et al . , 2004b ) . in addition , some carrier screening programmes are offered by gynaecologists or midwives in antenatal care . embedding in a healthcare setting can ensure adequate information provision to increase informed choice , a more optimal informed consent procedure , a medical follow - up if necessary and psychosocial counselling . an offer of carrier testing through the internet by commercial companies runs the risk of disconnecting the service completely from its usual embedding in a medically supervised context . when these companies started to offer carrier tests , they referred consumers to seek medical supervision in the established health care system at the consumer 's own discretion . pathway genomics suggested you should consult with a physician or other appropriate health care professional regarding the diagnosis , treatment and prevention of any disease or health condition ( http://www.pathway.com/more_info/terms_of_service accessed 04/05/2010 ) . at the start of offering their service , counsyl also sent out this message by underlying that people could order the test directly from their website to receive your kit immediately . everyone has a prescription : the acmg recommends that adults of reproductive age be offered carrier testing for cf and spinal muscular atrophy , two of the many conditions assayed by the universal genetic test . alternatively , you may get the test through your doctor ( https://www.counsyl.com/learn/easy/ accessed 04/05/2010 ) . counsyl recently changed its stance and since the beginning of may 2010 , testing can only be requested through a physician , who can be found , among others , via the list offered on counsyl 's website . the company also sends the results directly to the physician for interpretation , thereby , technically no longer selling ( but still advertising ) tests directly to consumers . in october 2010 , dna direct also changed its policy ; one can find on their website that they are no longer offering testing services directly to consumers ; but are now focusing their efforts on providing comprehensive yet easy - to - understand information and tools to consumers , physicians , hospitals , employers and health plans ( http://www.dnadirect.com/web/consumers accessed 04/11/2010 ) . pathway genomics has also , in the last few months , changed the way they offer tests ; consumers must now obtain a physician prescription and the company offers consumers the opportunity to send an email to the treating physician of the consumer directly from the company site in order to request the prescription . the offer through physicians may eliminate some of the concerns that were raised about information provision , but does not solve the issue about the appropriateness of the test provided . the absence of medical supervision for some dtc tests may compromise or fail to foster patient health especially in the case of carrier couples who may need intensive counselling regarding their reproductive choices , as well as concerning the risks for other family members . as preconceptional carrier screening programmes have an impact on healthcare budgets , careful consideration should be given to whether the expenses for such a programme are justified within the realm of the healthcare system ( modell and kuliev , 1991 ) . namely the option for and the cost of a specific screening programme might mean that other forms of screening can not be carried out . usually a balance should be made between the health gain obtained and the costs incurred . in the context of preconceptional carrier screening this has to be analysed carefully , as the primary goal of preconceptional carrier screening is enabling informed reproductive choice . furthermore , the identification of a high number of carriers or false positives may create a higher number of visits to medical doctors for more information , and thus leading to higher costs in follow - up activities . commercial companies underline that most users will pay for this test out of pocket and therefore , the health care system will not be burdened . since the need for follow - up information or tests is likely to happen in the traditional health care system , this is not completely true . it is foreseeable that companies testing for a whole list of disorders will create a higher number of patients visiting health services . for example , for the test panel offered by counsyl , the company foresees that 35% of the participating individuals would be carriers of at least one disease . furthermore , every mentioned company underlines the value of privacy and the possibility to perform the test outside the control of insurers or the healthcare system . they often play on people 's fear that if testing is conducted within the traditional health care system , third parties could access their genetic information and that this might lead to discrimination . as expressed by counsyl , no one will ever have access to your data without your express consent ( https://www.counsyl.com/about/privacy/ accessed 04/05/2010 ) . pathway genomics states that your dna and results belong to you and no one else . nobody should see your data or results unless you want them to ( http://www.pathway.com/more_info/dna_security accessed 04/05/2010 ) . in contrast , dna direct and counsyl want health insurers to be involved in their services . dna direct underlines that their services will be reimbursed by most insurers , although insurance companies may still ask for a letter of medical necessity which people with no a priori high risk may have . counsyl advances that for many people the test will be entirely free [ paid by the insurer ] ; for many others insurance will cover 70% or more of the cost ' . in contrast , dnatraits refuses any reimbursement : dnatraits does not accept reimbursement from insurance companies as this might limit our ability to ensure your privacy ( http://www.dnatraits.com/philosophy accessed 04/05/2010 ) . as it is to be expected that more companies will want their services to be reimbursed by insurance companies for their customers , it becomes crucial that carrier tests involve a required degree of usefulness , have a scientific basis and are voluntary in nature . the implementation of a preconceptional carrier test should result in the optimization of the advantages whilst reducing as much as possible the risks or adverse effects of the offer . therefore , it is crucial that the tests offered for important health problems are suitable and have a known predictive value . furthermore , it is important to ensure quality pre- and post - test information and counselling that can lead to informed decision - making for the participants . all potential negative psychosocial impacts resulting from participation in a carrier test should be avoided or minimized and the testing process should lead to a correct understanding of the test result . in our opinion , a commercial offer of dtc genetic carrier testing through the internet makes it difficult to meet all these criteria . the purpose of carrier screening is to enlarge people 's reproductive autonomy by enabling carrier couples to make informed reproductive decisions . in this paper , we have criticized dtc genetic testing companies that have included large numbers of disorders in the test panel , including less severe ones , as well as disorders for which only suboptimal tests are available according to the traditional screening criteria . it is , however , questionable whether commercial ( preconceptional ) carrier test providers can continue to exist using this business model as long as such genetic carrier tests are not integrated in larger , more comprehensive prenatal , or preferably in preconceptional , care settings ( which are , admittedly , not really established in general healthcare yet ) . the company university diagnostics ltd ( www.udlgenetics.com , only to be consulted on http://www.archive.org/ ) , for example , started to offer a service to discover cf carrier status , but has ceased to exist ( advisory committee on genetic testing , 1999 ) . furthermore , an analysis of the companies activities in this field shows that various genetic tests that were marketed online in recent years are no longer available for purchase . without the support of the healthcare system , it may be that only a relatively small percentage of the population will become aware of these services and will use them ( kolor et al . , 2009 ) . that being said , involving a physician in the dtc offer of genetic tests may not solve many of our concerns . if physicians are not well educated about which tests should be given based on specific criteria , they may simply become a pawn in the commercial companies attempt at increasing their market size . various recent events have created the expectation that regulatory oversight will increase in the near future ( borry et al . , 2010 ) . for example , the european society of human genetics recently endorsed a statement in which it recommended to ensure , among other issues , the quality of the testing services , the provision of pretest information and genetic counselling , a face - to - face consultation and oversight of this industry ( european society of human genetics , 2010 ) . as the government has a duty to protect citizens against the risks of unsound testing , the government can be expected to ensure that specific standards are being held and to promote the responsible provision and responsible use of specific genetic testing services to the public . if we agree that reproductive autonomy should be respected , carrier screening for various serious disorders should be available , but careful implementation is necessary in order to ensure optimization of the screening offer , counselling and follow - up . all other authors were involved in redrafting and revising of the paper and approved the final version of the manuscript for submission . is funded by the european commission fp7 marie curie initiative . l.h . and m.c.c . are financially supported by the centre for medical systems biology and the centre for society and genomics in the framework of the netherlands genomics initiative . funding to pay the open access publication charges for this article was provided by the section community genetics , clinical genetics & emgo institute for health and care research , vu university medical center , amsterdam .

recently , a number of commercial companies are offering preconceptional carrier tests directly - to - consumers . this offer raises a number of concerns and issues above and beyond those encountered with preconceptional tests offered within the traditional health care setting . in order to bring some of these issues to light and to initiate dialogue on this topic , this article discusses the following issues : the current offer of preconceptional carrier tests ( until the end of 2010 ) through online commercial companies ; the implications for the informed consent procedure and the need for good information ; the need for medical supervision and follow - up ; and the appropriate use of existing resources . the article concludes with some reflections about the potential sustainability of the offer of preconceptional carrier tests directly - to - consumers .

pubmed

substantial growth has occurred in the options for operative fracture treatment in the last few decades.1 this , in proportion to newer and better internal fixation devices or implants . specially designed intramedullary ( i m ) nails , and plates and screws for every bone and even parts of bones have helped increase treatment efficacy and union rates . titanium alloys and stainless steel are generally employed for the manufacture of orthopedic implants.1 after fracture union , the implant loses its purpose and continues to exist only as a foreign object inside the patient s body . removal of asymptomatic hardware is still debated , and there is a lack of clear guidelines.2 although there are several presumed benefits of implant removal , like functional improvement and pain relief , the surgical procedure can be challenging and may lead to complications such as neurovascular injury and refractures.2 there is wide country to country and surgeon to surgeon variations in the viewpoint on removal of hardware , with some advocating and many opposing routine removal.3 add to this the patients demands owing to their own perceptions and fears about the foreign device inside their body . in children , though , routine implant removal after fracture union is still standard procedure.4 implants may disturb function , and some theoretical long - term risks such as growth disturbance , foreign body reaction , chronic infection , and corrosion are used as arguments for removal . however , benefits should outweigh risks and removal should not require a more extensive operation than insertion.4 the consensus dilemmas over routine implant removal in adults notwithstanding , there are circumstances when removal of hardware does become necessary . these include pain and discomfort to the patient , infection and implant failure . in our center we designed this short study to investigate the commonest indications of implant removal , and to detect which implants were most likely to require removal . the study was conducted prospectively on patients admitted for removal of implants in the orthopedics department of a teaching hospital . adult patients aged 18 years or more who presented in the outpatient department ( opd ) with hardware related problems that necessitated removal was admitted . patients admitted over a period of 7-month starting february 2013 were included in the study . patients who had fixation devices intended to be removed after a definite interval to begin with , like percutaneous k - wires , external fixators and tarsal screws , were not included in the study . patients requiring removal of joint prostheses were also excluded from the study . at the time of admission , the potential risks of the operation and the possibility of non - favorable outcomes were explained to all patients . after admission , routine inpatient investigations were performed on all patients to evaluate their fitness for surgery . postoperatively , the patients were retained in the hospital for variable periods depending on the indication of removal and the condition of the wound . antibiotics were continued for longer duration in patients with infected hardware . at discharge , all the patients were strictly advised to protect the extremity for a variable length of time as demanded by the bone and the implant removed . they were followed in the opd for another 4 months and evaluated for symptom relief / persistence / new problems , and the data were collected . the data were analyzed by the authors and also by statistician using spss software and applying t - test , and results were compiled . eighty - seven patients fulfilled the inclusion criteria , but four did not return for follow - up . that left us with eighty - three patients on whom to conduct the study ( n = 83 ) . seventy - one patients were males ( 85.55% ) and 12 were females ( 14.45% ) . their ages ranged from 20 years to 78 years , and the mean age was 38 years . the reasons for removal of implants were found to lie in five categories : pain / discomfort / prominent hardware , infected hardware , implant failure , elective ( patient s insistence ) , and other reasons ( table 1 ) . distribution of cases thirty - three patients out of eighty - three had hardware pain or discomfort or prominence ( 39.75% ) . they ranged in age from 20 to 60 years ( mean age 31.6 years ) . the time since fracture fixation ranged from 4 months to 96 months ( average 38 months ) . the implants most commonly responsible in order of frequency were patella tension band wiring ( tbw ) ( n = 8) , olecranon tbw / plates ( n = 7 ) , distal humeral plates ( n = 6 ) , and femoral i m nails ( n = 4 ) . the mean duration of hospital stay in these patients was 5 days . at 4 months follow - up , 15 patients out of 34 reported complete relief of pain ( 44.11% ) . the average pain visual analog scale ( vas ) score decreased from 5.2 preoperatively to 2.1 postoperatively , which was statistically significant ( p < 0.05 ) . one had an ulnar nerve palsy postoperatively , which recovered ( chart 1 , figure 1a and b ) . ( b ) radiographs before and after removal of the implants twenty - four patients out of 83 ( 28.91% ) needed hardware removal because they had developed infection at the implant site a variable duration after osteosynthesis . their ages ranged from 26 years to 78 years ( mean 49.33 years ) , and the duration since first surgery varied from 2 months to 156 months ( mean 47.57 months ) . one ununited fracture was managed with external fixator ; the other was an infected olecranon which required repeat debridements followed by repeat osteosynthesis and flap coverage . in this group , the implants most commonly removed included distal tibial / ankle plates and screws ( n = 8) , proximal tibial plates ( n = 4 ) and olecranon plates ( n = 3 ) . after the removal , infection subsided in 21 patients out of 24 ( 87.5% ) . one of them had a refracture of the tibial shaft after sequestrectomy ( chart 2 ) ( figures 2 and 3 ) . distribution of infected hardware exposed and infected medial plates in the distal tibia in three patients infected and failed femoral locking nail eight ( 9.52% ) patients required implant removal and revision osteosynthesis for implant failure . their average age was 35 years ( 18 - 50 years ) , and the average time since the primary procedure was 7.6 months ( 2 - 12 months ) . these included 3 femoral i m nails , 2 distal tibial locked plates , 1 humeral shaft dynamic compression plate , and 2 patients with cannulated cancellous screws in the femoral neck ( chart 3 , figure 4 ) . distribution of failed hardware plate failure in the humeral shaft and proximal femur one patient during the routine course of his follow - up after plating of both forearm bones was found to have extensive bone resorption under the plates ( figure 5 ) . these plates were removed . on follow - up , there was no fracture or other complications . bone resorption beneath ulnar plate seventeen ( 20.48% ) patients had their implants removed on demand , despite being asymptomatic . during the course of their follow - up , three of these had persistent pain at the operated site . two developed superficial wound infections which prolonged their hospital stay but responded to intravenous antibiotics and wound lavage . distribution of implants removed on patient s wish the most consistently encountered obstacle during surgery was difficulty in removing the hardware from the bone . this was seen especially in locked plates of the distal humerus and forearm , with ingrowth of bone around the plate / screws . loss of contour ( rounding ) of the screw head slot was also commonly encountered preventing the engagement of the driver in the screw head . screw heads had to be cutoff to remove the plate in two patients because of this complication , and the shank left in the bone . in one patient who had presented for elective removal of an interlocked tibial nail , we failed to extract the nail despite best efforts . in another patient with a painful femoral nail , fortunately , we did not encounter any major vascular injury or iatrogenic fracture during the removal of any implant . one patient had an ulnar nerve neuropraxia after removal of distal humeral plates , which recovered . sequestrectomy was done , and the patient presented with a refracture in follow - up . these last two , and persistent pain in three previously asymptomatic patients were the only true complications in our series . femoral interlocking nail broken during removal overall , the most frequently removed implants in our series were distal tibial / ankle plates ( 14.45% of implants removed ) , femoral i m nails ( 13.25% ) , olecranon wires and plates ( 12.04% ) , and tibial i m nails and patellar tbws ( 9.53% each ) . pertinent to mention though that 6 of the 8 tibial nail removals were elective ( asymptomatic patients ) ( chart 5 ) . overall distribution of removed implants all implants except one removed in our series were made of stainless steel , and all were made by indian manufacturers . the issue of removing metallic implants used in fracture fixation has been oft discussed , and at length . popular opinion probably still is that implant removal should not be considered a routine procedure.2,3,5 although the ao - association for the study of internal fixation has published recommendations on the timing of hardware removal in recent fractures with uncomplicated healing ( table 2 ) , the clinical indications for implant removal are not well established , and few definitive data exist to guide whether routine implant removal is appropriate . furthermore , the surgical procedures for implant removal are fraught with risks of fracture , neurovascular injury , and infection . various arguments have been made from time to time to justify removal of hardware after fracture union , e.g. , metal allergy , corrosion , carcinogenesis and metal ion toxicity , but for none has concrete proof been produced.2 all the same , a lot of metal is removed from human bones every day . and that depicts that hardware does need to be removed in a significant subset of patients . large studies on implant removal in symptomatic patients are lacking , though many patients get their implants removed for one reason or another . our endeavor was to document the common indications for removal of internal fixation devices and highlight potential issues , even as most specialists are already well versed with them . timing of implant removal in uncomplicated fracture healing ; ao guidelines we found that majority of patients that needed removal of implant were men ( 85.5% ) . shrestha et al . in their retrospective series also found a male preponderance ( 189 out of 275 patients ) to the tune of 68.72%.6 their study , however , also included children . 30 of these ( 75% ) were males.7 there definitely seems to be a strong male preponderance in implant removal surgeries . the mean age of patients requiring removal for infection was higher ( 49.33 years ) than those with pain ( 31.6 years ) or implant failure ( 35 years ) . in our study , implant associated pain or discomfort was the most common reason necessitating removal ( 39.75% ) . brown et al . found that 31% patient sunder going open reduction and internal fixation of ankle fractures had persistent lateral pain.8 they also found that only 11 of 22 patients who got their hardware removed had improvement in the pain . prospectively studied 60 patients who had implant removal for hardware pain , and at 1 year follow - up all their patients were satisfied.9 although we did not primarily aim to evaluate the outcome after removal , all our patients had at least some relief in their hardware pain at 4 months follow - up . the next most common indication in our series was implant infection ( about 29% ) . some of these patients had been treated non - operatively for varying lengths of time to control the infection but without success . others presented for the first time at such a stage , e.g. , skin necrosis , that removing the implant was the only logical option . trampuz and widmer estimated that overall about 5% of all internal fixation devices become infected.10 they also impressed the role of biofilms in the resistance of pathogens to systemically administered antibiotics . only one was a delayed infection ( 2 - 10 weeks ) , a woman with an olecranon plate who developed skin necrosis and the plate had to be removed 2 months after surgery . all the others were late infections , caused by constant hematogenous seeding of the implant from skin , respiratory , dental and urogenital infections . the incidence of infections is likely to rise as more operations are performed by the day , and longevity increases translating into greater periods of possible bacterial implant seeding in the body . trampuz and widmer recommended stoppage of any antibiotics 2 weeks before the removal surgery , if possible , to get an accurate intraoperative tissue culture . they also suggested that the removed implant be sonicated in saline to dislodge microorganisms from its surface and the resultant sonicated fluid be sent for microbiologic examination . kukla et al . in a study of implants removed from the proximal femur ( dynamic hip screw and gamma nails ) found that the most common indications were avascular necrosis of the femoral head , deep chronic infections , shaft fractures , and screw cut - out.11 implant failure was the next most common indication in our series in symptomatic patients but it was less common than those in whom the hardware was removed at the patient s behest . reasons for failure of the implant include faulty implant ( poor quality ) , faulty operative technique , inadequate implant , delayed healing , and patient noncompliance . akhtar et al . cited the most common cause of failure as poor quality of the implant.12 peivandi et al . they recommended that credible and trusted implant brands should be used in fracture fixation.13 sharma et al . in a restrospective study of 41 failed upper and lower limb implants found that plate failure was more common than nail failure in the lower limb.14 in most cases in their series , the patient had a significant re - trauma causing failure of the implant . several studies have been carried out on the indications of removal of metalwork in asymptomatic patients . although most authors agree that routine removal should not be practiced , they also agree that there is a need for the development of concrete indication guidelines for implant removal . at the same time , there is a paucity of literature assessing the relative frequency of the usual indications of implant removal , viz . we believe that routine removal in asymptomatic patients should not be practiced , and if at all necessary , the removal should not require a larger procedure than the index operation . we also agree that implant removal surgeries are fraught with risks , including fractures , bleeding , nerve injuries and infection , and should be done only after explaining to the patient the possibility of all these complications . in addition to the possible new problems , the removal surgery may not entirely fulfill the intended purpose , e.g. , the pain may not completely go away , infection may not resolve , and additional surgeries may be required . all these factors must be borne in mind before embarking on such a process with high hopes of success . our study is limited by a small sample size and a short follow - up period . this may falsely favor titanium implants , although the probable reason for this is the low affordability of patients catered to by our center . more studies with greater number of cases and wider study dimensions are needed to produce concrete literature on the patterns of removal surgeries in symptomatic implants . we found that pain and implant prominence ( mechanical symptoms ) are the most common indications . other indications are implant failure , bone resorption due to excessive stress shielding and patient s will . the average age of patients requiring removal for infection is higher than that for other reasons . carefully done , the removal should be a safe procedure , and there is a low but definite possibility of complications . several factors like bone ingrowth and wear of the implant may make its removal difficult .

introduction : fracture fixation has become advanced with the advent of new and custom metal implants for each type of bone / fracture . after union though , the implant ceases to be important and may be removed . routine removal is advocated by some and opposed by others . nevertheless , some patients require removal of the hardware because of various implant - related problems . our study was aimed at identifying the most common causes for implant removal.objective:to investigate the common indications of orthopedic implant removal surgeries.methods:adult patients admitted for implant removal in our department were included in the study . they were operated in the next ot list . they were then followed for an average 4 months for resolutions of symptoms or appearance of new problems.results:a total of 83 patients were studied . 71 of them were males . the mean age was 38 years . the reasons for removal of implants were found to lie in five categories : pain / discomfort / prominent hardware , infected hardware , implant failure , elective ( patient s insistence ) , and other reasons . overall , the most frequently removed implants in our series were distal tibial / ankle plates ( 14.45% of implants removed ) , femoral intramedullary ( i m ) nails ( 13.25% ) , olecranon wires and plates ( 12.04% ) , and tibial i m nails and patellar tension band wirings ( 9.53% each).discussion and conclusion : the clinical indications of implant removal are not well established , and few definitive data exist to guide whether routine implant removal is appropriate . symptomatic hardware frequently needs removal . we found that pain and implant prominence ( mechanical symptoms ) are the most common indications . infection is the next most common , followed by hardware failure . other indications are implant failure , bone resorption due to excessive stress shielding and patient s will . removal is , however , not an easy surgery , and several factors such as bone ingrowth and wear of the implant may make its removal difficult .

pubmed

glaucoma , a neurodegenerative disease , is characterized by gradual loss of retinal ganglion cells ( rgcs ) and optic nerve atrophy.1 multiple factors are related to glaucoma , such as high intraocular pressure ( iop)2 and low cerebral spinal fluid pressure.3 mller cells are the major type of glial cells in the mammalian retina that can support and nourish retinal neurons , maintain extracellular ion homeostasis , glutamate recycling , and interaction in synaptic transmission.4,5 therefore , mller cells are involved in retinal function . investigations indicate that mller cells not only play an important physiological role , but they are also involved in multiple pathological retinal diseases such as glaucoma.68 it has been reported that reactive mller cells can aggravate retinal damage by releasing cytokines such as tumor necrosis factor- ( tnf-).9 growing evidence supports that increased glial production of tnf- contributes to the neurodegeneration in glaucoma.10 tnf- is a secreted inflammatory cytokine that is responsible for apoptosis , necrosis , and inflammation.11,12 tnf- is increased in the retina following ischemia or damage and other neurodegenerative disorders.1317 -aminoadipic acid ( aaa ) , a six - carbon homolog of glutamate , is a well - known compound that induces specific glial toxicity through blocking the glutamate uptake.1820 the use of aaa to interfere with glial influence on neuronal tissues is reported.21,22 however , the effects of aaa inhibition on mller cell gliosis in glaucoma are unknown . the current study aimed to determine if aaa treatment inhibits mller cell gliosis and protects against retinal abnormalities induced in a rat model of acute ocular hypertension ( aoh ) mimicking glaucoma adult male sprague dawley rats ( weight range , 200250 g , 810 weeks ) were used in this study in accordance with the association for research in vision and ophthalmology statement for the use of animals in ophthalmic and vision research and followed the people s republic of china animal care guidelines . this study was monitored and approved by the animal care committee of the capital medical university , scxk ( jing ) 2012 - 0001 . the animals were housed with a 12-hour light/12-hour dark cycle with standard chow and water . all surgical procedures were performed under anesthesia with intraperitoneal chloral hydrate ( 450 mg / kg ) and topical 0.5% proparacaine hydrochloride eye drops ( alcon , inc . , the iop was increased to 110 mmhg ( 14.63 kpa ) for 60 minutes by using an elevated 500 ml plastic container of sterile physiological saline connected to a 27-gauge needle placed in the anterior chamber of the eye . sham procedure eyes were treated similarly but without the elevation of the bottle ; hence , the normal iop was maintained . time - course examination was performed at 1 day , 3 days , and 5 days after acute iop elevation . animals were divided into sham operation ( ctrl ) group , aoh group , aoh + phosphate - buffered saline ( pbs ) control group , and aoh + aaa - treated group . aaa ( 250 g ) was injected into vitreous humor in 5 l at 50 g / l concentration after 3 hours of acute iop elevation according to the literature.23,24 tissues were fixed with 4% paraformaldehyde . eyeballs were cryoprotected in 30% sucrose overnight at 4c and then embedded in optimal cutting temperature compound ( sakura finetechinical co. ltd . , sections ( 14 m ) were incubated in 3% bovine serum albumin and 0.3% triton x-100 ( sigma - aldrich co. , st louis , mo , usa ) to block nonspecific binding and then incubated with primary mouse monoclonal antibodies against thy-1 ( 1:200 , rgc marker ; abcam , cambridge , uk ) , primary mouse monoclonal antibodies against glutamine synthetase ( gs ; 1:100 , mller glial cell maker ; abcam ) , rabbit polyclonal antibodies against glial fibrillary acidic protein ( gfap ; 1:200 , gliosis maker ; cell signaling technology inc . , danvers , ma , usa ) overnight at 4c , followed by incubation with secondary antibodies for 1 hour at room temperature . sections were mounted with fluoromount - g ( southernbiotech , birmingham , al , usa ) . slides were visualized using a confocal microscope ( leica microsystems , wetzlar , germany ) . the intensity of immunoreactivity from photographs was analyzed using image - proplus 6.0 ( media cybernetics inc . , silver spring , md , usa ) optical densities obtained from immunohistochemistry images were corrected by subtracting the average value of background noise from five image inputs . the terminal deoxynucleotidyl transferase - mediated uridine 5-triphosphate - biotin nick end labeling ( tunel ) assay was conducted according to the manufacturer s protocol . briefly , tissue sections were fixed in paraformaldehyde and were subsequently incubated in a tunel reaction medium . the sections were then mounted on microscope slides and examined under ultraviolet light using an epifluorescence microscope . retinas from sham operation group ( ctrl ) , aoh group , aoh + pbs control group , and aoh + aaa group were homogenized in lysis buffer and sonicated to dissolve the tissue completely . in all , 40 g of total proteins from each sample were loaded per lane for sodium dodecyl sulfate polyacrylamide gel electrophoresis . the proteins were transferred onto the nitrocellulose membrane ( schleicher & schuell , dassel , germany ) and the blocked nitrocellulose membrane was incubated with a primary rabbit polyclonal antibody against tnf- ( 1:1,000 ; abcam ) for 3 hours ( room temperature ) . after washing in tris buffered saline , with tween-20 ( tbst ) , the membranes were incubated with horseradish peroxidase - conjugated goat anti - rabbit or mouse igg prior to detection of the labeled proteins by using ecl - plus kit ( perkinelmer inc . , waltham , ma , usa ) followed by exposure of blots to x - omat ( kodak , rochester , ny , usa ) imaging film . the images were quantified using imagej software ( national institutes of health , maryland , usa ) . the gel doc-2000 imaging system ( bio - rad laboratories inc . , hercules , ca , usa ) was used to perform the quantitative analysis of western blot results . the -actin band density was expressed as 100% , and the other group was expressed as percentage of that from the control group . analysis of variance was used to determine the difference between tnf- and fluorescence intensity among three or more independent ( unrelated ) groups , followed by multiple comparisons using the student newman wallis test was used to determine the difference between tunel - positive cells and thy-1-positive rgcs among three or more independent ( unrelated ) groups , followed by multiple comparisons using the nemenyi test . statistical analysis was performed using sas 9.2 ( sas institute inc . , cary , nc , usa ) . adult male sprague dawley rats ( weight range , 200250 g , 810 weeks ) were used in this study in accordance with the association for research in vision and ophthalmology statement for the use of animals in ophthalmic and vision research and followed the people s republic of china animal care guidelines . this study was monitored and approved by the animal care committee of the capital medical university , scxk ( jing ) 2012 - 0001 . the animals were housed with a 12-hour light/12-hour dark cycle with standard chow and water . all surgical procedures were performed under anesthesia with intraperitoneal chloral hydrate ( 450 mg / kg ) and topical 0.5% proparacaine hydrochloride eye drops ( alcon , inc . , hnenberg , switzerland ) . the iop was increased to 110 mmhg ( 14.63 kpa ) for 60 minutes by using an elevated 500 ml plastic container of sterile physiological saline connected to a 27-gauge needle placed in the anterior chamber of the eye . sham procedure eyes were treated similarly but without the elevation of the bottle ; hence , the normal iop was maintained . time - course examination was performed at 1 day , 3 days , and 5 days after acute iop elevation . animals were divided into sham operation ( ctrl ) group , aoh group , aoh + phosphate - buffered saline ( pbs ) control group , and aoh + aaa - treated group . aaa ( 250 g ) was injected into vitreous humor in 5 l at 50 g / l concentration after 3 hours of acute iop elevation according to the literature.23,24 eyeballs were cryoprotected in 30% sucrose overnight at 4c and then embedded in optimal cutting temperature compound ( sakura finetechinical co. ltd . , tokyo , japan ) . sections ( 14 m ) were incubated in 3% bovine serum albumin and 0.3% triton x-100 ( sigma - aldrich co. , st louis , mo , usa ) to block nonspecific binding and then incubated with primary mouse monoclonal antibodies against thy-1 ( 1:200 , rgc marker ; abcam , cambridge , uk ) , primary mouse monoclonal antibodies against glutamine synthetase ( gs ; 1:100 , mller glial cell maker ; abcam ) , rabbit polyclonal antibodies against glial fibrillary acidic protein ( gfap ; 1:200 , gliosis maker ; cell signaling technology inc . , danvers , ma , usa ) overnight at 4c , followed by incubation with secondary antibodies for 1 hour at room temperature . sections were mounted with fluoromount - g ( southernbiotech , birmingham , al , usa ) . slides were visualized using a confocal microscope ( leica microsystems , wetzlar , germany ) . the intensity of immunoreactivity from photographs was analyzed using image - proplus 6.0 ( media cybernetics inc . , silver spring , md , usa ) . optical densities obtained from immunohistochemistry images were corrected by subtracting the average value of background noise from five image inputs . the terminal deoxynucleotidyl transferase - mediated uridine 5-triphosphate - biotin nick end labeling ( tunel ) assay was conducted according to the manufacturer s protocol . briefly , tissue sections were fixed in paraformaldehyde and were subsequently incubated in a tunel reaction medium . the sections were then mounted on microscope slides and examined under ultraviolet light using an epifluorescence microscope . retinas from sham operation group ( ctrl ) , aoh group , aoh + pbs control group , and aoh + aaa group were homogenized in lysis buffer and sonicated to dissolve the tissue completely . in all , 40 g of total proteins from each sample were loaded per lane for sodium dodecyl sulfate polyacrylamide gel electrophoresis . the proteins were transferred onto the nitrocellulose membrane ( schleicher & schuell , dassel , germany ) and the blocked nitrocellulose membrane was incubated with a primary rabbit polyclonal antibody against tnf- ( 1:1,000 ; abcam ) for 3 hours ( room temperature ) . after washing in tris buffered saline , with tween-20 ( tbst ) , the membranes were incubated with horseradish peroxidase - conjugated goat anti - rabbit or mouse igg prior to detection of the labeled proteins by using ecl - plus kit ( perkinelmer inc . , waltham , ma , usa ) followed by exposure of blots to x - omat ( kodak , rochester , ny , usa ) imaging film . the images were quantified using imagej software ( national institutes of health , maryland , usa ) . the gel doc-2000 imaging system ( bio - rad laboratories inc . , hercules , ca , usa ) was used to perform the quantitative analysis of western blot results . the -actin band density was expressed as 100% , and the other group was expressed as percentage of that from the control group . analysis of variance was used to determine the difference between tnf- and fluorescence intensity among three or more independent ( unrelated ) groups , followed by multiple comparisons using the student newman wallis test was used to determine the difference between tunel - positive cells and thy-1-positive rgcs among three or more independent ( unrelated ) groups , followed by multiple comparisons using the nemenyi test . statistical analysis was performed using sas 9.2 ( sas institute inc . , cary , nc , usa ) . values were reported as mean sd . a value of p<0.05 was considered as statistically significant . to determine the effects of aaa on rgcs in the aoh model , aoh was induced in sd rats and rats were intravitreally treated with pbs , aaa , aoh + pbs , and aoh + aaa . significantly reduced retinal thickness in the inner plexiform layer ( ipl ) and inner nuclear layer was observed after aoh induction versus control ( figure s1 ) . the mean values of tunel - positive cells per visual field were 0.10.3 in control ( figure 1a ) , 0.20.4 in aaa(figure 1b ) , 4.60.8 in aoh + pbs , and 1.70.7 in aoh + aaa . aoh induced the apoptotic death of cells in the ganglion cell layer(gcl ; figure 1c ) . in the aoh model , intravitreal treatment with aaa attenuated the tunel - positive rgcs more significantly than the intravitreal treatment with pbs ( p<0.05 ; figure 1d ) , indicating that aaa treatment rescued rgcs from the cell apoptosis induced in the aoh model . in addition , retinal sections were immunostained with antibody against thy-1 , a marker for rgcs.25 the mean values of thy-1-positive rgcs per visual field in ctrl , aaa , aoh + pbs , and aoh + aaa groups were 8.20.9 , 8.10.8 , 3.90.7 , and 60.6 , respectively . aaa treatment did not change the number of rgcs in normal conditions ( figure 2a and b ) . when compared with the ctrl group ( figure 2a ) , the number of thy-1-positive rgcs was decreased in the aoh + pbs group ( figure 2c ) , and aaa treatment significantly rescued thy-1-positive rgcs in aoh ( p<0.05 ; figure 2d ) , corresponding to aaa - attenuated apoptosis of rgcs in aoh ( aoh + pbs versus aoh + aaa , p<0.05 ; figure 1 ) . in the rat model of aoh , aaa rescued rgcs . to determine if aaa protects against aoh - induced rgc death through modulating mller cell gliosis , immonohistochemistry for gfap ( a marker for activated glial cells ) and gs ( mller cell - specific marker ) was conducted.26 mller cells cover the whole retina from the nerve fiber layer to the photoreceptor layer,27 and gfap was mainly expressed along nerve fiber layer and gcl and was less or absent at other layers in the control group ( figure s2 ) . in the rat model of aoh , colocalization of gfap and gs was observed across ipl ( figure s2 ) , indicating mller cell activation . after 1 day , 3 days , and 5 days of aoh induction in rat , gfap expression was also observed across ipl ( figure s3 ) , indicating mller cell gliosis . aaa treatment versus pbs control markedly reduced gfap immunoreactivity across ipl ( figure 3 ) , indicating attenuation in mller cell gliosis . as mentioned earlier , aaa rescued rgcs likely through the inhibition of mller cells gliosis in the rat model with aoh . activation of mller cells may induce tnf- production , leading to neurodegeneration in glaucoma.10 to determine if aaa modulates tnf- production , western blot was performed for the investigation of retinal protein levels of tnf-. significantly upregulated protein levels of retinal tnf- were observed in aoh versus sham operation ( ctrl ) group ( p<0.05 ; figure 4 ) . importantly , aaa treatment versus pbs control significantly attenuated tnf- levels in the retinas of the rat model of aoh ( p<0.05 ; figure 4 ) . to determine the effects of aaa on rgcs in the aoh model , aoh was induced in sd rats and rats were intravitreally treated with pbs , aaa , aoh + pbs , and aoh + aaa . significantly reduced retinal thickness in the inner plexiform layer ( ipl ) and inner nuclear layer was observed after aoh induction versus control ( figure s1 ) . the mean values of tunel - positive cells per visual field were 0.10.3 in control ( figure 1a ) , 0.20.4 in aaa(figure 1b ) , 4.60.8 in aoh + pbs , and 1.70.7 in aoh + aaa . aoh induced the apoptotic death of cells in the ganglion cell layer(gcl ; figure 1c ) . in the aoh model , intravitreal treatment with aaa attenuated the tunel - positive rgcs more significantly than the intravitreal treatment with pbs ( p<0.05 ; figure 1d ) , indicating that aaa treatment rescued rgcs from the cell apoptosis induced in the aoh model . in addition , retinal sections were immunostained with antibody against thy-1 , a marker for rgcs.25 the mean values of thy-1-positive rgcs per visual field in ctrl , aaa , aoh + pbs , and aoh + aaa groups were 8.20.9 , 8.10.8 , 3.90.7 , and 60.6 , respectively . aaa treatment did not change the number of rgcs in normal conditions ( figure 2a and b ) . when compared with the ctrl group ( figure 2a ) , the number of thy-1-positive rgcs was decreased in the aoh + pbs group ( figure 2c ) , and aaa treatment significantly rescued thy-1-positive rgcs in aoh ( p<0.05 ; figure 2d ) , corresponding to aaa - attenuated apoptosis of rgcs in aoh ( aoh + pbs versus aoh + aaa , p<0.05 ; figure 1 ) . in the rat model of aoh , aaa rescued rgcs . to determine if aaa protects against aoh - induced rgc death through modulating mller cell gliosis , immonohistochemistry for gfap ( a marker for activated glial cells ) and gs ( mller cell - specific marker ) was conducted.26 mller cells cover the whole retina from the nerve fiber layer to the photoreceptor layer,27 and astrocytes mainly locate along gcl . gfap was mainly expressed along nerve fiber layer and gcl and was less or absent at other layers in the control group ( figure s2 ) . in the rat model of aoh , colocalization of gfap and gs was observed across ipl ( figure s2 ) , indicating mller cell activation . after 1 day , 3 days , and 5 days of aoh induction in rat , gfap expression was also observed across ipl ( figure s3 ) , indicating mller cell gliosis . aaa treatment versus pbs control markedly reduced gfap immunoreactivity across ipl ( figure 3 ) , indicating attenuation in mller cell gliosis . as mentioned earlier , aaa rescued rgcs likely through the inhibition of mller cells gliosis in the rat model with aoh . activation of mller cells may induce tnf- production , leading to neurodegeneration in glaucoma.10 to determine if aaa modulates tnf- production , western blot was performed for the investigation of retinal protein levels of tnf-. significantly upregulated protein levels of retinal tnf- were observed in aoh versus sham operation ( ctrl ) group ( p<0.05 ; figure 4 ) . importantly , aaa treatment versus pbs control significantly attenuated tnf- levels in the retinas of the rat model of aoh ( p<0.05 ; figure 4 ) . glaucoma is a leading cause of irreversible vision loss characterized by progressive death of rgcs , and elevated iop is a major risk factor.28 a rodent model of aoh is well established for acute angle closure glaucoma , and it has been widely used to investigate the pathogenesis of death of rgcs.29 in the current rat model of aoh , we found induced cell apoptosis and decreased number of rgcs with iop induction versus sham operation control . importantly , treatment of aaa versus pbs control significantly attenuated rgc apoptosis and rescued the reduced number of rgcs , demonstrating protective effects on aoh retinas ( figure 5 ) . activated mller cells contribute to the progression of glaucoma.30 mller cells perform a multitude of important regulatory and supportive roles , including secretion of trophic factors , removal of metabolic waste , and neurotransmitter recycling.31,32 we found that mller cells were activated with induced gfap immunoreactivity in the rat model of aoh versus control retinas . gfap , an intermediate filament protein , is considered as a marker of reactive mller cell gliosis,27 which is not or less expressed in mller cells in normal retinas and expressed highly at ischemic,33 light - induced retinal degeneration,34 and retinal detachment.35 activation of mller cells so far was demonstrated to have both protective and detrimental effects.3638 especially early after injury , mller cell gliosis is believed to be neuroprotective and promotes the repair of neurons in response to injury,36 with gfap upregulation to provide additional structural integrity to the retina at the site of injury.39 however , activated mller cells also express tnf-,40 monocyte chemoattractant protein,41 and nitric oxide42 to induce death of rgcs . tnf- induction contributes to inflammation , apoptosis , and necrosis , leading to cell death . we observed that aaa treatment attenuated gfap immunoreactivity in mller cell processes across ipl and reduced retinal tnf- levels in the rat model of aoh . these findings suggested that aaa promoted survival of rgcs in the rat model of aoh , likely through the inhibition of aoh - induced mller cell gliosis and in turn downregulation of tnf- protein production . we found that aaa could effectively protect retina against loss of rgcs and apoptosis in aoh retinas through attenuating mller cell gliosis and downregulating tnf- production . these observations suggested that aaa might be a potential therapeutic target in the treatment of neurodegeneration in glaucoma . ( a ) reprehensive results of hoechst staining in rat retina after 1 d , 3 d , and 5 d of aoh ; ( b ) quantitative analysis of the retinal thickness . notes : the results showed that the thickness of ipl and inl layers decreased significantly following the reperfusion time after aoh treatment ( scale bar = 50 m ) . * p<0.05 compared with the control group ( n=4 per group ) . abbreviations : aoh , acute ocular hypertension ; ipl , inner plexiform layer ; inl , inner nuclear layer ; ctrl , control ; d , day ; gcl , ganglion cell layer ; onl , outer nuclear layer . note : strong expression of gfap was shown in the gs - positive mller cell after 5 d of aoh compared with the control group ( scale bar = 50 m ) . abbreviations : gs , glutamine synthetase ; gfap , glial fibrillary acidic protein ; aoh , acute ocular hypertension ; ctrl , control ; gcl , ganglion cell layer ; ipl , inner plexiform layer ; inl , inner nuclear layer ; onl , outer nuclear layer ; d , days . increased gfap immunoreactivity after aoh induction in rat retinas . notes : ( a ) immunofluorescent staining showed that aoh increased gfap immunoreactivity in the mller cell processes across ipl . abbreviations : gfap , glial fibrillary acidic protein ; aoh , acute ocular hypertension ; ipl , inner plexiform layer ; d , day ; pbs , phosphate - buffered saline ; aaa , -aminoadipic acid .

objectiveocular hypertension is an important risk factor for glaucoma . the purpose of this study was to investigate the gliotoxic effects of -aminoadipic acid ( aaa ) in a rat model of aoh and its underlying mechanisms.materials and methodsin the rat model of acute ocular hypertension ( aoh ) , intraocular pressure was increased to 110 mmhg for 60 minutes . animals were divided into four groups : sham operation ( ctrl ) , aoh , aoh + phosphate - buffered saline ( pbs ) , and aoh + aaa . cell apoptosis in the ganglion cell layer was detected with the terminal deoxynucleotidyl transferase - mediated uridine 5-triphosphate - biotin nick end labeling ( tunel ) assay , and retinal ganglion cells ( rgcs ) immunostained with thy-1 were counted . mller cell activation was detected using immunostaining with glutamine synthetase and glial fibrillary acidic protein . tumor necrosis factor- ( tnf- ) was examined using western blot.resultsin the rat model of aoh , cell apoptosis was induced in the ganglion cell layer and the number of rgcs was decreased . mller cell gliosis in the retinas of rats was induced , and retinal protein levels of tnf- were increased . intravitreal treatment of aaa versus pbs control attenuated these retinal abnormalities to show protective effects in the rat model of aoh.conclusionin the retinas of the rat model of aoh , aaa treatment attenuated retinal apoptosis in the ganglion cell layer and preserved the number of rgcs , likely through the attenuation of mller cell gliosis and suppression of tnf- induction . our observations suggest that aaa might be a potential therapeutic target in glaucoma .

pubmed

although endoscopic resection can be a curative procedure for such lesions 1 , it still remains technically challenging because of the high risk of severe adverse events ( saes ) including delayed perforation 2 . on the other hand , surgical treatment of such lesions often results in extensive resections , causing significant morbidity . recently , laparoscopic and/or endoscopic full - thickness resection ( ftr ) has been reported to be a useful technique for the treatment of nonampullary sdns 3 4 5 . despite their minimally invasive nature , all ftr procedures are associated with the possibility of intraperitoneal tumor dissemination . therefore , we developed an alternative ftr method , which we call laparoscopy - assisted endoscopic full - thickness resection with ligation device ( laeftr - l ) . herein we report on resection of nonampullary sdns using laeftr - l in 5 consecutive patients and an evaluation of the efficacy and safety of these procedures . from june 2015 to september 2015 , 5 patients underwent resection of nonampullary sdns using laeftr - l . written informed consent the set up for the laeftr - l procedure was previously described 3 . after mobilizing the duodenum from the retroperitoneum , a forward - viewing endoscope ( gif260j ; olympus , medical systems corp . , tokyo , japan ) was inserted into the duodenum and the target lesion was identified ( fig . endoscopic light was used to help the surgeon identify the tumor location . using an endoclip as a marker , the tumor ( including the seromuscular layer ) was ligated using a ligation device ( pneumo - activate evl device ; sumitomo bakelite corp . , the laparoscopist then temporarily sutured the seromuscular layer , after confirming the presence of a full - thickness aspirated sign ( fig . then , an endoscopist resected the targeted lesion under the ligation band using snare technique ( fig . 2b ) . finally , the specimen was retrieved intraluminally using the endoscope ( fig . despite the lack of a seromuscular layer , the provisional and additional sutures prevented exposure of the abdominal cavity to malignant cells . in addition , vessels were ligated during the closure of the defect in the duodenal wall . therefore , the laeftr - l procedure was completed in a closed manner without complications . endoscopic findings of laparoscopy - assisted endoscopic full - thickness resection with ligation device for superficial duodenal neoplasm . a a flat elevated lesion , measuring 15 mm in diameter , located at the opposite side of the ampulla of vater . b using an endoclip as a marker , the targeted lesion was ligated with a ligation device . e laparoscopic temporary suture secures patency of the duodenal wall . f resected lesion after administration of 0.1 % indigo carmine solution . laparoscopic findings of laparoscopy - assisted endoscopic full - thickness resection with ligation device for superficial duodenal neoplasm . a full - thickness aspirated sign , which helps the surgeon accurately identify the tumor location . inhibitors of gastric acid and protease secretion were administered to the patients during their hospitalizations . a normal diet was resumed approximately 2 days after the procedure , and the patients were discharged after a 1-week hospital stay . from june 2015 to september 2015 , 5 patients underwent resection of nonampullary sdns using laeftr - l . written informed consent the set up for the laeftr - l procedure was previously described 3 . after mobilizing the duodenum from the retroperitoneum , a forward - viewing endoscope ( gif260j ; olympus , medical systems corp . , tokyo , japan ) was inserted into the duodenum and the target lesion was identified ( fig . endoscopic light was used to help the surgeon identify the tumor location . using an endoclip as a marker , the tumor ( including the seromuscular layer ) was ligated using a ligation device ( pneumo - activate evl device ; sumitomo bakelite corp . , ltd . the laparoscopist then temporarily sutured the seromuscular layer , after confirming the presence of a full - thickness aspirated sign ( fig . then , an endoscopist resected the targeted lesion under the ligation band using snare technique ( fig . 1c , fig . 2b ) . finally , the specimen was retrieved intraluminally using the endoscope ( fig . despite the lack of a seromuscular layer , the provisional and additional sutures prevented exposure of the abdominal cavity to malignant cells . in addition , vessels were ligated during the closure of the defect in the duodenal wall . therefore , the laeftr - l procedure was completed in a closed manner without complications . endoscopic findings of laparoscopy - assisted endoscopic full - thickness resection with ligation device for superficial duodenal neoplasm . a a flat elevated lesion , measuring 15 mm in diameter , located at the opposite side of the ampulla of vater . b using an endoclip as a marker , the targeted lesion was ligated with a ligation device . e laparoscopic temporary suture secures patency of the duodenal wall . f resected lesion after administration of 0.1 % indigo carmine solution . laparoscopic findings of laparoscopy - assisted endoscopic full - thickness resection with ligation device for superficial duodenal neoplasm . a full - thickness aspirated sign , which helps the surgeon accurately identify the tumor location . inhibitors of gastric acid and protease secretion were administered to the patients during their hospitalizations . a normal diet was resumed approximately 2 days after the procedure , and the patients were discharged after a 1-week hospital stay . two of the 5 tumors were located at the duodenal bulb , and the others at the second part of the duodenum ( opposite to the ampulla of vater ) . the mean operative time was 173 minutes ( range 138 217 minutes ) . mean diameter of the resected specimen was 24 mm ( range 18 32 mm ) , and the mean tumor size was 9 mm ( range 7 11 mm ) . complete resection was achieved , and full - thickness resection was histologically confirmed in all cases ( fig . because the malignant potential of sdn is difficult to define from the preoperative endoscopic diagnosis 6 , complete en - bloc resection is necessary to achieve appropriate pathological evaluation and treatment . although conventional endoscopic mucosal resection ( emr ) could be a curative strategy for sdns , complete resection is challenging , especially for flat or non - lifting lesions . in recent years , the indications for esd however , delayed perforation in the duodenum , in particular , may cause potentially fatal peritonitis or retroperitoneal complications , requiring emergency open surgery to counter leakage of bile and pancreatic juices 8 . moreover the technical difficulty of this procedure is extremely high , due to the poor maneuverability of the endoscope and the thin wall . therefore , duodenal esd is not a standard clinical procedure . on the other hand , although short - term clinical outcomes were preferable , we need to consider the risk of intraperitoneal tumor dissemination , given the lack of data on long - term outcomes in patients undergoing conventional ftr procedures . compared with conventional ftr procedures , laeftr - l has two advantages . most importantly , the resected specimen can be collected intraluminally , avoiding contact with the peritoneum . shen et al . conducted an animal model study and revealed that pneumoperitoneum from laparoscopic surgery could be a risk factor for peritoneal dissemination of tumor cells 9 . because long - term outcomes of sdns treated with the conventional ftr method rarely have been investigated , peritoneal exposure of tumor cells should be avoided as far as possible . moreover , laeftr - l does not require proficient experience in esd . indeed , total operative time was not different from previous reports on conventional ftr for sdns 3 4 , and endoscopic resection time was even significantly shorter . tumor size is a major limiting factor when considering the indication for laeftr . in the current study , reported that esophageal subepithelial lesions no larger than 13 mm could be successfully resected by emr with a ligation device 10 . considering the size of the ligation device , lesions smaller than 15 mm in diameter may be candidates for this procedure . although additional studies are needed , application of a large oblique cap may enable en - bloc resection of larger lesions . in addition , tumor location is also important to decisions about the indication for laeftr - l . laeftr - l can not to be applied to lesions close to the ampulla of vater , because mobilization of these portions is technically difficult with laparoscopic procedures . in conclusion , laeftr - l may be a promising technique for the treatment of nonampullary sdns which can achieve complete resection without peritoneal exposure of tumor cells or saes . prospective studies are needed to further evaluate the efficacy and safety of laeftr - l procedures .

background and study aims : recently , laparoscopic and/or endoscopic full - thickness resection ( ftr ) has been reported to be a useful technique for the treatment of superficial duodenal neoplasms ( sdns ) . in the current study , we evaluated clinical outcomes in 5 consecutive patients who underwent resection of nonampullary sdns using laparoscopy - assisted endoscopic full - thickness resection with ligation device ( laeftr - l ) , which is an alternative ftr method developed to avoid peritoneal dissemination . using a snare technique with a ligation band , the duodenal lesions were easily resected . the provisional and additional sutures for the resected site prevented delayed perforation and bleeding and they also protected the abdominal cavity from direct exposure to malignant cells . complete resection could be achieved and ftr was histologically confirmed in all cases . the mean operation time was 173 minutes ( range 138 217 minutes ) . mean diameter of the resected specimen was 24 mm ( range 18 32 mm ) . no adverse events ( aes ) were observed . laeftr - l , which can achieve complete resection of nonampullary sdns without severe aes and peritoneal dissemination , could be a useful technique for the treatment of such lesions .

pubmed

congenital adrenal hyperplasia ( cah ) is caused by defects in various enzymes necessary for cortisol synthesis . 90 - 95% of cah cases are caused by 21-hydroxylase deficiency ( 21-ohd ) . in patients with 21-ohd , some of these precursors are shunted into the androgen synthetic pathway , causing signs and symptoms of androgen excess , including ambiguous genitalia in females and rapid somatic growth with accelerated skeletal maturation in both sexes1 ) . since the discovery of cortisone in the 1950s , cah has been managed effectively , and the clinical course of the disease has markedly improved2 ) . typical medical treatment consists of oral glucocorticoid and mineralocorticoid administration in order to compensate for adrenal steroid deficiencies and to suppress adrenal androgens . herein , we report four cases of patients who were diagnosed as having cah . they have been managed with hydrocortisone and fludrocortisone . thereafter , adrenal masses were found by abdominal computed tomography ( ct ) scan in all four cases . recent reports reveal a high apprearance of small tumors in patients with untreated cah and most adrenal masses in children with cah are benign3 - 5 ) . two of our four cases were managed with adrenalectomy because of increasing adrenal mass size and virilization . a male neonate who presented with severe vomiting at 15 days of life was brought to medical attention . however severe vomiting recurred after he was discharged and he was brought to kyunghee medical center . laboratory evaluation revealed a ph level of 7.36 , hco3 13 meq / l , base excess -10 meq / l , and na 109 meq / l . electrolytes were normalized and vomiting was resolved . thereafter the level of na was normalized . however at the age of 20 years , his height was 145 cm ( less than 3 percentile ) . abdominal ct performed at the age of 18 years ( july 30 , 2004 ) revealed the presence of multiple bilateral adrenal masses ( possibly adenoma ) . the size of his right adrenal mass was 2.41.3 cm and the size of left adrenal masses were 2.71.7 cm and 2.31.9 cm with adrenal hyperplasia . follow - up abdominal ct was performed at the age of 20 years ( fig . 1 ) . abdominal ct showed that both adrenal masses on both sides were increasing in size . the size of his right adrenal mass was 3.01.8 cm , and the size of his left adrenal masses were 3.32.4 cm and 3.02.6 cm . bilateral adrenalectomy was performed at the age of 20 years . a female neonate presented with ambiguous genitalia at birth suggesting salt - losing cah . when she was at the age of 3 years , vaginoplasty and clitoroplasty were performed . at the age of 11 abdominal ct performed at the age of 11 years showed a 3.54.0 cm mass in her right adrenal gland ( fig . hydrocortisone and fludrocortisone administration was carried out during follow - up and vaginoplasty and clitoroplasty were performed at the age of 4 years . when she was 11 years old and 12 years old , abdominal cts were performed due to her virilization including hirsuitism and masculine voice . a male neonate was admitted to medical attention at 1 month of age with poor oral intake and diarrhea . he was admitted to kyunghee medical center at 5 months of age with poor oral intake and vomiting . laboratory evaluation revealed a ph level of 7.35 , hco3 14 meq / l , base excess -9 meq / l , and na 119 meq / l . during follow - up , he was diagnosed with cah . after his medication had begun , the level of na was normalized . during follow - up , medication was stopped on his parents ' request and since the age of six , he showed secondary growth such as hyperpigmentation , hirsuitism , large penis , and pubic hair . abdominal ct performed at the age of 14 years revealed the presence of adrenal masses ( left adrenal gland : 2.01.4 cm & 1.21.3 cm , right adrenal gland : 1.11.2 cm ) . since there was no change in the size of adrenal mass , adrenalectomy was not performed . a male neonate who presented with severe vomiting at 15 days of life was brought to medical attention . however severe vomiting recurred after he was discharged and he was brought to kyunghee medical center . laboratory evaluation revealed a ph level of 7.36 , hco3 13 meq / l , base excess -10 meq / l , and na 109 meq / l . electrolytes were normalized and vomiting was resolved . thereafter the level of na was normalized . however at the age of 20 years , his height was 145 cm ( less than 3 percentile ) . abdominal ct performed at the age of 18 years ( july 30 , 2004 ) revealed the presence of multiple bilateral adrenal masses ( possibly adenoma ) . the size of his right adrenal mass was 2.41.3 cm and the size of left adrenal masses were 2.71.7 cm and 2.31.9 cm with adrenal hyperplasia . follow - up abdominal ct was performed at the age of 20 years ( fig . 1 ) . abdominal ct showed that both adrenal masses on both sides were increasing in size . the size of his right adrenal mass was 3.01.8 cm , and the size of his left adrenal masses were 3.32.4 cm and 3.02.6 cm . when she was at the age of 3 years , vaginoplasty and clitoroplasty were performed . at the age of 11 abdominal ct performed at the age of 11 years showed a 3.54.0 cm mass in her right adrenal gland ( fig . hydrocortisone and fludrocortisone administration was carried out during follow - up and vaginoplasty and clitoroplasty were performed at the age of 4 years . when she was 11 years old and 12 years old , abdominal cts were performed due to her virilization including hirsuitism and masculine voice . a male neonate was admitted to medical attention at 1 month of age with poor oral intake and diarrhea . he was admitted to kyunghee medical center at 5 months of age with poor oral intake and vomiting . laboratory evaluation revealed a ph level of 7.35 , hco3 14 meq / l , base excess -9 meq / l , and na 119 meq / l . during follow - up , he was diagnosed with cah . hydrocortisone and fludrocortisone medication was initiated . after his medication had begun , the level of na was normalized . during follow - up , medication was stopped on his parents ' request and since the age of six , he showed secondary growth such as hyperpigmentation , hirsuitism , large penis , and pubic hair . abdominal ct performed at the age of 14 years revealed the presence of adrenal masses ( left adrenal gland : 2.01.4 cm & 1.21.3 cm , right adrenal gland : 1.11.2 cm ) . since there was no change in the size of adrenal mass , adrenalectomy was not performed . cah describes a group of autosomal recessive condition in which deletions or mutations of the cytochrome p450 21-hydroxylase gene result in glucocorticoid and/or mineralocorticoid deficiency6 ) . in 95% of cases , epidemiology , genetics , pathophysiology , clinical features , and the management of cah will be reviewed as follows . the overall incidence of classical 21-ohd is 1:10,000 - 1:15,000 in most reported populations8 ) . the 21-hydroxylase gene cyp21a2 is located on chromosome 6p21.3 within the hla histocompatibility complex9 ) . two highly homologous 21-hydroxylase genes exist ; cyp21a2 and cyp21a1p , of which cyp21a2 codes for the active 21-hydroxlyase and cyp21a1p is an inactive pseudogene . more than 90% of mutations of the active cyp21a2 gene are generated by recombinations between the active and the inactive pseudogene10 ) . the pathophysiology of cah due to 21-ohd is closely linked to the degree of enzyme deficiency6 ) . the biosynthesis of cortisol is regulated by negative feedback on hypothalamic corticotropin - releasing hormone ( crh ) and pituitary adrenoconticotrophic hormone ( acth ) secretion . a defect in cortisol biosynthesis leads to a compensatory increase in hypothalamic and pituitary secretion of crh and acth . this stimulates the adrenal cortex and results in the pathological correlative of hyperplastic adrenal glands . the consequence of 21-ohd is a state of hypocortisolism , hypoaldosteronism , and hyperandrogenism combined with catecholamine deficiency and several metabolic disturbances . endogenous glucocorticoids are essential for the development and continuing regulation of the adrenal medulla , synthesizing epinephrine and norepinephrine . without cortisol , the organogenesis of the adrenal medulla is severely disturbed and the consequence is catecholamine deficiency , mainly comprising a lack of epinephrine11 ) . cah is classified as classic , the severe form , or non - classic , the mild or late - onset form . classic cah is subclassified as the salt - losing or non - salt - losing ( simple virilizing ) . non - classic cah is subclassified as the late - onset form or cryptic form . salt - losing cah is a severe form with a concurrent defect in aldosterone biosynthesis . non - classic cah may be asymptomatic or may be associated with signs of androgen excess developing during childhood or at puberty . the classic forms are characterized by a congenital decrease of cortisol and aldosterone secretion and an increase in androgen biosynthesis . all female infants with classic cah due to 21-ohd typically have ambiguous genitalia at birth because of exposure to high concentrations of androgens in the uterus . the internal genitalia are normal , including uterus , fallopian tubes , and ovaries ; wolffian duct structures are not present . boys with classic cah have no signs of cah at birth , except subtle hyperpigmentation and possible penile enlargement . thus , the age at diagnosis in boys varies according to the severity of aldosterone deficiency . up to 2/3 of patients suffering from classic 21-ohd have insufficient aldosterone biosynthesis leading to salt - losing episodes after birth and later on12 ) . boys with the salt - losing form typically present at 7 - 14 days of life with vomiting , weight loss , lethargy , dehydration , hyponatraemia , hyperkalaemia , and can present shock . patients with non - classic cah do not have cortisol deficiency , but instead have manifestations of hyperandrogenism , generally later in childhood or in early adulthood13 , 14 ) . manifestations include premature pubarche , tall stature , advanced bone age , menstrual irregularities , infertility , hirsutism and acne . cah due to 21-ohd can be diagnosed in newborn screening programs by screening for 17-hydroxyprogesterone in dried blood spots . today , most cases of classical 21-ohd are diagnosed with this tool . a high concentration of 17-hydroxyprogesterone in a randomly timed blood sample is diagnostic of classic 21-ohd . the gold standard for diagnosis of cah is a corticotropin stimulation test , with measurement of 17-hydroxyprogesterone at 60 min . a stimulated concentration of 17-hydroxyprogesterone higher than 45 nmol / l is diagnostic of 21-ohd . the aim of medical treatment in 21-ohd is to suppress adrenal androgens sufficiently without impairing growth , while allowing for normal pubertal development and fertility . in classic cah , glucocorticoids are given in doses sufficient to suppress excessive secretion of crh and acth without total suppression of the hypothalamic - pituitary - adrenal . physiological cortisol secretion rates are about 6 - 8 mg / m / day15 - 17 ) . to achieve these goals , the glucocorticoid doses have to exceed the physiological cortisol secretion rate , using doses of 12 - 18 mg / m fludrocortisone was administered in cases of salt - losing manifestation and elevated plasma renin activity , to decrease glucocorticoid doses , or according to the genotype19 ) . the use of fludrocortisone in patients with non - salt - losing classic cah allows management with lower doses of glucocorticoid and improves linear growth18 - 20 ) . infants with the salt - losing form of 21-ohd require mineralocorticoid ( fludrocortisone , usually 0.1 - 0.2 mg ) and sodium chloride supplements ( 1 - 2 g daily ; 1 g nacl contains 17 meq of sodium ) in addition to glucocorticoid treatment . treatment is indicated only for those with symptoms and aims to reduce hyperandrogenism18 - 20 ) . cah constitutes a continuum of disorders that affect patients throughout their lives ( table 2 ) . a variety of clinics involving close interaction between paediatric , reproductive , adult endocrinologists , and clinical psychologists experienced in psychosexual counselling will be necessary for patients with cah21 - 24 ) . the development of adrenal adenoma or carcinoma in patients with cah is rare ; the etiology is not clear . however , the incidence of adrenal masses appears to be higher in cah patients and in heterozygotes than in the general population3 ) . recent reports have revealed a high prevalence of small adrenal tumors in patients with untreated 21-ohd cah3 , 4 ) . histological types of adrenal tumor include adenoma , myelolipoma , and hemangioma25 ) . rarely , virilizing adrenal carcinoma has been found in patients with cah , but most adrenal masses in children with cah are benign5 ) . the pathogenesis of adrenal tumor in patients with 21-ohd cah is thought to be related to a consequence of acth hypersecretion , which results from the lack of glucocorticoid synthesis26 ) . avoidance of high steroid dose is likely to result in insufficient suppression of corticotropin stimulation27 ) . chronic poor compliance to therapy appears to be associated with the development of the tumor . therefore patients with 21-ohd cah should maintain steroid medication to avoid the appearance of adrenal tumor . follow - up imaging may help to discover the appearance of adrenal tumor in patients with long - term management of 21-ohd cah . there has been much improvement during the past 50 years in the understanding of cah , and the management with cah continues to improve . furthermore new medical strategies that offer the prospect of an improved outcome continue to evolve . however , patients with 21-ohd cah should maintain steroid medication to avoid the appearance of adrenal tumor , to improve its symptoms , and to normalize electrolyte disorder , particularly hyponatremia .

congenital adrenal hyperplasia ( cah ) caused by 21-hydroxylase deficiency is an autosomal recessive disease , which leads to cortisol and aldosterone deficiency and hyperandrogenism . typical medical treatment includes oral glucocorticoid and mineralocorticoid administration to suppress adrenal androgens and to compensate for adrenal steroid deficiencies . however , some patients stopped taking medicine without the doctor 's consent . among these patients , four cases of cah patients showing the presence of hyponatremia as an initial electrolyte disorder were found with adrenal adenoma . hypersecretion of adrenocorticotrophic hormone and chronic poor compliance to therapy appears to be associated with the development of the adrenal tumor . two cases were managed with adrenalectomy because of increasing adrenal tumor size and virilization . whereas the other two cases did not increase in size and were observed without adrenalectomy . therefore , it is important that patients with cah maintain steroid medication to avoid the appearance of adrenal tumor .

pubmed

self - evaluation of general health status has been associated with actual health in that what people report about their health has been shown to predict mortality . self - rated health ( srh ) as a single survey question developed by the world health organization ( who ) has been validated as a tool to predict mortality in populations with and without cardiovascular diseases [ 13 ] and functional ability [ 4 , 5 ] . asking participants to describe their overall health on a five - point scale ( ranging from excellent to poor ) has achieved popularity as a health - indicating tool in the united states ( us ) and other countries [ 6 , 7 ] . worldwide , noncommunicable diseases ( cancer , cardiovascular diseases , diabetes , and chronic lung diseases ) are largely attributed to four main behavioral factors : tobacco use , unhealthy diet , insufficient physical activity , and harmful alcohol use . poor health for persons with chronic diseases has been attributed largely to a lack of adherence to medical recommendations which include diet and physical activity . high consumption of fruits and vegetables resulted in a lower incidence of cardiovascular disease in nurses and health professionals in a 14-year follow - up study . reporting poor health may be due , in part , to poor management of chronic disease . symptoms of chronic diseases attributed to poor srh among a swedish population of middle - aged and older adults were tiredness / weakness , depression , and musculoskeletal pains . lifestyle behaviors , particularly diet and physical activity , can contribute to or help prevent cardiovascular diseases such as coronary heart disease , hypertension , and type 2 diabetes . diet and physical activity can have positive effects for persons with preexisting coronary heart disease , as well . reduction in chronic disease burden and increase in quality of life have been well documented with higher daily consumption of fruits and vegetables [ 13 , 14 ] and physical activity . the relative risk of cardiovascular disease events for persons with type 2 diabetes decreased with moderate to vigorous physical activity in 14-year follow - up studies for men and women even after adjusting for sociodemographic factors . moreover , srh , as an indicator of health and wellbeing , has been positively associated with fruits and vegetables intake [ 18 , 19 ] . eating at fast - food restaurants and high - fat diets were associated with poor / fair srh in african - americans and australian women , respectively . following dietary and physical activity recommendations has been associated with the prevention and lessening of the severity of cardiovascular diseases . dietary recommendations include half your plate being comprised of fruits and vegetables and cutting back on foods high in solid fats . physical activity guidelines specify being physically active most days of the week to attain 150 minutes of moderate or 75 minutes of vigorous physical activity per week . lack of physical activity and unhealthy dietary patterns place a burden on society due to lack of work productivity and increased health care costs . physical health components from the short form 12 ( sf-12 ) were associated with increased physical activity and healthier diet from baseline to five - year follow - up of a european cohort from the inter99 study . several studies have found an association with lifestyle behaviors and self - evaluation of health . in particular , regular physical activity is reported to be significantly associated with srh in various populations [ 2326 ] . adults from the us with and without cardiovascular diseases who reported engaging in healthy dietary and physical activity behaviors had a higher odds of reporting optimal srh ( excellent , very good , or good ) as compared to fair or poor . physical activity , besides lowering the risk for the development of chronic diseases , also improves the health outcomes of such disease conditions . therefore , the objective of this study was to examine the relationship between dietary and physical activity behaviors and srh . it was hypothesized that participants who reported lower fruits and vegetables intake and higher fatty - food intake and perform less physical activity will have srh poor / fair as compared to those in the good , very good , or excellent category , adjusting for covariates . this is a secondary data analysis of the living for health program ( florida heart research institute , miami , florida , us ) . the living for health program was conducted from 2008 to 2012 and collected information on health behaviors from adults 18 years old ( n = 9,453 ) . during these years , 190 health fair events were held in low income minority communities in miami - dade county to screen participants for cardiovascular disease risks . an accredited institutional review board ( irb ) provided a waiver of consent for the analyses . self - rated health assessment was added in the year 2012 and was answered by 2,108 individuals during the remainder of the living for health program . the final sample size ( n = 1,701 ) consisted of the participants who responded to all the questions included in this analysis . self - rated health was measured by the following question : how would you rate your current health state ? there were five possible responses : excellent , very good , good , fair , and poor . the responses were categorized into two as follows : ( 1 ) excellent / very good / good or ( 2 ) fair / poor for this study . the sociodemographic questionnaire collected information on age , gender , ethnicity , smoking , health insurance , and comorbidities . age was converted to a categorical variable ( 1830 , 3155 , and > 55 years old ) to assess differences between older and younger adults based on the demographics of the population . smoking status derived from the question do you smoke ( any tobacco product ) ? and was categorized as ( yes / no ) . there were insufficient participants to access differences for the original five possible categories for ethnicity . based on the distribution , ethnicity was collapsed into three categories : hispanic , non - hispanic black , and other . other category included white , american indian , pacific islander , indian , and asian . health insurance was categorized as a binary variable ( no insurance / yes insurance ) . presence of comorbidities ( yes / no ) was measured combining two questions : ( 1 ) has any medical professional ever told you that you have high glucose , high blood pressure , high cholesterol , coronary heart disease ) and ( 2 ) have you been prescribed medications for ( diabetes , hypertension , cholesterol , coronary heart disease ) ? a positive response to either question was categorized as having comorbidities . participant 's height and weight were measured and body mass index ( bmi ) was calculated as weight ( kg)/height ( m ) . bmi was classified as bmi : 18.529.9 m ( nonobese ) and bmi : 30 ( obese ) . there were < 1.0% of participants classified as underweight ( bmi < 18.5 ) ; these participants were included in as part of the nonobese group . a single question was used to measure fruits and vegetables intake : on a typical day in the past month , how many servings of fruits and vegetables did you eat ? examples of what a serving represents were provided with the question ( i.e. , 1 cup fresh , 1 medium size fruit , etc . ) . the five possible options were 5 or more per day , 4 a day , 3 a day , 2 a day , or 1 a day . the responses were categorized into three as follows : ( 1 ) 5 or more per day ; ( 2 ) 3 - 4 per day ; ( 3 ) 2 or less per day . fat intake was measured by the question : if you ate fast food in the last month , what type was it ? examples of high fat foods ( fried food , breaded items , taco salads , nachos , double burgers , pizza , hot dogs , croissant items , donuts , shakes , and cakes ) and low fat foods ( salads ( no creamy dressings ) , single burgers , grilled chicken , fruits , and yogurt parfaits ) were provided . from the five possible options , the responses were categorized into three as follows : ( 1 ) primarily low fat foods , mostly low fat or some high fat ; ( 2 ) both high fat and low fat foods about the same ; ( 3 ) mostly high fat , some low fat or primarily high fat foods . physical activity was measured with the question : in the last month , how often were you physically active ? ( performance of at least 30 minutes of fitness walking , cycling , jogging , swimming , aerobic dance , or active sports was considered physical activity ) . physical activity was collapsed from six possible options into three categories as follows : ( 1 ) 57 days a week ; ( 2 ) 14 days a week ; ( 3 ) 13 times a month or never to allow sufficient number in each category . differences between male and female participants were assessed with the chi - square test for categorical variables . logistic regression models were conducted to examine the relationship between the binary dependent variable , srh with fruits and vegetables intake , physical activity , and fat intake adjusting for confounding variables . age and gender differences were found in perceptions of srh [ 11 , 29 ] . poorer physical srh was also associated with lack of health insurance , minority status , and less education for a cohort of randomly selected kentucky adults . comorbidities and obesity are known factors of poorer health . since gender interacted with srh and with physical activity ( fruit and vegetable intake , fat intake , and physical activity ) a reduced model ( unadjusted ) and full model ( adjusted ) including covariates ( age , bmi , smoking status , ethnicity , health insurance , and comorbidities ) were conducted . all statistical analyses were performed using ibm statistical package for the social science version 19 ( spss inc . , chicago il , us ) . females as compared to males had a higher percent classified as obese ( 35.4% versus 27.0% , p = 0.001 ) , reported poor / fair srh ( 23.4% versus 15.0% , p < 0.001 ) , and were less physically active ( 33.9% versus 25.4% , p = 0.001 ) . participants who reported poor / fair srh as compared to those with excellent / very good / good were females ( 78.6% versus 67.9% , p < 0.001 ) ; older > 55 years ( 35.5% versus 29.4% , p = 0.024 ) ; obese ( 46.8% versus 29.2% , p < 0.001 ) ; non - hispanic black ( 38.0% versus 29.4% , p = 0.001 ) ; had comorbidities ( 61.4% versus 40.5% , p < 0.001 ) ; consumed 2 servings of fruits and vegetables per day ( 73.0% versus 59.3% , p < 0.001 ) ; had an intake of primary high fat foods / mostly high fat or some low fat foods ( 17.7% versus 10.8% , p < 0.001 ) ; and were less physically active ( 40.3% versus 29.0% , p < 0.001 ) ( table 2 ) . unadjusted logistic regression models for females showed that those who consumed 2 servings of fruits and vegetables per day were 2.2 times more likely to report poor / fair srh as compared to those who consumed 5 servings per day ( p = 0.001 , 95% ci 1.393.70 ) . logistic regression model adjusted for age , bmi , smoking , ethnicity , health insurance , and comorbidities showed that females who consumed 2 servings of fruits and vegetables per day were 2.1 times more likely to report poor / fair srh as compared to those who consumed 5 servings per day ( p = 0.003 , 95% ci 1.233.54 ) . there was a more modest and nonstatistically significant increase in the odds of poor / fair srh for men with the lowest level of daily fruits and vegetables consumption . unadjusted and adjusted models indicated that males who consumed 3 - 4 servings of fruits and vegetables per day were 2.6 and 2.8 times more likely to report poor / fair srh as compared to those who consumed 5 servings per day ( p = 0.031 , 95% ci 1.096.58 and p = 0.028 , 95% ci 1.127.35 , resp . ) . unadjusted logistic regression models of fat intake for females indicated that those who consumed mostly high fat foods were 1.6 times more likely to report poor / fair srh as compared to those who consumed primarily low fat foods ( p = 0.015 , 95% ci 1.1032.49 ) . females who consumed both high and low fat foods were 1.3 times more likely to report poor / fair srh as compared to those who consumed primarily low fat foods ( p = 0.041 , 95% ci 1.0131.83 ) . logistic regression models adjusted for age , bmi , smoking , ethnicity , health insurance , and comorbidities showed that females who consumed mostly high fat foods were 1.5 times more likely to report poor / fair srh as compared to those who had primarily low fat foods ( p = 0.036 , 95% ci 1.032.43 ) . similarly , females who consumed both high and low fat foods about the same were 1.4 times more likely to report poor / fair srh as compared to those who had primarily low fat foods ( p = 0.018 , 95% ci 1.072.01 ) . unadjusted logistic regression model for males indicated that those who consumed mostly high fat foods were 3.1 times more likely to report poor / fair srh as compared to those who consumed primarily low fat foods ( p < 0.001 , 95% ci 1.705.97 ) . the adjusted model showed that males who consumed mostly high fat foods were 3.2 times more likely to report poor / fair srh health as compared to those who consumed primarily low fat foods ( p = 0.001 , 95% ci 1.672.43 ) . unadjusted logistic regression model for females showed that those who performed 13 times a month or never of physical activity were 1.6 times more likely to report poor / fair srh as compared to those who performed 5 days per week of physical activity ( p = 0.003 , 95% ci 1.182.29 ) . logistic regression model adjusted for age , bmi , smoking , ethnicity , health insurance , and comorbidities showed that females who reported 13 times a month or never of physical activity were 1.6 times more likely to report poor / fair srh as compared to females who performed 5 days per week of physical activity ( p = 0.004 , 95% ci 1.172.35 ) . unadjusted logistic regression model showed that males who performed 13 times a month or never of physical activity were 1.8 times more likely to report poor / fair srh as compared to those who performed 5 days per week of physical activity ( p = 0.043 , 95% ci 1.023.40 ) . the association of physical activity and srh was no longer significant for males once adjustment variables were added ( p = 0.218 , or = 1.50 , 95% ci 0.792.49 ) ( table 4 ) . we found higher odds of poor / fair srh for males and females who consumed the lowest level of fruits and vegetables . no gender differences were found in a longitudinal cohort of canadian young adults who were more likely to report excellent srh if they had a high intake of fruits and vegetables as adolescents . similarly , dietary intake high in fiber was associated with more favorable srh as compared to lower fiber for a cohort of african - american adults , independent of gender . the presence of any chronic disease influences one 's perception of health and wellbeing [ 3335 ] . persons with diabetes may be more likely to consume more fruits and vegetables after their diagnosis . even though diagnosis with chronic disease could result in better health behavior , consuming fruits and vegetables at least five times per day was associated with optimal srh for both individuals with cardiovascular diseases and diabetes as well as for persons without these chronic illnesses . our sample population , who are approximately more than half - hispanic , may have a large proportion of first generation immigrants from caribbean and latin american countries . the perception of good health may differ based on country of origin . for example , foreign - born haitian americans had higher levels of perceived stress as compared to african - americans , but african - americans were more likely to rate their health as poor / fair compared to haitian americans . we found that both males and females , who consumed mostly high fat foods , were more likely to report poor / fair srh as compared to those consuming primarily low fat foods . consistent with our results , collins et al . reported higher intake of fat and saturated fat to be associated with poorer srh for a large cohort of australian women . frequency of eating at fast - food restaurants was positively associated with poor srh for a cohort of african - americans . our results contradicted barreto and de figueiredo who reported lower odds of consuming fatty meat and whole milk for those diagnosed with at least one chronic disease and even lower for those diagnosed with two or more chronic diseases compared to absence of disease in a spanish population . they suggested that medical advice to modify lifestyle behavior may be a probable confounder of diet and health when comparing persons with and without chronic disease . similarly , the odds of having poor health increased twofold in a cohort of greek nurses making an effort to avoid fatty foods in their diet . a strong association between physical activity and srh for females was observed in our study . females who engaged in less physical activity were more likely to report poor / fair srh as compared to those who were more physically active . several studies found physical activity to be related to srh regardless of gender for a national sample , for a cohort of older adults , and for african - american adults with a high proportion of chronic diseases . our findings on gender differences in physical activity levels and srh are in agreement with several other studies [ 3841 ] . as expected , the likelihood of rating health as poor / fair was substantially higher for women , as compared to men . considering the impact of physical activity on health status , variation of the effect of physical activity according to gender could also be associated with different proportions of srh . these distinctions could be explained by the biological and sociocultural environment inequalities between men and women . for instance , it was found that gender roles and reduced access to resources and social conditions , such as safe environment , do not foster regular physical activity among women [ 40 , 41 ] . while women generally tend to rate their health worse in health and behavioral studies , this is consistently in line with lack of physical activity . of utmost relevance of the role of physical activity in health is the gradient effect of the levels of physical activity on srh , as demonstrated by evidence from several past studies [ 4244 ] . participants who were highly active and very highly active were twice as likely to have excellent srh as compared to those who were less physically active . the main objective of our study was to assess the relationship of indicators of modifiable cardiovascular disease risk factors as follows : dietary and physical activity lifestyle and their relationship to srh . we examined srh in a half - hispanic population with non - hispanics who were predominately black and we found significant differences in ethnicity for srh . however , it is noteworthy to mention that ethnic differences were adjusted in the final models . a cohort of older hispanics rated their health poorer than non - hispanic whites yet had a lower mortality rate after a 16-year follow - up , based on data from the health and retirement study and adjusting for demographics , socioeconomic status , health status , and health behaviors at baseline . furthermore , level of acculturation in hispanic americans was associated with srh whereby srh of the more acculturated matched native non - hispanic whites and blacks . self - rated health differences were found within african - americans over time that were based on differences in age , education , smoking , and morbidity ( angina , congestive heart failure , diabetes , and kidney disease ) , having been hospitalized in the year prior to baseline , depressive symptoms , mobility limitations , and initial self - rated health . this was a large sample of adults from miami dade county , florida , primarily minorities , half of whom were from a diverse hispanic population . to our knowledge , this is the first study to investigate the relationship between lifestyle behaviors and srh for a population largely comprised of hispanics with a considerable proportion of blacks . nevertheless , there are some limitations . causality of lifestyle behaviors with srh could not be established due to the single time point . other factors that may have influenced srh such as socioeconomic status , neighborhood , housing situation , and psychosocial factors were not collected in this study . modifiable lifestyle behaviors known to reduce cardiovascular disease risk , low fruits and vegetables intake and high fat intake , were associated with poor / fair srh in males and females . poorer srh was significantly associated with low physical activity in females only , and low physical activity was also more common in women than men in this study sample . these results indicate that gender differences may have implications in designing and monitoring lifestyle interventions to prevent cardiovascular diseases .

background . lack of adherence to dietary and physical activity guidelines has been linked to an increase in chronic diseases in the united states ( us ) . the aim of this study was to assess the association of lifestyle behaviors with self - rated health ( srh ) . methods . this cross - sectional study used self - reported data from living for health program ( n = 1,701 ) which was conducted from 2008 to 2012 in 190 health fair events in south florida , us . results . significantly higher percent of females as compared to males were classified as obese ( 35.4% versus 27.0% ) , reported poor / fair srh ( 23.4% versus 15.0% ) , and were less physically active ( 33.9% versus 25.4% ) . adjusted logistic regression models indicated that both females and males were more likely to report poor / fair srh if they consumed 2 servings of fruits and vegetables per day ( or = 2.14 , 95% ci 1.303.54 ; or = 2.86 , 95% ci 1.127.35 , resp . ) and consumed mostly high fat foods ( or = 1.58 , 95% ci 1.032.43 ; or = 3.37 , 95% ci 1.672.43 , resp . ) . the association of srh with less physical activity was only significant in females ( or = 1.66 , 95% ci 1.172.35 ) . conclusion . gender differences in health behaviors should be considered in designing and monitoring lifestyle interventions to prevent cardiovascular diseases .

pubmed

the role of endoscopic injection of dextranomer / hyaluronic acid copolymer ( dx / ha ) has expanded , as an alternative to open surgery , to a treatment option for observation protocols using prophylactic antibiotics . deflux has been fda approved since 2001 as a treatment option for grade 24 vesicoureteral reflux ( vur ) in single systems with no accompanying urological malformations in children above one year of age . the operative ease of technique , short procedure , and recovery time as well as repeatability of injections have popularized the technique with surgeons and parents alike . reports of expanded use to patients of all ages and grades of vur even in the presence of duplication , ureterocele , or other lower tract anomalies are published worldwide [ 1 , 2 ] . in a statement of the aua board of directors from october 2007 , it was determined that : it is the current position of the american urological association that endoscopic injection of the dextranomer / hyaluronic compound deflux is an option in the management of pediatric vesicoureteral reflux ( vur ) [ 3 , 4 ] . the initial success rate was quoted as 68% and is still mentioned on the current website of the manufacturing company ( q - med , scandinavia inc ) . in the meantime , the original subureteric injection technique ( sting ) has evolved to the intraureteric injection technique introduced by the atlanta group . using this hydrodistention - implantation technique ( hit ) , the authors reported success rates of 89% . however , the reported worldwide success rates differ considerably ( 55%89% [ 7 , 8 ] ) . unpublished data from our institution show an overall radiographic success rate of 75% for patients and 84% for renal units after the first injection . although the success rates continuously improve , the numbers of most institutions still differ significantly from the traditional 98% for open reimplants . since up to 25% of patients will have remaining vur after dx / ha injection , we still perform voiding cystograms 2 months after surgery . positional instillation of contrast ( pic ) cystography was developed to detect occult vur in patients with recurrent urinary tract infections ( uti ) after a negative standard voiding cystogram . it has a higher sensitivity than standard voiding cystogram ( vcug ) and can detect occult vur . parents can be advised that in case of a positive pic , dx / ha injection can be performed during the same anesthesia . in the current study , we compared two different pic techniques and evaluated two different scenarios : first , whether pic cystography or high - pressure cystograms were useful to detect de novo contralateral vur in the case of preoperative unilateral vur and second , whether pic cystography , when performed intraoperatively immediately after dx / ha injection , can predict a successful surgical outcome by correlating the results with a standard 2-month postprocedure vcug . a retrospective chart review was performed on all intraoperative cystograms performed at the time of dx / ha injections at our institution from 20052007 . dx / ha injections were performed for all grades of vur and included complex cases such as duplicated systems . in case of preoperative unilateral reflux , a pic cystogram was performed on the contralateral unaffected side before dx / ha injection . if positive , bilateral injection took place . the injections were done using the hit technique as described by kirsch et al . . dx / ha was injected until a sufficient mound and good ureteral coaptation was observed . at this point the outflow portion of the 10 french off set cystoscope ( wolf , germany ) was placed at the bladder neck filling the bladder with contrast material ( cystografin ) to 100 cm water pressure until voiding occurred around the cystoscope ensuring complete bladder filling . the second group underwent standard pic cystography with the outflow portion of the cystoscope directed at the ureteral orifice at a 100 cm water pressure for 5 seconds ( figure 2 ) . radiographic views for both tests were performed in anterior - posterior and lateral as well as upper tract views . the intraoperative cystogram was noted to be positive if any grade of vur was present ( figure 3 ) . in cases of a positive study on the contralateral side or for remaining vur after injection , the patients were left on prophylactic antibiotics and followed up at 4 weeks postoperatively with an ultrasound and 2 months postoperatively with a vcug . the correlations of the intra- and postoperative study as well as the detection of de novo vur were noted . altogether 116 patients and 177 renal units ( ru ) were treated and available for evaluation . out of the 116 patients , new contralateral vur ( de novo ) for the 34 preoperative unilateral refluxing patients was found intraoperatively in 27 ru ( figure 4(c ) ) . the high - pressure intraoperative cystogram ( group 1 ) was performed in 86 rus and the standard pic ( group 2 ) in 91 rus . there was no statistically significant difference between groups 1 and 2 for the detection of de novo vur ( figure 4(b ) ) . for both groups , only 5 ru were found to have remaining vur intraoperatively after dx / ha injection ( 2 in group 1 and 3 in group 2 ) . postoperative pain management , if necessary , consisted of ibuprofen or acetaminophen according to parental preference . in all patients , the postoperative us at four weeks were negative for hydronephrosis or any other new abnormality . the dx / ha deposits were seen in the bladder in all cases . at 2 months postoperative standard vcug detected 20 ru ( 23% ) to be positive for vur in group 1 and 16 ru ( 18% ) positive for vur in group 2 . altogether 36 ru were positive , for an allover success rate of 80% for ru and 75% for patients ( figure 4(a ) ) . the grade of vur , age and gender of patient as well as amount of deflux injected did not influence the result . vesicoureteral reflux remains a common disease , and especially for lower grades the prospect of spontaneous resolution is good . but , the data on the value of daily prophylactic antibiotics remain controversial , and most parents are concerned about their long - term use . the discussion becomes evident by the current multi - institutional nih - funded rivur study , which aims to evaluate the role of prophylactic antibiotic in vur . in addition to prolonged antibiotic use , parents and patients alike dread the yearly necessary voiding cystogram to check for resolution . therefore , to avoid the daily antibiotics and the yearly vcug , many parents consider surgical treatment options . the endoscopic injection of dx / ha is an attractive management alternative for parents . when presenting the surgical options , all parents are enthusiastic about the minimal invasive nature of the procedure with low complication possibilities and fast recovery times . however , the 20%25% lower success rate of the endoscopic procedure in comparison to the open surgery is a concern . although dx / ha can be reinjected , it requires further testing , anesthesia , and time spent away from school or work . therefore , it would be highly desirable to develop a method to check the success of the deflux injection intraoperatively . pic cystography is used to detect occult vur in patients with a negative standard voiding study who suffer from recurrent utis . according to rubenstein , the procedure is a 100% sensitive and 91% specific in identifying vur and can , therefore , detect occult vur . recently confirmed the findings in 5 patients . in a study on 39 patients with febrile utis and negative voiding studies from 4 different institutions , pic cystograms were performed and vur identified in 82% of these patients . additionally , the authors noted that orifices that were patulous , laterally displaced , or positive for hydrodistention were much more likely to be positive for vur than normal orifices . also performed intraoperative pic cystograms and found that 23% of patients had a positive , postoperative vcug despite a negative intraoperative pic study , and palmer confirmed these results . the authors concluded that pic cystography was not useful to raise the success rate of deflux injection . in the current study , we used two different cystogram techniques : the standard pic study and a high - pressure cystogram , filling the bladder until irrigation fluid leaked around the cystoscope . even with this aggressive cystogram technique only an 80% correlation between a negative intraoperative cystogram and postoperative cystogram was achieved . the success rate of 75% for patients and 80% for ru could not be improved . a relative wound edema might increase the coaptation of the ureteral orifice , temporarily giving a negative intraoperative study result . other possibilities include decrease in the size of the injected dx / ha deposit over time , also thought to be responsible for long - term failure after dx / ha injection . this phenomenon has been noted during cystoscopy at the time of the planned second injection of deflux after initial failure . the dx / ha deposits tend to migrate medially and toward the bladder neck causing long - term failure of injection . elmore et al . reported the onset of new contralateral vur after dx / ha implantation in approximately 13% of 126 patients with preoperatively unilateral vur . in the current study , twenty seven of these 34 patients demonstrated a positive pic cystography intraoperatively and were successfully injected with dx / ha . none of these de novo detected refluxing units were positive for vur in the postoperative cystograms . a negative intraoperative cystogram correlates with the postoperative vcug in only 80% . considering that the success rates of dx / ha injections are commonly between 7580% , the addition of intraoperative cystograms can not replace postoperative studies . in patients treated with unilateral vur , pic cystography can detect occult reflux and prevent postoperative contralateral new onset of vur .

the endoscopic injection of dx / ha in the management of vesicoureteral reflux ( vur ) has become an accepted alternative to open surgery . in the current study we evaluated the value of cystography to detect de novo contralateral vur in unilateral cases of vur at the time of dx / ha injection and correlated the findings of immediate post - dx / ha injection cystography during the same anesthesia to 2-month postoperative vcug to evaluate its ability to predict successful surgical outcomes . the current study aimed to evaluate whether an intraoperatively performed cystogram could replace postoperative studies . but a negative intraoperative cystogram correlates with the postoperative study in only 80% . considering the 7580% success rate of dx / ha implantation , the addition of intraoperative cystograms can not replace postoperative studies . in patients treated with unilateral vur , pic cystography can detect occult vur and prevent postoperative contralateral new onset of vur .

pubmed

the accuracy to detect fetal pathology depends on the expertise of the sonographer and some limitations inherent to this imaging technique . descent of the fetal head and progressive ossification of the skull bones towards the end of the pregnancy , maternal obesity and oligohydramnios are all well - known limitations affecting the sensitivity to detect central nervous system pathology . the first reported use of fetal magnetic resonance imaging ( mri ) dates from 1989 . advances in mri technology during the last 2 decades and better knowledge of fetal pathology , have both led to an increasing role for mri in the diagnostic work - up of prenatal anomalies . most fetal mri studies reported so far , have addressed the additional role of mri in the diagnosis and characterization of fetal anomalies detected on ultrasound , with emphasis on central nervous system pathology . in this manuscript we discuss the role of mri as an adjunct to ultrasound for cases diagnosed with ventriculomegaly . the descriptive term cerebral ventriculomegaly is used when the maximal diameter of the atrium of the lateral ventricles measured in a plane perpendicular to the midsagittal section of the brain exceeds 10 mm . the prevalence of ventriculomegaly ranges between 0.3 - 1.5 and the severity can be classified in mild ( 10 - 12 mm ) , moderate ( 12 - 15 mm ) and severe ( > 15 mm ) , as illustrated in fig . 1 ( valsky et al . , 2004 ; another classification system is based on measuring the thickness of the overlying brain parenchyma in the category with an atrial diameter of > 15 mm , i.e. > 3 mm thickness indicates a moderate ventriculomegaly and 3 mm a severe form ( levine et al . , 2003 ) . measurement of the lateral ventricle is subject to errors owing to an off - axis image plane of a section , an angled measurement , or improper choice of ventricular boundary giving risk to false - positive test results . therefore , a precise definition of the reference planes and anatomical landmarks is important to avoid inaccurate measurements and facilitate imaging follow - up comparisons ( levine et al . , 2008 ) . ventriculomegaly has a wide range of causes and can roughly be divided in three categories : ( 1 ) an imbalance between the production and absorption of cerebrospinal fluid , of which the obstructive form is most frequently observed , ( 2 ) abnormal cerebral development such as partial or complete agenesis of the corpus callosum and neuronal proliferation / migration disorders and ( 3 ) destructive disease processes leading to loss of neuronal tissue by vascular insults or infectious pathogens . unilateral ventriculomegaly is more often seen in destructive processes , whereas developmental anomalies are more characterized by bilateral broadening of the ventricles ( girard et al . , 2003 ) . the reported incidence of additional anomalies goes up to 70 - 85% , strongly depending on the number of fetuses included in the study and the distribution of the severity of the cerebral ventriculomegaly observed in the study population ( huisman et al . , 2002 ; the term isolated ventriculomegaly is used in case no other structural anomalies are seen at the time of diagnosis . isolated and/or unilateral ventriculomegaly , in particular the mild form , has a lower incidence of peri- and postnatal morbidity and mortality ( senat et al . , 1999 fetal mri is mainly performed on 1.5 tesla scanners . to date , no adverse health effects have been demonstrated neither fetal nor maternal after short term exposition to electromagnetic fields as used in clinical mri . the haste sequence ( half fourier acquired single shot turbo spin echo ) is nowadays most used and combines short acquisition times ( 1 image in less than 1 second ) with a good signal - to - noise ratio , good t2 contrast and an acceptable spatial resolution ( slice thickness of 3 mm ) . t1 weighted imaging is often used to detect hemorrhages or calcifications . t1-weighted imaging sequences have a lower signal to noise ratio , require longer scanning times ( up to 18 seconds per slice ) and are hence much more susceptible to fetal and maternal motion . novel scanning sequences include the use of diffusion weighted imaging and diffusion tensor imaging for a more functional analysis of the developing brain ( guimiot et al . , 2008 ; mri scans have a big field of view and can be obtained in any given plane . the major limitations of fetal mri are the impact of fetal motion on the image acquisition and its relatively low spatial resolution , in particular compared to ultrasound . contra - indications for mri are the same as for non - pregnant patients ( claustrophobia , metallic brain clips , pacemaker implant , ) . opposed to ultrasound , mri has an excellent contrast resolution which enables to differentiate easily between gray and white matter . mri also allows to directly visualize the cortical region and the fossa posterior , without sonographic limiting factors such as maternal obesity , the amount of amniotic fluid , fetal head position or the acoustic shadows of the skull bones . knowledge of the normal appearance and maturation of the developing cerebral gyri and sulci is helpful in the appropriate diagnosis and counseling of anomalous fetuses . at 14 weeks the sylvian fissure and callosal sulcus are respectively visible at 16 and 18 weeks , and the central sulcus is not reliable seen until 24 - 25 weeks of gestation . weeks , numerous new sulci and gyri develop and by the age of 32 - 35 weeks secondary gyri are visible throughout the cerebral cortex ( levine and robson , 2005 ) . an important role in the prenatal diagnostic work - up of ventriculomegaly is the detection and characterization of additional cerebral anomalies ( kubik- huck et al . , 2000 ; , 2002 ; launay et al . , 2002 ; levine et al . , 2003 ; valsky et al . , 2004 ; mehta and levine , 2005 ; zimmerman and bilaniuk , 2005 ; glenn and barkovich , 2006 ; benacerraf et al . , 2007 ; morris et al . the incidence of additional malformations detected on mri following normal ultrasound findings varies greatly between several reported studies and depends on the severity of ventriculomegaly , the expertise of the sonographer and the selected patient population . percentages of detection range between 5% for mild ventriculomegaly and up to 50% for moderate and severe cases ( valsky et al . , 2004 ; salomon et al . , numbers should be interpreted with caution , because of the systematic lack of postnatal / postmortem ( imaging ) correlates , the uncertainty between the time span between prenatal ultrasound and mri in several studies , the a priori knowledge of ultrasound findings by the fetal mri specialist and the lack of data investigating the sonographic detection / confirmation of the fetal anomalies following the mri scan . address several items why the added value of mri is difficult to statistically demonstrate : an overenthusiastic attitude towards new technologies , comparing a first line routine ultrasound exam with a mri exam in a tertiary centre , the lack of description of the ultrasound technique and checklist , suboptimal or insufficient ultrasound images in the reported studies and no information with respect to the time span between the ultrasound and mri exam ( malinger et al . , 2004 ) . at last , it is very important to stress that the interobserver agreement , i.e. the interpretation agreement between 2 observers using the same modality , for both imaging modalities can vary greatly . in a study of levine et al . , in which the agreement between experts using the same imaging modality was investigated , overall consensus was reached with respect to normal and abnormal findings in only 60% of the cases for ultrasound and in 53% for mri ( levine et al . , 2008 ) . the numbers even drop below 50% for the detection of cerebellar and gyral anomalies . important to note is that the readers in this study could not indicate their uncertainty in each finding , but were only allowed to indicate the presence or absence of an anomaly . given the often subtle findings of fetal central nervous pathology , such as cerebellar of gyral anomalies , this probably has led to an overestimate of the discrepancies . another important finding is that the level of subspecialty training of the individuals interpreting the mri examination , in particular the involvement of pediatric neuroradiologists , significantly improved the interobserver agreement . below , we will discuss some disease entities often associated with ventriculomegaly , with emphasis on the added value of mri ( launay et al . , 2002 ; glenn and barkovich , 2006 part 2 ; benacerraf et al . , 2007 ) . mri is a very sensitive technique to detect small deposits of intraventricular blood , which may suggest an intraventricular hemorrhage as cause of the hydrocephaly ( fig . mri has also a high accuracy to detect corpus callosum dysgenesis and allows to screen for additional malformations such as cortical anomalies , periventricular of subependymal nodular heteropia and interhemispheric cysts ( fig . accurate characterization of those additional anomalies is important in terms of prognosis and parental counseling . mri is equally good for a morpho- and volumetric assessment of the fossa posterior in case of suspicious ultrasound findings . it allows a good evaluation of the vermis , the cerebellar hemispheres , the pedunculi , the brainstem , the 4th ventricle , the tentorium cerebelli and the retrocerebellar space , which enables to detect developmental anomalies such as vermis hypoplasia , dandy walker malformations , chiari malformations and other fossa posterior malformations ( fig . a recurring indication for ventriculomegaly reported in the literature , is the detection of cortical malformations , such as polymicrogyria ( fig . 5 ) , the excellent tissue contrast of mri and the absence of bony interference , makes this technique ideal for cortical evaluation . congenital infections such as cytomegalovirus and toxoplasmosis , are also good candidates to refer for mri , in particular to detect pathological white matter changes and cortical malformations ( fig . focal loss of brain tissue , hemorrhages and sequelae of ventriculitis can also easily be detected on mri . on the basis of reported studies , fetal mri has a limited role over ultrasound in assessing the size of the cerebral ventricles , except for cases where fetal position and calvarial ossification cause problems . in the second trimester , in particular the 20 - 24 weeks group , mri is useful to detect small foci of brain hemorrhage or callosal anomalies in fetuses diagnosed with cerebral ventriculomegaly ( griffiths et al . , 2010 ) . later in pregnancy ( over 25 weeks ) , mri adds information about normal and pathological cortical development and maturation , depicts fossa posterior anomalies and is a more sensitive imaging modality to detect white matter pathology ( griffiths et al . , 2010 ; yi et al . , referring prenatal cases of ventriculomegaly for fetal mri is a topic of debate , not only due to questions related to the diagnostic accuracy of mri and its added value , but also because of practical issues such as the availability of mri , the cost and available expertise of fetal radiologists . most studies reported in the literature advocate the added value of mri in cases of moderate and severe ventriculomegaly , not only to confirm the severity , but mainly to further detect and characterize additional anomalies . in cases of mild unilateral ventriculomegaly , mri might demonstrate additional findings which are difficult to detect with sonography such as leucomalacy and neuronal migration disorders . in both categories ,

the indication for fetal magnetic resonance imaging ( mri ) remains a subject of debate , partly because of questions concerning its diagnostic accuracy compared to ultrasound , partly because of practical factors such as accessibility , high costs and available expertise . most studies advocate an added value for mri in cases diagnosed with central nervous system pathology . mri is a good modality to detect small foci of brain hemorrhage , to depict callosal anomalies , to add information about normal and pathological cortical development , and is a more sensitive imaging method to detect white matter pathology . this manuscript discusses the role of mri as an adjunct to ultrasound for cases diagnosed with cerebral ventriculomegaly .

pubmed

lung cancer is the third most common cancer diagnosed and the most common cause of cancer - related death among both men and women in the united states ( us ) . the american cancer society estimates that in 2012 , 226,160 patients ( men , 116,470 ; women , 109,690 ) will be diagnosed with lung cancer and 160,340 ( men , 87,750 ; women , 72,590 ) will die from the disease . lung cancer is often diagnosed at an advanced stage of disease , as early - stage disease is often asymptomatic . the stage distribution of lung cancer cases diagnosed in 1999 - 2006 included in the surveillance , epidemiology and end results ( seer ) program were : distant metastasis at diagnosis , 56% ; regional metastasis , 22% ; localized , 15% ; and unstaged , 8% . stage of disease was a strong risk factor for survival , with advanced disease having much lower five - year survival compared to early disease . from 1999 - 2006 , the seer program reported that the five - year relative survival was 3.5% for distant stage at diagnosis , 24% for regional stage and 52.9% for localized stage . bone is one of the most frequent sites of metastasis in patients with lung cancer . these osteolytic lesions undermine the structural integrity of bone placing lung cancer patients at an increased risk for skeletal - related events ( sres ) such as pathological fractures , spinal cord compression and severe pain requiring radiotherapy or surgery for bone lesions.[68 ] these skeletal complications result in impaired mobility and reduced quality of life adding substantially to the overall burden of disease in patients with lung cancer . data are limited on the occurrence and outcomes of bone metastasis and sres among elderly patients who develop lung cancer . a few studies have reported results pertaining to the occurrence of bone metastasis and of sres among lung cancer patients with bone metastases , but none have focused on older patients . we therefore undertook this study to quantify the impact of bone metastasis and sres on mortality among older lung cancer patients by analyzing a combined population - based cancer registry and the medicare claims database . the seer program , supported by the national cancer institute ( nci ) , collects cancer incidence and survival data from population - based cancer registries from selected geographic areas . during the time period of the present study , the seer program covered 14 to 26% of the us population from 9 to 13 geographic areas . the seer data included information on patient demographics , date of cancer diagnosis , tumor stage and other characteristics at diagnosis and summary information on treatments received soon after diagnosis . the medicare program , administered by the centers for medicare and medicaid services , covers 97% of us population ages 65 years . medicare data included demographic information on beneficiaries and claims data from hospitals and other institutional and non - institutional providers . the medicare claims data included dates of service , diagnosis codes ( international classification of diseases ninth revision ( icd-9 ) ) and procedure codes ( current procedural terminology ( cpt ) . we identified a cohort of patients aged 65 years or older and having a seer record of a new diagnosis of lung cancer between july 1 , 1999 and december 31 , 2005 and used seer and medicare data to determine possible occurrence of bone metastasis , sres and deaths occurring through december 31 , 2006 . we required each subject to have a baseline period of at least six months of full fee - for - service medicare coverage before his / her lung cancer diagnosis date . the claims data from this baseline period provided information on the presence of comorbidities , and on the presence of possible bone metastasis and sres at diagnosis ( based on icd-9 codes ) . we excluded patients who did not have both medicare part a and part b coverage and those who were enrolled in a medicare advantage plan during the baseline period because we did not have complete medicare claims data on the services they may have received . in all analyses , we censored subjects if they died or lost full fee - for - service coverage before the end of 2006 ( e.g. , if their coverage changed such that we did not have access to all of their medical claims ) . bone metastasis , with or without evidence of sres , was the time - dependent exposure variable of main interest in our analyses . throughout this paper , we apply the term bone metastasis and sres to patients who have claims - based evidence of these conditions . we used the following medicare claims data as evidence of bone metastasis : a ) at least one inpatient claim with an icd-9 diagnosis code of 198.5 ( secondary malignant neoplasm of bone and bone marrow ) as the primary or secondary discharge diagnosis ; b ) at least one outpatient claim with a diagnosis code of 198.5 , paired with a code for procedures used to diagnose or treat bone metastasis ; or c ) at least one outpatient physician evaluation and management claim with a diagnosis code of 198.5 ( above algorithm available on request ) . we classified subjects as having concurrent bone metastasis at the time of diagnosis if they had a claim in the month of the lung cancer diagnosis or in the preceding month . among subjects without concurrent bone metastasis at diagnosis , we identified new bone metastasis as the earliest occurrence of one of the above claims patterns at any time during follow - up . for the purpose of this study , we defined sres as one or more of the following conditions , occurring concurrently with or after the first bone metastasis : fractures , radiotherapy to bone , surgery to bone and spinal cord compression . we identified sres using combinations of diagnosis and procedure codes from inpatient and/or outpatient claims ( codes available on request ) . we classified an sre as concurrent with bone metastasis if it occurred within 30 days before or after the earliest bone metastasis date and as subsequent to bone metastasis if it occurred at any time more than 30 days after the bone metastasis date . we excluded from the analysis patients with claims suggesting bone metastases occurring more than 30 days prior to the lung cancer diagnosis ( n = 570 ) . the excluded patients comprising of 2% of overall patients with bone metastases had similar demographic characteristics as the bone metastases patients included in the analysis but were more likely to have unstaged and unspecified histology . we obtained information on date of death using the combined seer registry and medicare claims data . we used the concordant date of death in these two sources , if there was agreement between them . if the seer date of death was missing , we used the medicare date of death . from the seer data , we obtained information on age , gender , race / ethnicity and stage at cancer diagnosis . from the medicare claims data we computed each person 's charlson comorbidity score on the basis of icd-9 diagnosis codes in his / her inpatient records for the 17 medical conditions [ table 1 ] comprising the charlson index in the 12-month period prior to the month of cancer diagnosis . in computing the charlson index , we used the approach described by romano et al , and updated by quan et al . characteristics of patients with lung cancer , total cohort and according to bone metastasis status : seermedicare , july 1 , 1999-december 31 , 2006 descriptive analyses of the cohort included frequency distributions and median values , where applicable . we computed the proportion of patients with lung cancer having claims - based evidence of bone metastasis at one year from diagnosis for the overall cohort of lung cancer cases and for subgroups specified by tumor stage at diagnosis . for these computations , we selected patients diagnosed with lung cancer from july 1 , 1999 through december 31 , 2005 , so that these patients would have a potential observation period of at least one year post lung cancer diagnosis . we used cox regression to estimate mortality hazards ratios ( hrs ) and 95% confidence intervals ( cis ) among patients with bone metastasis with or without sres , compared to patients without bone metastasis , adjusting for covariates . we also estimated mortality hrs for subgroups specified by initial tumor stage , age , gender and race / ethnicity at cancer diagnosis . confounders were categorical forms of age at cancer diagnosis ( 65 - 69 , 70 - 74 , 75 - 79 , 80 - 84 , 85 + ) , gender ( male , female ) , race ( white , african american , hispanic american , other ) , stage at lung cancer diagnosis ( distant , localized , regional , unstaged ) , and charlson comorbidity score ( 0 , 1 , 2 , 3 + ) . this study was approved by the uab institutional review board and by the nci seer - medicare program . the seer program , supported by the national cancer institute ( nci ) , collects cancer incidence and survival data from population - based cancer registries from selected geographic areas . during the time period of the present study , the seer program covered 14 to 26% of the us population from 9 to 13 geographic areas . the seer data included information on patient demographics , date of cancer diagnosis , tumor stage and other characteristics at diagnosis and summary information on treatments received soon after diagnosis . the medicare program , administered by the centers for medicare and medicaid services , covers 97% of us population ages 65 years . medicare data included demographic information on beneficiaries and claims data from hospitals and other institutional and non - institutional providers . the medicare claims data included dates of service , diagnosis codes ( international classification of diseases ninth revision ( icd-9 ) ) and procedure codes ( current procedural terminology ( cpt ) . we identified a cohort of patients aged 65 years or older and having a seer record of a new diagnosis of lung cancer between july 1 , 1999 and december 31 , 2005 and used seer and medicare data to determine possible occurrence of bone metastasis , sres and deaths occurring through december 31 , 2006 . we required each subject to have a baseline period of at least six months of full fee - for - service medicare coverage before his / her lung cancer diagnosis date . the claims data from this baseline period provided information on the presence of comorbidities , and on the presence of possible bone metastasis and sres at diagnosis ( based on icd-9 codes ) . we excluded patients who did not have both medicare part a and part b coverage and those who were enrolled in a medicare advantage plan during the baseline period because we did not have complete medicare claims data on the services they may have received . in all analyses , we censored subjects if they died or lost full fee - for - service coverage before the end of 2006 ( e.g. , if their coverage changed such that we did not have access to all of their medical claims ) . bone metastasis , with or without evidence of sres , was the time - dependent exposure variable of main interest in our analyses . throughout this paper , we apply the term bone metastasis and sres to patients who have claims - based evidence of these conditions . we used the following medicare claims data as evidence of bone metastasis : a ) at least one inpatient claim with an icd-9 diagnosis code of 198.5 ( secondary malignant neoplasm of bone and bone marrow ) as the primary or secondary discharge diagnosis ; b ) at least one outpatient claim with a diagnosis code of 198.5 , paired with a code for procedures used to diagnose or treat bone metastasis ; or c ) at least one outpatient physician evaluation and management claim with a diagnosis code of 198.5 ( above algorithm available on request ) . we classified subjects as having concurrent bone metastasis at the time of diagnosis if they had a claim in the month of the lung cancer diagnosis or in the preceding month . among subjects without concurrent bone metastasis at diagnosis , we identified new bone metastasis as the earliest occurrence of one of the above claims patterns at any time during follow - up . for the purpose of this study , we defined sres as one or more of the following conditions , occurring concurrently with or after the first bone metastasis : fractures , radiotherapy to bone , surgery to bone and spinal cord compression . we identified sres using combinations of diagnosis and procedure codes from inpatient and/or outpatient claims ( codes available on request ) . we classified an sre as concurrent with bone metastasis if it occurred within 30 days before or after the earliest bone metastasis date and as subsequent to bone metastasis if it occurred at any time more than 30 days after the bone metastasis date . we excluded from the analysis patients with claims suggesting bone metastases occurring more than 30 days prior to the lung cancer diagnosis ( n = 570 ) . the excluded patients comprising of 2% of overall patients with bone metastases had similar demographic characteristics as the bone metastases patients included in the analysis but were more likely to have unstaged and unspecified histology . we obtained information on date of death using the combined seer registry and medicare claims data . we used the concordant date of death in these two sources , if there was agreement between them . if the seer date of death was missing , we used the medicare date of death . from the seer data , we obtained information on age , gender , race / ethnicity and stage at cancer diagnosis . from the medicare claims data we computed each person 's charlson comorbidity score on the basis of icd-9 diagnosis codes in his / her inpatient records for the 17 medical conditions [ table 1 ] comprising the charlson index in the 12-month period prior to the month of cancer diagnosis . in computing the charlson index , we used the approach described by romano et al , and updated by quan et al . characteristics of patients with lung cancer , total cohort and according to bone metastasis status : seermedicare , july 1 , 1999-december 31 , 2006 we computed the proportion of patients with lung cancer having claims - based evidence of bone metastasis at one year from diagnosis for the overall cohort of lung cancer cases and for subgroups specified by tumor stage at diagnosis . for these computations , we selected patients diagnosed with lung cancer from july 1 , 1999 through december 31 , 2005 , so that these patients would have a potential observation period of at least one year post lung cancer diagnosis . other analyses , described below , were unrestricted with regard to length of observation . we used cox regression to estimate mortality hazards ratios ( hrs ) and 95% confidence intervals ( cis ) among patients with bone metastasis with or without sres , compared to patients without bone metastasis , adjusting for covariates . we also estimated mortality hrs for subgroups specified by initial tumor stage , age , gender and race / ethnicity at cancer diagnosis . confounders were categorical forms of age at cancer diagnosis ( 65 - 69 , 70 - 74 , 75 - 79 , 80 - 84 , 85 + ) , gender ( male , female ) , race ( white , african american , hispanic american , other ) , stage at lung cancer diagnosis ( distant , localized , regional , unstaged ) , and charlson comorbidity score ( 0 , 1 , 2 , 3 + ) . this study was approved by the uab institutional review board and by the nci seer - medicare program . the overall cohort of 126,123 patients were predominantly white ( 85% ) with a median age at diagnosis of 75 years and a median follow - up of 0.6 years [ table 1 ] . eighty - one percent of patients had non - small cell lung cancer . during the study period , 24,820 ( 19.8% ) patients had claims - based evidence of bone metastasis either concurrently with the diagnosis of lung cancer diagnosis ( n = 9,523 ) or during follow - up ( n = 15,297 ) . the median time from cancer diagnosis to bone metastasis was 5.4 months among patients without a bone metastasis at diagnosis . patients with , compared to those without , a bone metastasis were more likely to have distant stage disease ( 72% vs. 41% ) and to have died by the end of the study period ( 95% vs. 82% ) [ table 1 ] . the two groups ( patients with and without bone metastasis ) were similar with regard to age , race / ethnicity , gender , year of diagnosis , histology , comorbidity score and length of follow - up . figure 1 displays the proportion of patients with evidence of bone metastasis at one year post lung cancer diagnosis , according to stage at diagnosis , among the 126,123 patients ( localized , n = 23,821 ; regional , n = 30,365 ; distant , n = 59,319 ; unstaged , n = 12,618 ) diagnosed with lung cancer from july 1 , 1999 through december 31 , 2005 . at one year post diagnosis of lung cancer , the proportion with evidence of bone metastasis was 21% for all stages combined and was 38% for distant , 12% for regional , 11% for unstaged and 5% for localized stages at primary cancer diagnosis . when restricted to patients with non - small cell lung cancer , the proportion with evidence of bone metastasis at one year post diagnosis was 21% for all stages combined and was 39% for distant , 11% for regional , 13% for unstaged and 5% for localized stages . proportion of 126,123 patients with lung cancer who had evidence of bone metastasis within 1 year by stage of the 24,820 patients with a possible bone metastasis , 12,665 ( 51% ) had evidence of a concurrent ( n = 11,015 , 44% ) or subsequent ( n = 1,650 , 7% ) sre ( data not displayed in a table ) . among the 12,665 patients with an sre , most ( n = 10,598 , 84% ) presented with only one skeletal complication at the first diagnosis of an sre . of the 10,598 presenting with a single skeletal complication , 8,357 ( 79% ) had radiotherapy to bone , 1,509 ( 14% ) experienced a fracture , 645 ( 6% ) had spinal cord compression and 87 ( 1% ) had surgery to bone . of 12,665 patients with sre , 2,067 ( 16% ) had more than one skeletal complication at the first diagnosis of an sre . of these 2,067 patients , 969 ( 47% ) had radiation to bone plus fracture , 617 ( 30% ) had radiation to bone plus spinal cord compression , 183 ( 9% ) had radiation to bone plus fracture plus spinal cord compression , 125 ( 6% ) had fracture plus spinal cord compression ; all other combinations accounted for < 3% each . overall , 81% of patients with sre had radiotherapy to bone either diagnosed as the only sre or in combination with other sres . among the 2,481 patients who developed a fracture , regardless of whether it was an sre by itself or in combination with other sres , 1,625 ( 65% ) had a fracture coded as pathological . the most common site of pathological fracture was the spine ( n = 991 ) , followed by hip ( n = 290 ) , femur ( n = 185 ) , humerus ( n = 141 ) , tibia / fibula ( n = 14 ) , and distal radius / ulna ( n = 4 ) . table 2 displays hrs for death in relation to possible bone metastasis and sres , adjusted for age at cancer diagnosis , race / ethnicity , gender , stage at cancer diagnosis and charlson comorbidity score . the hr for risk of death was 2.4 ( 95% ci = 2.4 - 2.5 ) , both for patients with bone metastasis but no sre and for patients with bone metastasis plus sre , compared to patients without bone metastasis . stage - specific analyses indicated that hrs associated with bone metastasis complicated with sre ranged from 2.0 ( 95% ci = 1.9 - 2.0 ) for distant stage disease at cancer diagnosis to 7.3 ( 95% ci = 6.8 - 7.8 ) for localized disease at diagnosis , and bone metastasis without sre ranged from1.9 ( 95% ci = 1.9 - 2.0 ) for distant stage at diagnosis to 6.1 ( 95% ci = 5.7 - 6.5 ) for localized stage table 3 . hazard ratio ( hr ) for death in relation to bone metastasis and other factors among patients with lung cancer : seer - medicare , july 1 , 1999-december 31 , 2006 adjusted hazard ratio ( hr ) for death in relation to bone metastasis and skeletal - related factors ( sres ) among men with lung cancer , by stage at diagnosis : seer - medicare , july 1 , 1999-december 31 , 2006 hrs for death in relation to bone metastasis in white patients were similar to those for african american patients ( white : hr = 2.4 , 95% ci = 2.4 - 2.5 ) ; african american , hr = 2.3 , 95% ci = 2.2 - 2.4 ) ( data not displayed in a table ) . hrs for death among men and women were also similar with regard to bone metastasis ( men , hr = 2.4 , 95% ci = 2.4 - 2.5 ; women , hr = 2.5 , 95% ci = 2.4 - 2.5 ) . we found a positive association between bone metastasis and with bone metastasis complicated with sre and mortality in all categories of age groups ( 65 - 69 , 70 - 74 , 75 - 79 , 80 - 84 , 85 + ) . patients with , compared to those without , a bone metastasis were more likely to have distant stage disease ( 72% vs. 41% ) and to have died by the end of the study period ( 95% vs. 82% ) [ table 1 ] . the two groups ( patients with and without bone metastasis ) were similar with regard to age , race / ethnicity , gender , year of diagnosis , histology , comorbidity score and length of follow - up . figure 1 displays the proportion of patients with evidence of bone metastasis at one year post lung cancer diagnosis , according to stage at diagnosis , among the 126,123 patients ( localized , n = 23,821 ; regional , n = 30,365 ; distant , n = 59,319 ; unstaged , n = 12,618 ) diagnosed with lung cancer from july 1 , 1999 through december 31 , 2005 . at one year post diagnosis of lung cancer , the proportion with evidence of bone metastasis was 21% for all stages combined and was 38% for distant , 12% for regional , 11% for unstaged and 5% for localized stages at primary cancer diagnosis . when restricted to patients with non - small cell lung cancer , the proportion with evidence of bone metastasis at one year post diagnosis was 21% for all stages combined and was 39% for distant , 11% for regional , 13% for unstaged and 5% for localized stages . proportion of 126,123 patients with lung cancer who had evidence of bone metastasis within 1 year by stage of the 24,820 patients with a possible bone metastasis , 12,665 ( 51% ) had evidence of a concurrent ( n = 11,015 , 44% ) or subsequent ( n = 1,650 , 7% ) sre ( data not displayed in a table ) . among the 12,665 patients with an sre , most ( n = 10,598 , 84% ) presented with only one skeletal complication at the first diagnosis of an sre . of the 10,598 presenting with a single skeletal complication , 8,357 ( 79% ) had radiotherapy to bone , 1,509 ( 14% ) experienced a fracture , 645 ( 6% ) had spinal cord compression and 87 ( 1% ) had surgery to bone . of 12,665 patients with sre , 2,067 ( 16% ) had more than one skeletal complication at the first diagnosis of an sre . of these 2,067 patients , 969 ( 47% ) had radiation to bone plus fracture , 617 ( 30% ) had radiation to bone plus spinal cord compression , 183 ( 9% ) had radiation to bone plus fracture plus spinal cord compression , 125 ( 6% ) had fracture plus spinal cord compression ; all other combinations accounted for < 3% each . overall , 81% of patients with sre had radiotherapy to bone either diagnosed as the only sre or in combination with other sres . among the 2,481 patients who developed a fracture , regardless of whether it was an sre by itself or in combination with other sres , 1,625 ( 65% ) had a fracture coded as pathological . the most common site of pathological fracture was the spine ( n = 991 ) , followed by hip ( n = 290 ) , femur ( n = 185 ) , humerus ( n = 141 ) , tibia / fibula ( n = 14 ) , and distal radius / ulna ( n = 4 ) . table 2 displays hrs for death in relation to possible bone metastasis and sres , adjusted for age at cancer diagnosis , race / ethnicity , gender , stage at cancer diagnosis and charlson comorbidity score . the hr for risk of death was 2.4 ( 95% ci = 2.4 - 2.5 ) , both for patients with bone metastasis but no sre and for patients with bone metastasis plus sre , compared to patients without bone metastasis . stage - specific analyses indicated that hrs associated with bone metastasis complicated with sre ranged from 2.0 ( 95% ci = 1.9 - 2.0 ) for distant stage disease at cancer diagnosis to 7.3 ( 95% ci = 6.8 - 7.8 ) for localized disease at diagnosis , and bone metastasis without sre ranged from1.9 ( 95% ci = 1.9 - 2.0 ) for distant stage at diagnosis to 6.1 ( 95% ci = 5.7 - 6.5 ) for localized stage table 3 . hazard ratio ( hr ) for death in relation to bone metastasis and other factors among patients with lung cancer : seer - medicare , july 1 , 1999-december 31 , 2006 adjusted hazard ratio ( hr ) for death in relation to bone metastasis and skeletal - related factors ( sres ) among men with lung cancer , by stage at diagnosis : seer - medicare , july 1 , 1999-december 31 , 2006 hrs for death in relation to bone metastasis in white patients were similar to those for african american patients ( white : hr = 2.4 , 95% ci = 2.4 - 2.5 ) ; african american , hr = 2.3 , 95% ci = 2.2 - 2.4 ) ( data not displayed in a table ) . hrs for death among men and women were also similar with regard to bone metastasis ( men , hr = 2.4 , 95% ci = 2.4 - 2.5 ; women , hr = 2.5 , 95% ci = 2.4 - 2.5 ) . we found a positive association between bone metastasis and with bone metastasis complicated with sre and mortality in all categories of age groups ( 65 - 69 , 70 - 74 , 75 - 79 , 80 - 84 , 85 + ) . we found that about 19.8% of patients 65 years or older with newly diagnosed lung cancer included in the seer program from july 1999 to december 2005 presented with or subsequently had evidence of a possible bone metastasis as indicated by medicare claims . sres were common , occurring in 51% of these patients . among patients with a potential follow - up time of one year post lung cancer diagnosis , the proportion with bone metastasis was 21% overall and 38% for distant stage of lung cancer . having a possible bone metastasis was associated with mortality among patients with lung cancer ; the association was similar for bone metastasis complicated with an sre and for bone metastasis without an sre . the skeleton is a frequent site of metastasis in patients with non - small - cell lung cancer which is the most common type of lung cancer . about 85% of lung cancer cases included in the seer program have a non - small cell histology . in our data , bone metastasis may be asymptomatic at onset and thus , are likely to be under - diagnosed resulting in inaccurate staging of lung cancer at diagnosis . studies report that the prevalence of bone metastasis among patients who report bone pain is almost similar to patients who did not report bone pain ( 27 - 32% vs. 16 - 27% ) . prior to 1991 , several studies found skeletal metastases in non - small cell lung cancer to range from 8% to 34%.[2531 ] with the advent of newer imaging techniques such as the positron emission tomography ( pet ) in the 1990s , bone metastasis has been noted to range from 24% to 30% for non - small cell lung cancer . the preceding results are consistent with our finding that 21% of medicare non - small cell lung cancer patients had possible bone metastasis within a year of lung cancer diagnosis . , the seer program reports that 56% of lung cancer patients were classified as having distant stage at the time of disease diagnosis . in the present study , bone metastases are common in patients with distant stage of lung cancer . in the present study , we found that 38% of patients with distant stage lung cancer had possible bone metastasis within a year of their cancer diagnosis . this result is consistent with findings from other studies which have reported that 30 - 40% of lung cancer patients with distant metastatic disease had a bone metastasis at diagnosis or subsequently developed a bone metastasis . in addition , autopsies of patients dying of lung cancer indicated that up to 30 - 40% had evidence of metastatic bone disease . in the present study , we found that 51% of patients with possible bone metastasis had one or more sres . this result agrees with findings from a retrospective observational study using data from a health insurance claims database in the us which reported that sres occurred in 55% of 534 lung cancer patients with bone metastasis . clinical trials of lung cancer have found that sres occurred in 48% to 50% of patients with bone metastasis . in our study , radiotherapy ( 81% of patients with sre ) to bone was the most common sre followed by fracture , spinal cord compression and surgery to bone . we observed that among patients with fractures , 65% ( or 13% of patients with sre ) had a pathological fracture with the spine as the most common site of occurrence followed by pelvis and extremities . other studies report that 7 - 10% of non - small lung cancer patients with sres developed pathological fractures . our results indicated that having a bone metastasis was associated with mortality among lung cancer patients . the lack of an additional effect of sre on mortality among patients with bone metastasis may be due to the short survival time in lung cancer patients compared to other tumor types . our companion studies of breast and prostate cancer , cancers with relatively longer survival time , found stronger associations for bone metastasis complication by sre than for bone metastasis without sre . our finding of a stronger association of bone metastasis and for bone metastasis complicated with sre for patients with localized stage lung cancer than for those with distant metastatic lung cancer at diagnosis may reflect the low mortality among patients with early stage disease and no bone metastasis . the strengths of the study include the use of the large population - based seer cancer registry data with pathologically confirmed cancer and the focus on lung cancer in the elderly . the combined cancer registry and medicare data provide a unique opportunity to evaluate the clinical progress of lung cancer . other advantages of the seer data include the continuity of data over time and the quality control measures instituted to ensure completeness of case ascertainment in the participating cancer registries . the main limitations of our study stem from the characteristics of the seer - medicare data . our study was limited to patients aged 65 years or older diagnosed with lung cancer between 1999 and 2005 , who had medicare part a and b coverage and who resided in one of the seer geographic areas . medicare data do not include claims of health maintenance organization enrollees , care provided in other settings like the veterans administration , reimbursement of covered services not captured by the medicare data such as out - of - pocket expenditures or coverage provided by medigap , or long term care at home or nursing homes . therefore , our results will not be generalizable to patients less than 65 years of age and may not represent patients aged 65 years and older who resided in geographic areas not included in the seer program who had only had medicare part a coverage or who were enrolled in medicare advantage plan . second , we have not validated the procedures used to identify bone metastasis in this study . we identified possible bone metastasis from inpatient claims or outpatient claims paired with selected procedure codes , an approach designed to reduce false positives . despite this approach it is also possible that we may have missed true cases in instances where claims were not obtained from the relevant facilities or where claims were obtained but not coded as bone metastasis . third , it is possible that the mortality differences we noted for bone metastasis or bone metastasis plus sre compared to those without these outcomes may be due to other confounding factors such as treatment factors or concurrent comorbid conditions that were not included in our analysis . finally , the data do not include information on functional status of patients , a predictor of outcomes . in conclusion , the present study found that having a bone metastasis as indicated by medicare claims , was associated with mortality among patients with lung cancer . we found no difference in mortality between patients with bone metastasis complicated by sre and patients with bone metastasis but without sre .

background : to quantify the impact of bone metastasis and skeletal - related events ( sres ) on mortality among older patients with lung cancer.materials and methods : using the linked surveillance , epidemiology and end results - medicare database , we identified patients aged 65 years or older diagnosed with lung cancer between july 1 , 1999 and december 31 , 2005 and followed them to determine deaths through december 31 , 2006 . we classified patients as having possible bone metastasis and sres using discharge diagnoses from inpatient claims and diagnoses paired with procedure codes from outpatient claims . we used cox regression to estimate mortality hazards ratios ( hr ) among patients with bone metastasis with or without sre , compared to patients without bone metastasis.results:among 126,123 patients with lung cancer having a median follow - up of 0.6 years , 24,820 ( 19.8% ) had bone metastasis either at lung cancer diagnosis ( 9,523 , 7.6% ) or during follow - up ( 15,297 , 12.1% ) . sres occurred in 12,665 ( 51% ) patients with bone metastasis . the hr for death was 2.4 ( 95% ci = 2.4 - 2.5 ) both for patients with bone metastasis but no sre and for patients with bone metastasis plus sre , compared to patients without bone metastasis.conclusions:having a bone metastasis , as indicated by medicare claims , was associated with mortality among patients with lung cancer . we found no difference in mortality between patients with bone metastasis complicated by sre and patients with bone metastasis but without sre .

pubmed

fiber - reinforced posts have superior advantages as compared with cast posts , generally regarding their esthetics , physical properties and their modulus of elasticity , which is similar to root dentin.1 posts are generally cemented with resin cements . proper selection of resin cements influence the retention of posts and the success of restoration.2,3 the studies indicate that failure of glass fiber - reinforced post - and - core restorations often occurs because of debonding at the post - resin interface and/or resin - dentin interface as a result of bond deficiencies.4,5 self - adhesive resin cements are the least investigated group among the resin cements . application steps are reduced by removing the pre - treatment procedures of the dentin.6 the organic matrix consists of newly developed phosphoric acid methacrylate . the adhesive mechanism is based on the chemical reaction between the hydroxyapatite crystals of the tooth and phosphoric acid monomers.7 self - adhesive cements are also resistant to moisture and able to release fluoride comparable to glass ionomer cements.8 also , they have satisfactory esthetic and mechanical properties.9 because of these positive features , they are used for the adhesive cementation of fixed prostheses and posts . removing the smear layer that contains gutta - percha remnants , microorganisms , and infectious dentin is necessary for the penetration of the adhesive system and resin cement into the dentin tubules.10,11 self - adhesive resin cements can not completely remove the smear layer.12,13 this remaining smear layer could cause a lower bond strength for self - adhesive resin cements as compared with an etch and rinse adhesive system.14 several chemical substances that are used as irrigants during the biomechanical preparation can remove the smear layer and alter the characteristics of the dentin substrate . sodium hypochlorite ( naocl ) has the capacity to dissolve organic tissues and neutralize toxic products and also has an antimicrobial action15 whereas ethylene diamine tetra acetic acid ( edta ) chelates calcium ions and promotes dentin demineralization and smear layer removal.16 sodium hypochlorite , ethylene diamine tetra acetic acid , orthophosphoric acid ( h3po4)10,17 and different laser systems18 are used to increase the micromechanical retention of the cement by removing the smear layer . no study has been conducted on comparing traditional irrigation systems and photodynamic diode laser performance on the bond strength of self - adhesive cements using fiber posts . lasers can be used to vaporize tissues , remove and modify the smear layer and eliminate residual tissue in the main canal.19,30 diode lasers can make alterations on dentin and may increase the bond strength to root canal dentin.20 alfredo et al.20 found increased bond strength using with 980 nm diode laser and attributed to the alterations caused by the laser on dentin surface , such as fissures and topographic changes . laser treatment can be a valuable instrument for the removal or modify of the smear layer in root dentin , as a debridement device during post space preparation . the aim of this study was to compare the effect of the photodynamic diode laser and other smear layer removal methods on bond strength of the self - adhesive cement . the null hypothesis was that no significant differences would be found among push - out bond strength values after different root canal surface treatments . in this study , 55 maxillary central incisors , which were extracted due to periodontal problems , were used . the teeth were cleaned of both calculus and soft tissues and stored in physiological saline before proceeding . the crowns of each tooth were removed at the level of the cemento - enamel junction with a high - speed diamond bur ( zr diamonds , brasseler gmbh , lemgo , germany ) under water spray . the working length of each root canal was established 1 mm short of the apical foramen with a size 15 k - type file ( mani inc . , the canals were prepared with a rotary system ( x - smart , dentsply , maillefer , ballaigues , switzerland ) . all teeth were instrumented using a set of rotary instruments ( pro taper , dentsply , switzerland ) to the size of f3 ( multi tapered # 30 ; finishing file ) by the same operator . the canals were irrigated at every change of instrument with 2 ml 5% hypochlorite solution and 5 ml for the final irrigation . the prepared canals were filled with matching tapered gutta percha points ( dentsply , maillefer , france ) and a resin sealer ( ah plus , dentsply , konstanz , germany ) . the coronal aspect of the gutta percha was removed with a heated probe ( gutta cut , vdw gmbh , munich , germany ) . after storing the teeth in a humid medium for 1 week at 37 , 14 ( diameter 1.4 mm ) glass fiber reinforced composite post ( snowlight post , abrasive technology , oh , usa ) with matching drill . after the post space preparations , the teeth were randomly divided into 5 experimental groups of 11 teeth each . the post spaces were treated with the following : group 1 : 5-ml 0.9% physiological saline ( nacl ) , with a disposable syringe for 1 minutegroup 2 : 5-ml 5.25% sodium hypochlorite ( naocl ) , with a disposable syringe for 1 minutegroup 3 : 5-ml 17% edta with a disposable syringe for 1 minutegroup 4 : etching with 37% orthophosphoric acid for 30 secondsgroup 5 : photodynamic diode laser ( helbo theralite laser , helbo photodynamic systems gmbh & co. , germany ) . group 1 : 5-ml 0.9% physiological saline ( nacl ) , with a disposable syringe for 1 minute group 2 : 5-ml 5.25% sodium hypochlorite ( naocl ) , with a disposable syringe for 1 minute group 3 : 5-ml 17% edta with a disposable syringe for 1 minute group 4 : etching with 37% orthophosphoric acid for 30 seconds group 5 : photodynamic diode laser ( helbo theralite laser , helbo photodynamic systems gmbh & co. , germany ) . bacteria - sensitive , light - active dye solution helbo endo blue ( phenothiazine-5-ium , 3 , 7-bis ( dimethylamino)- , chloride ) ( helbo photodynamic systems gmbh & co. , germany ) photosensitizer was applied for 1 minute into the post spaces and rinsed with water in group 5 . after rinsing in group 5 , a laser probe ( helbo 3d endo probe , helbo photo - dynamic systems gmbh & co. , germany ) was placed near the apex and irradiated for 1 minute with helbo theralite laser with a wavelength of 660 nm , an output power of 75 mw . the laser was delivered through a flexible fiber optic tip curved at an angle of 60. the fiber design permits a 3-dimensional exposure of the area , emitting light at the tip and from the lateral sides , thus leading to even light distribution both vertically and horizontally . ( r = 1.4 mm ) snowlight posts were luted with self - adhesive resin cement ( clearfil sa cement , kuraray medical inc . , japan ) . each root was sectioned perpendicular to its long axis to create 1 mm thick specimens with a 0.3 mm thickness slow - speed diamond saw ( buehler , usa ) at the coronal third of the root . one section of the 1 mm thick specimen was prepared from each tooth ( fig . push - out bond strength was tested using a universal testing machine ( ags - x , schimadzu co. , kyoto , japan ) at a crosshead speed of 1 mm / min until post debonding occurred . the specimens were loaded with a 1 mm diameter cylindrical tip in an apical - coronal direction to push - out the post toward the wider part of the root slice to avoid the taper limitation . the loads at failure were recorded as n and the bond strengths were expressed in mpa ; n was divided by the area of the bonded interface , which was calculated using the following formula : a = 2rh ( where a is the area of the bonded interface , = 3.14 , r is the post radius , and h is the thickness of the specimen in mm ) . the debonded specimens investigated under the stereomicroscope at original magnification 40 for the failure interface as between dentin - cement , post - cement and both dentin - cement and post - cement ( fig . 3 and fig . one tooth from each group was processed for sem analysis to observe the changes on root canal surfaces after the post space treatments . the two halves of the tooth were dehydrated and coated with gold for sem analysis . all calculated bond strengths were analyzed using one - way analysis of variance ( anova ) and tukey honestly significant differences ( hsd ) tests with spss 16.0 ( spss , inc . , chicago , il , usa ) ( =.05 ) . the mean values and standard deviations of the test groups are presented in table 1 . bond strength values showed that the higher results were obtained for the helbo theralite laser and 37% orthophosphoric acid groups . although nacl , naocl , edta , and h3po4 groups showed post - cement and cement - dentin failure both ( mixed failure ) ; helbo theralite laser showed post - cement failure at the cross sectional image of the dentin discs ( fig . the null hypothesis was rejected because the results of this study revealed that different smear layer removal methods from root canal dentin affect the bond strength of a fiber post cemented with adhesive resin to dentin ( p<.05 ) . the smear layer produced by post space preparation should be removed according to some authors owing to the increase of antibacterial effects21,22 and also adhesive effectiveness23 as it becomes porous and the authors insisted that the smear layer causes a weak adherent interface between cement and dentin . on the other hand , other authors insist upon retaining smear layer for adaptation of the materials to root surface.24,25 interestingly , in photodynamic diode laser group , bond strength was higher than the other groups except h3po4 group without the removal of smear layer . it may arise from the fact that collagen fibrils structure does not change after lasing . by conserving most of their intrafibrillar mineral , they are more stable and less affected by dehydration , which improves the infiltration of the luting agent . sodium hypochlorite increases the penetration of the monomers into the dentinal tubules by removing organic components , and breaks down to sodium chloride and oxygen . this is because broken down oxygen inhibits interfacial polymerization of resin bonding materials.24,26 on the other hand , 5% naocl alone did not remove the smear layer as effectively as found in other studies.27,11 also the residual naocl solution affect the change in redox potential of the bonding agent.11 others have found naocl and nacl could not remove the smear layer , which can act as a diffusion barrier on the dentin and reduce dentin permeability for bonding agents.2,28 results from another study with 5.25% naocl and 0.9% saline did not increase bond strength of luting cement,29 as shown in this study . a study conducted by takeda et al.30 mentioned that 17% edta alone was not effective in removing smear remnants as it did in this study ; in the same study , authors used 6% orthophosphoric acid to remove the smear layer , but in contrast to this study , it was not useful . , other authors showed that 37% orthophosphoric acid improved retention by eliminating dentin mineral content.31 dual irrigation methods , such as a combination of 17% edta and 5.25% naocl are effective methods for removing the smear layer,32 but this method results in sporadic erosion and dissolution of the dentin.33,34 moreover , it was shown that these solutions did not have a good ability in preparing complicated root canals.35 - 37 therefore , dual irrigation methods were not used for this study . when the push - out tests were examined , generally , failures of bonding were observed between resin and dentin.38,39 in the present study , only the diode laser group showed post - cement failure , the other groups showed mixed failures ( fig . 3 and fig . it was shown that using the photodynamic diode laser in post space increased the bond strength of resin cement to dentin interface , as compared with other surface treatment methods . it is important to improve the bonding effectiveness of the resin and dentin for post restoration success with removal of the smear layer and formation of the resin - dentin interdiffusion zone.2,40 on the contrary , demiryrek et al.27 used acetone based agent , which could not remove the smear layer and sealer remnants effectively on radicular dentin surfaces , to treat post space treatment and found to be more effective compared with edta , citric acid and orthophosphoric acid . it was concluded that removal of the smear layer and opening of dentinal tubules are not recommended when a self - etching / self - priming adhesive system is used.27 laser devices have been used in root canal treatment since the early 1970s,41 but their acceptance has been very slow . general causes of dissatisfaction have included thermal damage caused by the laser photonic energy.42 therefore , the photonic energy should be at its lowest possible level to eliminate thermal damage . it has been reported that as the diode - laser treatment ( = 810 nm ) partially opened dentinal tubules , signs of fusions and temperature increase at the root surfaces were observed.43 on the other hand , a study conducted by altundasar et al.42 after irradiation with a 980-nm diode laser ( gaalas ) ( with 2 times more power ) had opposite results and two - fold increase in temperature at the root surface was observed . to avoid the temperature rise , lower irradiation power was used for this study ( = 660 nm ) , and it was enough to improve bonding effectiveness at the cement - dentin interface . the results of this study suggest the following : ( 1 ) naocl ( 5.25% ) and naci ( 0.9% ) were not effective in removing the smear layer when used as a final irrigation . ( 2 ) according to the sem evaluation , orthophosphoric acid ( 37% ) and edta ( 17% ) were more effective methods in removing the smear layer than naocl , naci irrigants , and helbo theralite laser . however , helbo theralite laser and orthophosphoric acid were more effective at cement - dentin interface than edta for bond strength of self - adhesive cements . therefore , it may be more effective to modify the smear layer when a self - adhesive system is used .

purposethe purpose of this study was to compare the effect of a diode laser and traditional irrigants on the bond strength of self - adhesive cement.materials and methodsfifty - five incisors extracted due to periodontal problems were used . all teeth were instrumented using a set of rotary root canal instruments . the post spaces were enlarged for a no.14 ( diameter , 1.4 mm ) snowlight ( abrasive technology , oh , usa ) glass fiber reinforced composite post with matching drill . the teeth were randomly divided into 5 experimental groups of 11 teeth each . the post spaces were treated with the followings : group 1 : 5 ml 0.9% physiological saline ; group 2 : 5 ml 5.25% sodium hypochlorite ; group 3 : 5 ml 17% ethylene diamine tetra acetic acid ( edta ) , group 4 : 37% orthophosphoric acid and group 5 : photodynamic diode laser irradiation for 1 minute after application of light - active dye solution . snowlight posts were luted with self - adhesive resin cement . each root was sectioned perpendicular to its long axis to create 1 mm thick specimens . the push - out bond strength test method was used to measure bond strength . one tooth from each group was processed for scanning electron microscopic analysis.resultsbond strength values were as follow : group 1 = 4.15 mpa ; group 2 = 3.00 mpa ; group 3 = 4.45 mpa ; group 4 = 6.96 mpa ; and group 5 = 8.93 mpa . these values were analysed using one - way anova and tukey honestly significant difference test ( p<.05 ) . significantly higher bond strength values were obtained with the diode laser and orthophosphoric acid ( p<.05 ) . there were no differences found between the other groups ( p>.05).conclusionorthophosphoric acid and edta were more effective methods for removing the smear layer than the diode laser . however , the diode laser and orthophosphoric acid were more effective at the cement dentin interface than the edta , therefore , modifying the smear layer may be more effective when a self - adhesive system is used .

pubmed

in recent years , concerted efforts to identify , describe , and quantify the bacterial communities of the mammalian gastrointestinal tract have begun to bear fruit . many international consortia and collaborations have been established to answer the question : who is there? hot on the heels of this is the query : what are they doing there? this second , crucial , and rather more complex question is yet to be satisfactorily tackled , but fascinating glimpses of what lies ahead have been caught . the human microbiome project in the usa and the metagenomics of the human intestinal tract ( metahit ) consortium in europe are only two of the major initiatives with the purpose of characterizing the microbial communities that inhabit multiple sites in the human body ( skin , nasal , oral , urogenital , and intestinal flora ) and to look for correlations between the changes in the microbiome and prevalence of diseases . collaborations to investigate if there is a common core of bacterial species shared among all or the majority of human beings have also been initiated , resulting in some interesting paradigms . in the uterus , the developing fetus is believed to be completely devoid of bacterial flora . the same can not be said of bacterial influence on the growing fetus . upon birth , the intestinal tract of the microbiota is first acquired in the birth canal during delivery and then through breast milk . fecal microbial profiles of infants show a striking similarity to maternal vaginal and breast milk bacterial profiles ( 4 ) . during the course of early childhood , microbial composition changes with age and diet ( 5 , 6 ) . from the evolutionary point of view , the character of gut microbiota strongly depends on the main nutritional source , separating bacterial profiles of omnivores , carnivores , and herbivores ( 7 ) several groups have been studying the convergence and variability of the human gut microbiome . these studies were normally performed on a small group of healthy individuals . however , even though the sample size has been minimal , there were some major accomplishments , setting the scene for further large - scale metagenomic approaches . one initial observation was that the human gut flora belongs mainly to only two phyla firmicutes ( mostly represented by clostridia ) and bacteroidetes , with a smaller representation of bacteria belonging to proteobacteria and actinobacteria ( 8) . the second observation brought about the discovery of archaea , mostly represented by the methanogenic methanobrevibacter smithii ( 8 , 9 ) . what is interesting is that the diversity of archaea is but a fraction of the expansiveness of the bacterial species colonizing the gut . nevertheless , it has been argued that archaea may yet contribute to the health status of the human gut , due to the unique nature of their metabolic prowess ( 10 ) . the composition of the bacterial flora seems to strongly depend on the environment and the maternal input during delivery and upon breast feeding . in humans , members of the same family were shown to have bacterial communities that were more similar to each other than to unrelated individuals ( 11 ) . moreover , this feature was host genotype independent because there was no significant difference detected in the degree of similarity between samples collected from mono- and dizigotic twins . this likely suggests that the environment and maternal flora , rather than genetic factors alone , have greater impact on establishing bacterial communities in a new individual . this observation confirmed the statement that the largest amount of variability between the gut bacterial flora could be explained by intersubject differences ( 8) . recent second generation , high throughput studies have revealed more details about the human bacterial communities . one major study specifically aimed at assessing whether a common bacterial core shared by all or at least a majority of humans could be determined ( 12 ) . fecal material collected from a large cohort of individuals in different countries was subjected to metagenomic analysis . the bacterial groups that were present in the samples in vast numbers were bacteroidetes and dorea / eubacterium / ruminococcus . only 18 species were present in all individuals examined , while 57 species were seen in more than 90% of samples and around 50% shared 75 species . although these numbers are higher than routinely observed , they are indicative of the diversity of the human microbiome , while at the same time emphasizing the extent of the interindividual differences . to obtain a better picture of the role or function of the bacterial communities , failing this , concerted efforts have been made to first identify genes that are encoded by intestinal microbiota and to place their relevance within the context of gut function . more than 3 million open reading frames ( orf ) of bacterial genes have been , thus , identified , potentially coding for as many genes . however , the majority of these genes could only be found in very few samples , again confirming the huge variability between individuals . only around 300,000 genes were found in more than half of the individuals queried , denoting commonly shared microbial genes . each individual subject carried around 500,000 bacterially encoded genes , among which the majority were rare genes , shared by less than half of the group sampled ( 12 ) . around 2.5% of the microbially encoded genes could be assigned to the functional group of enzymes involved in carbohydrate metabolism , and again these sets of genes were more similar within individuals of the same family ( 11 ) . attention has also been drawn to the minimal gut microbiome genes that are present in most bacteria and code for functions that are necessary for a bacterium to survive in the gut . this set of genes can be divided into housekeeping genes ( amino acid synthesis , central carbon metabolism , and protein complexes such as rna / dna polymerases ) and those involved in gut - specific functions ( fermentation and adhesion ) . however , large portions of the orf / genes were of unknown functions ( 12 ) . on the other hand , efforts have been made to identify the minimal gut metagenome coding for functions involved in the homeostasis of the whole ecosystem , which are present in most individual host samples . most of these common bacterial genes are involved in the digestion of complex sugars and their subsequent fermentation . these genes were coded not by the most abundant bacteria but rather by species present in low number , suggesting that even a relatively small bacterial group might be crucial for the establishment of a well functioning gut , as exemplified by archaea . one must , however , note that the minimal gut microbiome and minimal gut metagenome are not mutually exclusive , and there are many genes that will belong to both groups , being necessary both for the survival of the bacterium as well as for gut function . a higher degree of redundancy at the gene level rather than taxonomic level suggests that the core microbiome should exhibit shared bacterial functions that need to operate to ensure survival and successful symbiosis in the gut ( 11 ) . in a more recent study , inching closer to marrying microbial profiles and defined functions , arumugam et al . ( 2011 ) have proposed a relatively new concept in the classification of the gut microbiome ( 13 ) . in this study , using stool samples representing different ethnicities , countries , and continents , the authors chanced on a perplexingly simple finding : despite a large and dynamic bacterial community , the combination of the microbes in each individual appeared to be systematically organized into clusters termed enterotypes . even more surprising was that only three enterotypes were identified based on different clusters of bacterial species . . a unique set of properties have been assigned to each enterotype , notably that there is no geographical demarcation , and further , no correlation between presence of a given enterotype with sex , age , or body mass index ( bmi ) could be shown . the three enterotypes can be identified and viewed on various levels prevalence of bacteria , methods of harvesting energy , and overrepresentation of certain enzymatic pathways . for instance , enterotype 1 is dominated by bacteroides that derive energy mostly from fermentation of sugars and is enriched in genes coding for biotin biosynthesis . the second enterotype is driven by prevotella and can be characterized by enrichment of genes coding for thiamine biosynthesis and harvesting energy from biodegradation of mucin glycoproteins . enterotype 3 is mostly enriched in ruminococcus , also known to be able to degrade mucins . this enterotype is , however , enriched in genes coding heme biosynthesis in contrast to the prevotella enterotype . the phylogenetic structure in this instance appears to be indicative of specific functional properties ( summarized in fig . the contemporary concept of enterotypes was given a boost recently when another study independently confirmed the existence of two enterotypes in a population of 98 individuals ( 6 ) . the study further found that the enterotype was associated with long - term diet as shown by transient feeding experiments . hence , although significant changes in the microbiota can occur rapidly on dietary change , no temporary shifts in enterotypes are forthcoming , that is the enterotype does not seem to budge easily . the human gut flora has recently been classified as belonging to one of three different clusters termed enterotypes . this diagram postulates that properties of the microbiome unique to each enterotype ( hatched - hexagon , hatched - triangle and hatched - rhombus ) give rise to enterotype - specific effects . each enterotype likely also harbors common essential functions that may then facilitate bacterial survival in the gut . the role of the individual 's immune system , nutritional input during early childhood , lifestyle , and the exposure to other gut microbial ecologies during the formative years of the microbiome may play a role in the establishment of the individual 's enterotype . the use of genetically identical individuals ( twin registries ) , patient cohorts , and materials collected from longitudinal studies may aid in answering some of these queries . finally , although exciting , the enterotype postulate does require further , robust proof for universal acceptance and use . it is altogether heartening to see that the findings of this study do not preclude the existence of additional enterotypes . the results obtained from analyzing the bacterial communities of the human gut and their roles in host physiology suggest transience in the gut microbiota , mechanisms of which remain to be discovered ( 4 , 6 ) . the 16s rna - based methods of identification still have a number of limitations including incomplete coverage of the sequenced gene fragments and problems of correct alignment to known bacterial genomes . one predictable problem in trying to fuse function and identity is of course aligning the bacterial presence with respective gene expression patterns . this conundrum has yet to be seriously tackled , although limited steps to redress this problem are underway . ( 2011 ) in a metatranscriptomic study of 10 volunteers found microbial mrna expression to have a stable representation of genes involved in nutrient processing , energy harvest , and biosynthesis of cellular components . finally , this study also found expression of small rnas that may have higher regulatory properties . although in no way conclusive , this study highlights an additional criterion of gut health assessment to complement with the concept of enterotype that is presently available only at the metagenomic level ( 14 ) . to facilitate rapid , easy , and non - invasive procurement of samples , fecal material the fecal microbiota is used to infer the entire gut microflora , and the lack of information about the potential difference between specific anatomical sites and viability of the bacteria , although glaringly obvious , is usually ignored . microbiota composition has been to compare microbial communities residing on intestinal mucosal surfaces with those found in the feces . the conclusions from such studies point toward variabilities in microbial composition between sample sites , indicating that looking at bacterial communities in the fecal sample may not be as representative for the gut microbiome as we would like to see it ( 8) . the results obtained from fecal - derived studies have , nevertheless , given added credence to the long - held view that different physiological properties may be influenced by different microbial manifestations , for example varied microbiomes or enterotypes . this may lend itself to some plasticity in the bacterial clusters , while retaining core enzymatic and biochemical functions that will reflect the individual enterotypes . the current definition of enterotypes will no doubt undergo further refinement over the next few years . this is likely to provide a better picture of microbial properties that impact on health status , xenobiotic , and nutritional metabolism and perhaps act as an indicator of disease susceptibilities . ideally , this information may in the future aid the choice of preferred therapeutic modules for selected diseases . although our current knowledge of enterotypes is far from complete , the potential uses of this new microbiome classification are tantalizing and set a standard to which the disease - associated bacterial flora or microbiome may be compared . the results obtained from analyzing the bacterial communities of the human gut and their roles in host physiology suggest transience in the gut microbiota , mechanisms of which remain to be discovered ( 4 , 6 ) . the 16s rna - based methods of identification still have a number of limitations including incomplete coverage of the sequenced gene fragments and problems of correct alignment to known bacterial genomes . one predictable problem in trying to fuse function and identity is of course aligning the bacterial presence with respective gene expression patterns . this conundrum has yet to be seriously tackled , although limited steps to redress this problem are underway . ( 2011 ) in a metatranscriptomic study of 10 volunteers found microbial mrna expression to have a stable representation of genes involved in nutrient processing , energy harvest , and biosynthesis of cellular components . finally , this study also found expression of small rnas that may have higher regulatory properties . although in no way conclusive , this study highlights an additional criterion of gut health assessment to complement with the concept of enterotype that is presently available only at the metagenomic level ( 14 ) . to facilitate rapid , easy , and non - invasive procurement of samples , fecal material the fecal microbiota is used to infer the entire gut microflora , and the lack of information about the potential difference between specific anatomical sites and viability of the bacteria , although glaringly obvious , is usually ignored . microbiota composition has been to compare microbial communities residing on intestinal mucosal surfaces with those found in the feces . the conclusions from such studies point toward variabilities in microbial composition between sample sites , indicating that looking at bacterial communities in the fecal sample may not be as representative for the gut microbiome as we would like to see it ( 8) . the results obtained from fecal - derived studies have , nevertheless , given added credence to the long - held view that different physiological properties may be influenced by different microbial manifestations , for example varied microbiomes or enterotypes . this may lend itself to some plasticity in the bacterial clusters , while retaining core enzymatic and biochemical functions that will reflect the individual enterotypes . the current definition of enterotypes will no doubt undergo further refinement over the next few years . this is likely to provide a better picture of microbial properties that impact on health status , xenobiotic , and nutritional metabolism and perhaps act as an indicator of disease susceptibilities . ideally , this information may in the future aid the choice of preferred therapeutic modules for selected diseases . although our current knowledge of enterotypes is far from complete , the potential uses of this new microbiome classification are tantalizing and set a standard to which the disease - associated bacterial flora or microbiome may be compared . the former is a program to extract , preserve , and build energy reserves , whereas the latter will identify and eliminate elements detrimental to the long - term survival of the organism . in superorganisms such as ourselves ( and other eukaryotes ) , the resident microbiota is able to influence both types of metabolic programs . here , we would like to discuss the current conundrums facing researchers exploring the microbial basis of adipocity . we think that it is wise to discuss the phenomenal progress made thus far , and emphasize the legitimate concerns that still plague the interpretation of the results . the role of the microbiota in regulation of host energy balance and metabolism can be studied at various levels . by introducing a semi - synthetic high fat diet ( hfd ) , containing a large amount of fat as well as sugars , one can induce weight gain and subsequent development of metabolic diseases ( 1517 ) . the composition of the bacterial flora can then be monitored simultaneously with changes in the animal 's body weight . one very powerful tool to address this question is germ - free mice raised in an environment completely devoid of bacteria . by simply comparing the physiology of germ - free mice with that of conventionally raised animals more interrogative methods rely on using germ - free mice that have been colonized with one specific species of bacteria ( monocolonization ) , a whole group of bacteria , or the entire bacterial flora isolated from conventionally raised animals ( coventionalization ) . experiments have shown that germ - free animals seem to be protected from diet - induced obesity ( 18 ) . however , this protective effect was later shown to be strongly dependent on the sugar compositions , that is type of sugar of these diets , not just the amount of fat - derived calories ( 19 ) . this may well be because germ - free animals lack the bacterial enzymes needed to digest polysaccharides , leading , therefore , to a lower calorie intake . complex polysaccharides are processed in the gut and fermenting microbes produce short - chain fatty acids ( scfas ) . butyrate , propionate , and acetate are scfas that can be directly used by colonocytes as an energy source or be further transported to the liver where they can be used as substrates for lipid synthesis ( 20 , 21 ) . the levels of scfa can be measured directly in the caecum but more often than not fecal material is used because it is readily available . however , one must remember that neither ceacal nor feacal levels of scfas are necessarily true indicators of the amounts produced in the gut because they are continuously used and absorbed by colonocytes . genes involved in scfa synthesis were identified in several metagenomic screenings as being overrepresented and highly abundant in the gut flora , strongly suggesting that carbohydrate fermentation is one of the most conserved and important functions of the gut microbiome ( 9 , 12 ) . genes governing scfa biosynthesis were also shown to be stably represented in at least one metatranscriptomic analyses ( 14 ) . given these observations , a series of experiments were performed to show that the ability of carbohydrate fermentation together with the production of scfas was linked to induction of obesity . when germ - free mice were colonized either by whole bacterial flora ( 22 ) or by saccharolitic fermenting bacteria such as bacteroidetes thetaiotamicron together with m. smithii , which facilitates fermentation ( 23 ) , an increase in body weight and adiposity was observed . the effect of fermenters on host adiposity was strongly reduced in mice lacking the major scfa receptor , gpr41 ( g - protein coupled receptor 41 ) , hence suggesting a link between scfa producer and host cell signaling ( 23 ) . in other studies , introduction of hfd in conventionally raised animals was accompanied by a shift toward fermenters in gut flora . this manifested as the appearance of bacterial genes taking part in absorption , transportation , and utilization of simple sugars as well as polysaccharide fermentation leading to scfa production . the authors then referred to this phenomenon as an obesity - associated microbiome with increased capacity for energy harvest ( 24 ) . animals on hfd had a microbial community that was characterized by a general decrease in microbial diversity and a phylogenetic shift from bacteroidetes toward firmicutes ( 24 ) . this could further be attributed to an extensive bloom of one of the families within firmicutes , namely mollicutes , belonging to erysipelotrichaceae ( 25 , 19 ) . here , it appears that noticeable phylogenetics shifts need not be a result of changes of many different species but rather the selective overgrowth of one or two members . the change in the proportional abundance of two major bacterial phyla was then confirmed in another set of experiments . however , in this instance , although the obese mice , as before , had more firmicutes , the levels of fecal scfas were not significantly different ( 26 ) . intriguingly , one study showing a decrease in fecal scfa content in obese mice , compared to lean ones , has also been published ( 19 ) . a more detailed approach to determine the pathways and routes of scfa absorption and use in these mouse models would shed more light on the relationship between hfd , scfa , and the microbiome . one of the most intensely studied models of obesity and metabolic syndrome are animals with disrupted leptin signaling . this is achieved in two ways , either by using mice lacking leptin receptor ( lep mice ) or by deleting the gene for the hormone itself ( ob / ob mice ) . . both genotypes of leptin signalling deficiency are characterized by uncontrolled food intake that results in morbidly increased body weight and development of metabolic problems ( 27 , 28 ) . comparison of intestinal microbiota compositions of ob / ob mice and their lean wild - type littermates revealed a decrease in bacteroidetes and a corresponding increase in firmicutes in obese animals , accompanied by a general decline in microbial diversity in this group ( 29 ) . this shift was probably responsible for the observed increase in abundance of bacterial genes involved in sugar metabolism in another study ( 24 ) . it is tempting to speculate that the increased food intake in those animals favored colonization by bacteria that had a higher metabolic capacity to extract energy , thanks to which both host and microbiota can benefit from otherwise lost calories . interestingly , the phenotype was somewhat transferable : wild - type germ - free mice colonized with flora from ob / ob mice ( 24 ) as well as mice that received hfd ( 25 ) were better at storing fat compared to those colonized with a normal flora . apart from effects on host metabolism arising through changes in the gut microbial community or the abundance of metabolites such as short chain fatty acids , the microbiome can also interact with the host tissues by tweaking host gene expression . among the many recently discovered genes transcriptionally regulated by bacteria is fasting - induced adipose factor ( fiaf ) also known as angiopoietin - like protein 4 ( 22 , 30 ) . fiaf is a lipoprotein lipase ( lpl ) inhibitor that in effect blocks lipid storage mediated through lpl activity . 2008 showed that fiaf mrna in epithelial cells increased in the presence of bacteria such as enterococcus faecalis ( 30 ) . a putative mechanism through the activity of the metabolically important nuclear receptor peroxisome proliferator - activated receptor gamma ( ppar ) was suggested to govern this increase . in a recent study , increases in the serum level of fiaf caused by administration of lactobacillus paracasei ssp paracasei f19 were associated with reduced weight gain in hfd - fed mice ( 31 ) . however , other studies reported lowered levels of mrna expression of fiaf in the intestinal tissue on bacterial exposure ( 22 , 19 ) . these seemingly confounding results may be explained as reflecting two different situations : ( 1 ) in which exposure of mice to the whole bacterial community results in a decrease in intestinal fiaf levels ; ( 2 ) , while introduction of a single defined bacterial species has the opposite result . because e. faecalis ( 30 ) or f19 ( 31 ) seem to be able to upregulate fiaf expression , it is , therefore , tempting to speculate that certain bacterial species could have a therapeutical role in weight management . another probiotic strain lactobacillus gasseri sbt2055 ( lg2055 ) was shown to impact health in small human cohort study . volunteers with higher bmi and visceral fat when consuming formula milk containing lg2055 lost weight and had reduced abdominal adiposity . this suggests that it has a positive effect on human metabolic health ( 32 ) . the mechanism(s ) of action is unknown , but another study in rodents postulated that milk fermented by lg2005 was able to restrict dietary fat absorption in rat intestine ( 33 ) . in humans , however , the correlation between changes in the gut flora and weight gain are much less clear because all the confounding factors ( sex , age , diet , exercise , living environment , antibiotic treatments , disease history , and finally genetic heterogeneity ) have to be taken into account . some groups claim that the shift from bacteroidetes toward firmicutes that was observed in mice was also associated with weight gain in humans ( 34 ) . a later study outrightly presents the opposite finding showing a lower ratio of firmicutes to bacteroidetes in overweight subjects ( 35 ) . in other reports , the decrease in the abundance of bacteroidetes could be confirmed , but no difference in firmicutes was observed , instead a bloom of actinobacteria carrying the majority of genes overrepresented in obese volunteers was apparent ( 11 ) . in another study , no significant differences were seen in the proportion of firmicutes versus bacteroidetes in relation to bmi ( 36 ) . ( 2010 ) also suggested that rather than major phylogenetic shifts , discrete , yet metabolically functional changes at the species level may occur in response to dietary changes similar to that observed for mollicutes in mice ( see above ) ( 35 ) . presently , we do not fully understand the role of microbiota in the regulation of host adiposity . in mice , the situation in human looks much more complicated , and it is very difficult to reach a consensus on the influence of a specific group of microbiota on changes in body weight . the variability seen in different studies built on microbial composition may be due to a multitude of factors , including diet , volunteer cohort , duration of study , methods of sample preparation , and storage as well as methods of detection . additionally , concentrations of bacterial metabolites such as scfa might vary due to increased microbial production , changes in absorption , shifts in microbial cross feeding patterns , or even the rate of transit ( reviewed in ( 37 , 38 ) ) . due to the complex nature of the gut environment , it is quite unlikely that a single factor , such as a phylogenetic change in the microbioata , could be responsible for the development of obesity . rather , changes in eating habits may favor bacterial species that now have to accommodate to a different nutritional environment . using germ - free mice , faith et al . ( 2011 ) have attempted to formulate a model to predict microbiota changes that are dependent on diet ( 38 ) . varying the abundance of 10 defined bacterial species that were introduced to the guts of germ - free mice that in turn received different refined diets ( protein , sugars , complex carbohydrate , and fat rich ) , faith and coworkers were able to infer a relationship between diet and microbiota . it appeared that in general , bacterial presence relied on availability of a single component in the food , indicating that the bacterial community may predominantly depend on the diet and can be changed by manipulating the type of food ingested . within this limited study at least however , as the authors rightly point out , one challenge ahead is to determine how the complex interplay between the human microbiome and the inordinate variability of the human diet affects parameters of gut health . presently , it is difficult to show whether the changes in the bacterial flora preceded weight increase , or if the changes that occur in the presence of each bacterial species are strongly associated with the ingested food . introducing more processed foods and antibiotics has led and will lead in the future to the reduction in the diversity of the microbial flora , simply because there will be a gradual decrease in nutrients available for microbial use ( reviewed in ( 39 ) ) . it seems likely that a balanced bacterial flora is needed for maintaining good health , and perturbing this balance could disrupt controlled energy harvest leading to problems such as obesity . inflammatory bowel disease ( ibd ) is an example of a complex disease postulated to arise following a disharmonious relationship between the immune system and the commensal flora . genetic , microbial as well as dietary and environmental cues together seem to play a role in the development of pathology . there are two major forms of ibd ulcerative colitis ( uc ) affecting mostly colon and crohn 's disease ( cd ) affecting colon as well as the distal part of the small intestine ( reviewed in ( 40 , 41 ) ) . previous studies have demonstrated that intestinal dysbiosis , characterized mainly by altered bacterial composition and loss of diversity , is strongly associated with the development of the disease ( 4244 , 12 ) . there are two major models for experimental colitis , chemically induced colitis , and spontaneous colitis occurring in genetically modified animals . two methods to chemically induce colitis are extensively used : the administration of either dextran sulphate sodium ( dss ) in drinking water or 2 , 4 , 6-trinitrobenzenesulfonic acid intrarectally . both chemically induced and spontaneous models have advantages and disadvantages that have been extensively reviewed elsewhere ( 45 ) . one posit that perturbed balance between the commensals bacteria and the host may play a role in the development of obesity is equally applicable to the pathogenesis of ibd . studies performed on animals depleted of certain genes involved in immune regulation of gut homeostasis show that specific genetic mutations harbored by the host may cause an uncontrolled growth of otherwise underrepresented bacteria , which leads to development of colitis ( reviewed in ( 46 ) ) . in recent studies , this altered microbiota was transmitted to wild - type animals that then develop the disease , suggesting that the microbiota retained the disease - causing shift that can be transmitted between individuals ( especially from mother to pups ) ( 47 , 48 ) epithelial cells have the ability to sense microbes by recognizing the microbe - associated molecular patterns ( mamps ) . several families of receptors expressed both on the cell surface as well as in the cytoplasm are involved in recognition of those mamp molecules , such as toll - like receptors ( tlrs ) ( 49 ) , nucleotide - binding oligomerization domain ( nod ) , ( 50 ) and nod - like receptors ( nlrs ) ( 51 , 52 ) . in this review , we would like to briefly highlight the involvement of all three of these recognition molecules in the pathogenesis of ibd , and how this may be connected to shifts in microbial communities . ligands recognized by tlrs are not only specific for pathogens but are also present on commensal microbiota , and it remains to be understood how the host distinguishes danger signals and homeostatic signals . most tlrs use an adaptor molecule myd88 to transmit the signals within the cell ( 49 ) . myd88- , tlr2- , and tlr4-deficient mice show a more severe phenotype of dss - induced colitis compared to their wild - type littermates . similarly , animals with depleted microbiota ( following treatment with antibiotics ) show increased susceptibility to experimental colitis . on the other hand , administration of tlr4 or tlr2 ligands , in microbiota - depleted animals , was able to ameliorate the disease to a level observed in animals harboring normal flora . these results strongly suggest that commensal microbiota play an important role in stimulating tlrs and setting the threshold of immune response needed for homeostasis ( 53 ) . additionally , spontaneous colitis observed in conventionally raised , but not germ - free il10 mice is dependent on myd88 signaling . deletion of myd88 in il10 animals abolishes tlr signaling and is protective against the development of the disease . these results suggest that tlr signaling , although necessary for establishing and maintaining balance between pro- and antiinflammatory signals , might also be responsible for development of the disease if other factors , such as il10 , are missing or when the balance is otherwise disrupted ( 54 ) . in addition to tlrs , microbial molecules can be recognized by members of the nod family that reside in the cytoplasm mutations in the nod2 gene have been associated with increased susceptibility to ibd , especially cd , but the precise role of this protein in the pathogenesis of the disease remains unknown ( 55 ) . nod2 mice display abnormal paneth cell functions and develop symptoms similar to cd ( 56 , 57 ) . microbial samples coming from patients harboring mutations in the nod2 gene display a shift in bacterial composition not seen in individuals not carrying these mutations ( 58 ) . additionally , nod2 animals harbor a microflora that is different from that of wild - type animals ( 59 ) . the third important group of defense molecules is nlrs that form cytoplasmic complexes known as inflammasomes that are able to sense both endo- and exogenic stress or damage - associated signals ( reviewed in ( 60 ) ) . apoptosis - associated speck - like protein ( asc ) , into multiprotein complexes that activate caspase 1 and subsequent cleavage of il-1b and il-18 ( 61 , 62 ) . mice that lack components of the inflammasome ( nlrp6 , asc ) or downstream targets of active complex ( caspase 1 , il-18 ) suffer from more severe dss - induced colitis than their wild - type littermates ( 48 ) . inflammasome loss resulted in the development of more colitogenic microflora that could be transferred to wild - type mice that were cohoused or cofostered with knock - out mice , resulting in more severe phenotype in these animals . two groups of bacteria were associated with the colitogenic microbiome : family prevotellaceae in the phylum bacteroidetes as well as a genus belonging to tm7 phylum . both groups are capable of disrupting the mucus barrier through its degradation and have been previously associated with development of ibd and periodontal disease . the prevotella cluster has been detected on mucosal surfaces taken from patients suffering from uc or cd but not in the guts of healthy subjects ( 63 ) . 2006 also highlighted the abundance of the members of the prevotella genera in mucosal biopsy specimens from uc patients ( 64 ) . on the species level , prevotella denticola is much more prevalent in subjects suffering from periodontitis than in healthy subjects ( 65 ) . antibiotic resistant bacteria belonging to prevotella genus has also been identified among periodontal bacteria ( 66 ) . tm7 phylum members ( gram - positive , non - cultivable bacteria ) could be found in equal numbers in biopsies obtained from uc , cd , and non - ibd patients ; however , the composition of the cloned 16s rrna sequences differed significantly between diseased and healthy samples ( 67 ) . one of the clones showed very high similarity to a clone suggested to play a causative role in periodontal disease ( 68 ) , and many members of that phylum have been associated with oral inflammation ( 69 , 65 , 70 ) . hence , there appear to be commonalities in mucosal inflammation , be it manifesting in the oral or intestinal cavities . klebsiella pneumoniae and proteus mirabilis as highly associated with colitogenic phenotype of t - bet rag2 mice ( 71 ) . these animals harbor a deficiency in innate immunity relying on t - cells responses , and they spontaneously develop colitis ( referred by authors as t - bet rag2 uc truc mice ) ( 47 ) . the colitic phenotype was transmittable to cohoused or cross - fostered wt mice , akin to the inflammasome - deficient mice discussed above ( 71 ) . neither of the identified species was , however , able to cause colitis when transferred to germ - free mice , indicating a need for other bacterial signals for a full - blown inflammatory reaction . klebsiella pneumoniae together with other drug - resistant enterobacteriacae has been previously reported to be present in oral cavities of patients with periodontal disease . it is postulated that the hydrolytic enzymes produced by these bacteria play a role in the destruction of the tooth - supporting tissues ( 72 ) . frank et al . ( 2007 ) identified additional shifts in microbiota in uc and cd patients compared to non - ibd samples ( 73 ) . uc and cd patients had fewer sequences representative of bacteroides and lachnospiraceae ( a subgroup of firmicutes ) and an overrepresentation of proteobacteria and also bacillus another subgroup of firmicutes . in the small intestine , proteobacterial sequences were more abundant and bacillus sequences less so , in uc and cd patients , relative to non - ibd samples . the bacterial profiles belonging to some cd and uc patients were clustered separately from the non - ibd samples and were characterized among the others by a decreased bacterial load . ibd patients recruited to this study harbored on average 25% less bacterial genes than healthy subjects , and principal component analysis clearly separated ibd patients from healthy subjects and cd patients from uc patients . one speculation , built on the hygiene hypothesis , is that decreasing diversity of the bacterial ecology may be a causative factor in diseases such as ibd . a new player in ibd pathogenesis is atg16l1 ( autophagy - related 16-like 1 ) that codes for a protein that is a part of a large complex necessary for autophagy a process in which cell components are targeted to degradation . the disease variant of the gene can be found in around 50% of european population and confers a twofold increase in disease susceptibility ( 7476 ) . people harboring such mutations also have altered microbial flora ( 58 ) . in mice with deleted atg16l1 , a reduced capacity for autophagy is seen ( 77 , 78 ) . on viral infection with a murine norovirus ( mnv ) , agt16l1 mice display additional features with strikingly abnormal paneth cells , resulting in aberrant packing and exocytosis of antimicrobial peptides . on dss treatment , these mice developed colonic ulcers with various hallmarks of cd - associated lesions . administration of antibiotics in these mice prevented the abnormal dss - induced pathologies suggesting the necessity of the microflora to induce a phenotype resembling cd ( 79 ) . interestingly , although antibiotics have been used to treat cd and uc , it seems to be more effective in the case of the former , as they could also prevent the relapse of the disease ( 80 ) . apart from the inflammatory disorder , ibd also predisposes to colorectal cancer ( crc ) . around 18% of patients changes in bacterial composition seen prior to the manifestation of colitis may also be associated with the pathology of colorectal cancer because truc mice ( described above ) spontaneously develop colonic dysplasia and rectal adenocarcinoma , resembling human colonic cancers associated with ibd ( 83 ) . toxin produced by this bacterium ( bft ) was identified as being a causative factor for both colitis and very early tumorigenesis in the apc mouse model of colon cancer ( harboring a heterozygous mutation in the apc gene ) ( 84 ) . bft acts as an oncogene by cleaving e - cadherin , a tumor suppressor protein triggering the nuclear localization of b - catenin and colonic cells proliferation , through the activation of the wnt pathway . it also activates the nf-b signaling pathway in the cells that results in secretion of proinflammatory cytokines . interestingly , btf exerts its oncogenic functions via the th17 cells ( 84 ) , the development of which is solely dependent on the presence of commensal bacteria in the gut ( 85 ) . hence , the pathologies linked to klebsiella ( 71 ) and b. fragilis appear to be dependent on the presence of the whole microbial community . although many bacterial species or groups are associated with the pathology of ibd , other prokaryotic partners may play protective roles . interestingly , b. fragilis implicated earlier in crc may have protective effects in infectious colitis caused by helicobacter hepaticus . further research could link the protective effects of psa with the function of il10 in response to commensal bacteria . psa was shown to not only prevent but also cure experimental colitis in mice , by actively inducing mucosal tolerance ( 87 ) . the presence of whole bacterial flora , as well as the fraction of fermenting microbiota , has also been suggested to play a protective role against colitis and helps exclude lethal invasions of pathogenic bacteria . germ - free animals are characterized by a more severe phenotype of dss - induced colitis compared to conventionally raised animals ( 88 , 89 ) . this phenotype could be reverted on conventionalization of gf animals , or by the administration of acetate in drinking water prior to dss treatment . g - coupled receptor 43 ( gpr43 ) is one of the cell surface receptors that is activated on binding of acetate . gpr43 mice showed greater morbidity and reduced potential to overcome the disease ( 89 ) . acetate produced by certain species of bifidobacteria has also been found recently to protect mice from colitis and subsequent death caused by infection with escherichia coli o157:h7 ( 90 ) . this clearly indicates that intestinal bacterial metabolites , garnered through microbial processing of available nutrients , do have a significant effect on gut health . in conclusion , recent studies have prompted researchers to peer deeply into the altered microbial composition in patients suffering from ibd . this may be associated with a general reduction of bacterial diversity as well as shifts toward certain bacterial groups and the appearance of specific , hitherto unidentified species . the changes might be driven by environmental cues and genetic predispositions . as elegantly shown by cadwell et al . ( 2010 ) , ibd is a very complex disease and to understand it , we have to look at more than one factor and be able to link microbial changes , presence of viruses with environmental conditions , such as diet and small changes in the host dna , that are not able to cause a disease when present alone ( 79 ) . this observation only serves to emphasize the importance of understanding the cross talk between different microbial communities thriving in the intestinal milieu . the dynamic communities of intestinal bacterial biofilms are likely to play a major role in setting the stage for host microbe interactions and subsequent physiological outcomes . the vast numbers of bacteria in the gut have over the years been postulated to directly extend their realm of influence beyond the immediate gut environment . here , we briefly list the role of the gut flora liver function , glucose metabolism , and the control of behavior . the gut microbiota has been attributed a role in the development of non - alcoholic fatty liver disease ( nalfld ) ( 91 , 92 ) . urinary metabolites of the nafld - susceptible mice strain 129s6 when fed hfd were enriched in microbiota - derived methylamines : dimethylamine , trimethylamine ( tma ) , and trimethylamine - n - oxide ( tmao ) . tma is synthesized exclusively by symbiotic bacteria ( 93 ) and can be further transformed into tmao by the microbiota themselves or at least in humans by the liver enzyme fmo3 . the production of methylamines by microbiota results in decreased bioavailability of choline for the host and seems to trigger nafld in mice . ( 2011 ) , who could link high levels of tmao in plasma with increased risk for cardiovascular disease in humans ( 92 ) . the interdependence between microbiota , metabolism , and inflammation as well as the host genotype has also been elegantly shown . mice lacking tlr5 suffer from spontaneous colitis ( 94 ) and at the same time have higher body weights ( 95 ) than their wild - type littermates . interestingly , the increase in weight could be attributed to higher chow consumption and indeed , the trait disappeared when mice were subjected to antibiotic treatment . the obese phenotype could be transferred from tlr5 animals to wild - type germ - free mice through the gut flora , suggesting that microbiota may be a factor responsible for hyperphagia and increased body weight in tlr5 mice . hyperphagia is also very much associated with satiety signals emanating from the gut and directed to the brain . increasingly , the gut flora is being recognized as a factor in the regulation of the central nervous system ( cns ) . germ - free mice display increased motor activity and reduced anxiety , compared with conventionally raised ones . this phenotype could be reversed if germ - free animals were exposed to bacteria early in life ( 96 ) . lactobacillus rhamnosus has been recently shown to possess antiinflammatory properties and has been suggested as a candidate to interfere with the cns signaling through the vagus nerve , which allows communication between the viscera and the brain . this probiotic was shown to reduce plasma levels of stress - induced corticosterone as well as anxiety- and depression - related behavior . these results strongly suggest that modulation of intestinal microbiota in healthy animals may lead to neurochemical changes affecting animal behavior ( 97 ) . these examples are telling in that they have attracted the attention of researchers who have traditionally been working away from the gut environment to seriously consider the gut microbial milieu when studying a variety of diseases ( see fig . the scope of this review unfortunately precludes the discussion of microbiome as a source or target for therapies and treatments of various ailments ( reviewed in ( 98 ) ) the gut microbiota can affect a variety of host responses . in this schematic diagram , host genotype , the environment , and diet the microbiota exerts its effects on the intestine through bacterial signaling molecules and metabolic products ( microbiome ) . components of the microbiome can also enter the circulation , be transported to various organs , and potentiate multiple effects in these organs ( gray arrow ) . at the same time , the gut brain axis can circumvent intestinal absorption and may allow the microbiome to directly affect the brain . once touted as the forgotten organ ( 99 ) , the microbiota is slowly beginning to reveal many of its secrets . having been under close scrutiny for many decades , the gut microbiota is now said to dictate important traits in the human host . future research delineating properties of the oral microflora should desist from chasing purely metagenomic queries . the oral cavity has one distinct advantage over the gi tract : accessibility , a trait that should be exploited to the fullest . crucially , information thus accrued would be important to those of us aspiring to understand the workings of the microbiota in the gi tract and beyond . painstakingly , slow research of yesterday that lay much of the groundwork for today 's ideas has given way to meta - analyses of high throughput findings . nevertheless , as we begin to answer the questions of who is there and what are they doing there, we inadvertently arrive at the inquiry how and why the superorganism functions . herein lies the potential to alter the course of many a pathology . what is obvious is the notion that microbial influence permeates into nearly every aspect of our existence . as a component species of a superorganism , we should now universally acknowledge the importance of the microbial world in which we live and , crucially , that which lives on us . there is no conflict of interest in the present study for any of the authors .

the gut microbiota consists of trillions of prokaryotes that reside in the intestinal mucosa . this long - established commensalism indicates that these microbes are an integral part of the eukaryotic host . recent research findings have implicated the dynamics of microbial function in setting thresholds for many physiological parameters . conversely , it has been convincingly argued that dysbiosis , representing microbial imbalance , may be an important underlying factor that contributes to a variety of diseases , inside and outside the gut . this review discusses the latest findings , including enterotype classification , changes brought on by dysbiosis , gut inflammation , and metabolic mediators in an attempt to underscore the importance of the gut microbiota for human health . a cautiously optimistic idea is taking hold , invoking the gut microbiota as a medium to track , target and treat a plethora of diseases .

pubmed

onychomycosis is a chronic fungal infection of the nail , characterized by nail discoloration , thickening , and defect . the involved pathogens are dermatophytes , yeasts ( candida ) , and nondermatophytes.1 dermatophytes such as trichophyton rubrum and trichophyton mentagrophytes are the most common agents of onychomycosis , accounting for 50%90% of cases , with prevalence of the infection higher in toenails than in fingernails.2 candida and nondermatophytic molds are frequent in countries with hot and humid climate.3 if untreated , nail fungal infections can extend to other nails and potentially cause further complications , especially in at - risk patients . aging and reduced peripheral circulation are among the major risk factors for onychomycosis ; diabetes weakens the immune function , favoring slow nail growth , thickening , and susceptibility to infections and nail injury.47 onychomycosis sometimes is associated with nail psoriasis , favored by abnormal capillary units and onychodystrophy.8 differential diagnoses of onychomycosis include chronic nail eczema , bacterial infections , traumatic onychodystrophies , pachyonychia congenita , nail bed tumors , yellow nail syndrome , and idiopathic onycholysis.9 the treatment of onychomycosis is sometimes difficult because the predisposing factors are not influenced by therapy.10,11 onychomycosis is usually treated with topical or systemic antifungals.12,13 local treatment requires strong patient adherence because the treatment time is generally long ; moreover , onychomycosis - related subungueal hyperkeratosis is generally thick and this limits the penetration of the antimycotic drug . consequently , long - term treatments can discourage or induce stopping of the therapy before healing is achieved . the cochrane meta - analysis of crawford et al14 reports limited success in the treatment of onychomycosis with topical agents . although many therapeutic options are available , new drugs with a greater safety and relatively rapid efficacy are required . one of the most used topical drug for nail psoriasis is tazarotene.15,16 its efficacy is classically related to its immunomodulating properties and anti - inflammatory activity , with consequent inhibition of the infective process.17 tazarotene is also successfully used for acne treatment , given its anti - inflammatory and immune - modulating effectiveness that protect keratinocytes from infection by propionibacterium acnes.17 epidermal penetration of tazarotene depends on its ability to reduce hyperkeratinization , favoring keratinocyte differentiation , as also documented by the reduction of expression of hyperproliferation - associated keratins 6 and 16.18 the latter appear dysregulated in genetic diseases , such as pachyonychia congenita and focal palmoplantar keratoderma.18 as a matter of fact , fungal infections could exacerbate or predispose an individual to pachyonychia congenita.19,20 in light of our previous studies reporting the clinical efficacy and the simplicity of the use of tazarotene in the nails of psoriatic patients,15,16 we investigated its effect on distal and lateral subungual onychomycosis in an open preliminary study of immunocompetent patients and its fungistatic activity in vitro . we report an observational open clinical trial in 15 patients affected by onychomycosis of the toenail treated with topical tazarotene ( zorac / tazorac ; allergan inc , irvine , ca , usa ) 0.1% gel in an off - label use . the enrolled patients were of both sexes and aged between 14 years and 70 years ( table 1 ) . patients eligible were those affected by distal and lateral subungual onychomycosis without associated skin or systemic diseases , nave or in washout for other treatments . exclusion criteria were immunosuppression , treatment for other systemic diseases , and the presence of other diseases causing nail dystrophy such as psoriasis , eczema , and lichen planus . the drug was applied once a day on the affected nail plates and nail folds for 12 weeks ( end point ) . we evaluated onycholysis , nail bed discoloration , and subungueal hyperkeratosis ( thickening of the subungueal region ) at baseline and end point , assigning the following semiquantitative scores for onycholysis and discoloration : absence ( 0 ) , 0%25% ( 1 ) , 25%50% ( 2 ) , and > 50% ( 3).14 for subungual hyperkeratosis , the scores were as follows : absent ( 0 ) , 1 mm ( 1 ) , 2 mm ( 2 ) , and > 3 mm ( 3).21 we also evaluated changes of the affected area comparing baseline and end point appearance of the affected target areas , as reported.22 briefly , the response rate was assessed by the analysis of digital photographs of the affected nails taken at the baseline and at the end of the study.23 data were analyzed by means of analysis of variance and student s t - test for continuous data and by mann whitney test for ordinal data ; p - values < 0.05 were considered statistically significant . mycological cultures and potassium hydroxide stains were also performed on nail samples taken by scraping , clipping , or curetting the affected nail site and subungual region at the baseline and at the end point . staining of fungi was performed with lactophenol cotton blue solution ( sigma - aldrich , st louis , mo , usa ) . fungistatic activity of tazarotene in vitro was investigated by disk diffusion assay ( kirby bauer method).24 briefly , 1 g of 0.1% tazarotene gel was dissolved in 3 ml of physiological solution in a sterile tube . to test the effect , the fungi analyzed were candida albicans , c. glabrata , t. mentagrophytes , and t. verrucosum . all statistical analysis was performed using the statistical package spss16.0 ( spss inc , chicago , il , usa ) . mycological cure in target nails ( ie , those with mycological confirmation of onychomycosis at baseline ) was observed in subungual samples collected at the first follow - up visit ( 4 weeks ) in 40% of patients , and complete cure with complete clinical healing in all patients was observed at the end point ( 12 weeks ) . direct microscopy and in vitro laboratory culture of the sampled material collected after 12 weeks were negative for infections . representative clinical images of four patients at baseline and end point after tazarotene treatment are reported in figure 1 . therapeutic results were good , with no side effects except for mild erythema on the perionychia in two patients ; nail growth and tropism were also restored . as shown in figure 2 , the percentage of affected area and the proportion of affected patients decreased ( p<0.001 ; figure 2a and b ) . onycholysis , subungual hyperkeratosis , and discoloration completely disappeared in all patients at the end point ( p<0.04 ; figure 2c ) . the nails were completely healed at the follow - up visits up to 6 months . patients fungal culture test11 was performed at baseline ( figure 3 ) ; 66.7% of patients were positive for t. rubrum ( figure 3a ) , 13.3% for t. mentagrophytes , 13.3% for e. floccosum , and 6.7% for t. tonsurans . disk diffusion assay ( figure 3b and c ) document the inhibition of t. mentagrophytes growth , starting with 2 ml of tazarotene solution , which became almost complete with 5 ml ( figure 3b ) , as well as the reduced growth of c. albicans ( figure 3c ) starting with 1 ml of tazarotene solution . similar results were observed with plates with c. glabrata and t. verrucosum , with greater prominence in the presence of 4 ml and 5 ml of the solution ( not shown ) . in this study , we have reported the therapeutic efficacy of tazarotene 0.1% gel for the topical treatment of distal and lateral subungual onychomycosis . t. rubrum , t. mentagrophytes , and e. floccosum are the most common agents implicated in onychomycosis.12 these dermatophytes are hyaline - septated molds , whose growing fungal hyphae penetrate into the stratum corneum of the skin and nails . the fungal infection produces keratinolytic proteases , creating a gap in the living cells.25 some fungi have developed the ability to digest keratinous debris in soil and the faculty of parasitizing keratinous tissues in animals.25 compared to the normal skin , the nail apparatus has a different immunological profile , a strong innate immunity , and an increased expression of antimicrobial peptides.26 on the other hand , the nail apparatus has a lower level of major histocompatibility complex class-1a antigen expression , production of immunosuppressive agents , anomalies of antigen - presenting cells , and inhibition of natural killer cell activity,27 all aspects that favor onychomycosis . successful topical treatment of nail onychomycosis is fraught with difficulty due to several factors , including slow nail growth , the physical presence of nail plate interfering with nail bed access , and the difficulty in finding compounds with adequate pharmacologic profile for nail penetration.28 these difficulties are reflected in the high incidence of recurrence after topical therapeutic clearance.29 at present , the most frequently used topical therapies for onychomycosis are amorolfine 5% , ciclopirox 8% nail lacquer , and tioconazole 28% solution , which need to be applied for prolonged periods and require strong adherence by the patients.13 amorolfine 5% nail lacquer is effective as monotherapy for the treatment of onychomycosis , but with limited efficacy when distal nail involvement is greater than 50%.30 up to june 2014 , ciclopirox was the only topical treatment approved by the us food and drug administration for onychomycosis in the us.28 the action of ciclopirox differs from that of other topical antifungal drugs , which generally act through ergosterol inhibition . the high affinity of ciclopirox for trivalent metal cations results in the inhibition of the metal - dependent enzymes responsible for the degradation of peroxides within the fungal cell.31 efficacy of ciclopirox 8% as a topical monotherapy ( ranged from 20% to 36% ) has been disappointing and limited to the mildest cases of toenail onychomycosis.28 for the remaining cases , a combined therapy with a systemic antifungal is recommended.28 nevertheless , oral antifungals have general adverse effects , including intestinal and respiratory complaints.32 tioconazole is an imidazole antifungal agent recommended for local infections caused by candida28 and can be used for the topical treatment of pachyonychia . efinaconazole 10% and tavaborole 5% solutions are new topical antifungals specifically developed for the treatment of dermatophytic onychomycosis . in phase iii clinical trials , both newer agents were applied once daily for 48 weeks without concomitant nail debridement . reported mycologic cure rates with efinaconazole are superior to that achieved with ciclopirox 8% nail lacquer.33 in our study , complete healing after topical treatment with tazarotene was obtained at the first follow - up visit after 4 weeks in 40% of patients and complete healing in all patients after 12 weeks , with a rapid and progressive amelioration of all evaluated clinical parameters . sometimes , the ineffectiveness of topical treatment is due to the subungual strong hyperkeratosis , which prevents drug penetration.34 hyperkeratosis also represents one of the most frequent features of nail psoriasis , and nail psoriatic patients also suffer from onychomycosis.35 in our patients , tazarotene has proven quite effective in reducing hyperkeratinization in fungus - infected nails . concerning the therapeutic effects of tazarotene , our results in vitro document a direct fungistatic activity . tazarotene is a synthetic third - generation retinoid derived from vitamin a , which has proved to be beneficial in modulating keratinocyte proliferation and in reducing inflammation . tazarotene is used for the treatment of acne , nonmelanoma skin tumors , and inflammatory skin diseases such as psoriasis.3638 in particular , tazarotene s effect is mediated by the intracellular upregulation of cellular retinoic acid - binding protein - ii expression,39 which transactivates nuclear retinoic acid receptors ( rars ) , in particular rar and rar , by binding to specific dna sequences on their promoter gene regions , leading to reduced proliferation of normal and neoplastic cells and favoring differentiation and apoptosis.3638,40 the reduction in the excessive v - akt murine thymoma viral oncogene homolog 1 activation supporting cell growth41 may also explain the beneficial effects of tazarotene on ungual hyperkeratosis . moreover , tazarotene can exert a protective action on the microvascular bed against fungal infection , similarly to that reported for psoriasis patients.16 finally , the activity of tazarotene against inflammation through immunomodulation may induce the blocking of fungal keratinolytic proteases , thus contributing to its efficacy . a limitation of our preliminary study results is the absence of a randomized placebo control group . nevertheless , a recent analysis published in 2014 reveals that in 21 randomized controlled trials of dermatophytic nail onychomycosis , present in the pubmed database , placebo cure varied from 8.7% to 1.2% , documenting that the pooled placebo cure mean rate is very low.42 our preliminary results documented the safety and rapid efficacy of topical treatment with tazarotene on distal and lateral subungual onychomycosis , thus representing an alternative new promising therapeutic approach . treated and cured patients were followed every 4 weeks , with no evidence of recurrence after 6 months . we also documented the fungistatic activity of tazarotene in t. mentagrophytes , t. verrucosum , c. albicans , and c. glabrata , supporting the theoretical assumption about its direct action on fungus replication . our clinical evidence of efficacy suggests that tazarotene could be useful in those patients with treatment - resistant forms of onychomycosis or in those with evident enhanced nail thickening , wherein the therapeutic reducing action of the retinoid can be providential . further studies concerning the specific effects of tazarotene and in a larger cohort of patients with a longer follow - up are needed to confirm the efficacy in terms of relapse and reinfection .

backgrounddistal and lateral onychomycoses are the most frequent forms of onychomycosis , causing subungual hyperkeratosis that usually limits local penetration of antimycotic drugs . tazarotene exerts anti - inflammatory and immune - modulating activities toward both infective agents and damaged keratinocytes . given the well - documented efficacy of tazarotene on hyperkeratotic nail psoriasis , we investigated its therapeutic use in onychomycosis.patients and methodswe designed a preliminary open clinical trial in patients affected by distal and lateral subungual onychomycosis of the toenails and verified the fungistatic activity of tazarotene in vitro . fifteen patients were treated with topical tazarotene 0.1% gel once per day for 12 weeks . mycological cultures and potassium hydroxide stains of nail samples were performed at the beginning and at the end of the study . treatment was considered effective when clinical healing and negative mycological culture were obtained . onycholysis , nail bed discoloration , and subungual hyperkeratosis were measured using standardized methodologies and analyzed by means of mann whitney test and analysis of variance . fungistatic activity of tazarotene was evaluated by disk diffusion assay.resultssix patients ( 40% ) reached a mycological cure on target nail samples already after 4 weeks of treatment . complete clinical healing and negative cultures were reached in all patients at week 12 , with a significant improvement of all clinical parameters of the infected nails . disk diffusion assay after 48 hours of incubation with tazarotene solution showed a central area of inhibition in all examined fungal cultures.conclusionour results documented a good clinical outcome using topical tazarotene 0.1% gel in distal and lateral subungual onychomycosis and its fungistatic activity of tazarotene in vitro . the majority of patients appeared cured at a 6-month follow - up . the efficacy and safety of tazarotene must be confirmed on a larger number of patients , although already documented in nail psoriasis patients often affected by onychomycosis .

pubmed

cardiac resynchronisation therapy ( crt ) has become an important treatment for patients with heart failure and left ventricular ( lv ) dyssynchrony [ 13 ] . however , clinical non - response to crt is reported in 2535 % of patients . although a large variety of causes for a suboptimal response have been cited , most attention has been focused on the selection of patients eligible for crt , and less on inadequate delivery of crt therapy . however , even with growing experience and improved materials and tools , the optimal position can not always be reached in one of the tributaries of the coronary sinus . this can be due to the absence of suitable side branches in the posterolateral area , coronary vein stenosis , lead instability , high stimulation threshold , phrenic nerve stimulation , or a combination of the above [ 57 ] . we studied the acute haemodynamic response ( ahr ) of the implanted system and alternative left ventricular endocardial pacing sites in patients clinically not responding to crt . we asked patients who remained in new york heart failure class iii or iv despite at least 6 months of crt and adequate medical therapy and with no therapeutic options left to undergo an acute haemodynamic study in search of improving existing crt , as previously described [ 8 , 9 ] . twenty - four non - consecutive patients , 23 males , age 72.8 9.1 years , mean ejection fraction 22.5 7.1 % , agreed to undergo this test ( table 1 ) . the ecg prior to implantation showed left bundle branch block ( lbbb ) in 12 , non - lbbb in 4 , and right ventricular ( rv ) pacing in 8 patients . table 1characteristics of clinical non - responders ( 24 patients)patientgenderage ( years)nyha classicm / dcmejection fraction ( % ) qrs morphologyqrs width ( ms)pt . 01m56ivdcm13lbbb135pt . ivicm17rvp210average23m/1f72.8 9.13.2 0.36dcm22.5 7.112lbbb/8rvp175 22patient characteristics of 24 clinical non - responders to crt . nyha class new york heart association class , dcm dilated cardiomyopathy , icm ischaemic cardiomyopathy , lbbb left bundle branch block , non - lbbb non left bundle branch block , pt . characteristics of clinical non - responders ( 24 patients ) patient characteristics of 24 clinical non - responders to crt . nyha class new york heart association class , dcm dilated cardiomyopathy , icm ischaemic cardiomyopathy , lbbb left bundle branch block , non - lbbb non left bundle branch block , pt . a 6f multipurpose angioplasty guiding catheter was introduced into the lv after a standard transseptal catheterisation . in one patient , the guiding catheter was introduced through the radial artery following coronary angiography . through this guiding catheter , endocardial pacing and measurements of lvdp / dtmax were accomplished by roving a medtronic 6416 temporary bipolar screw - in lead ( medtronic inc . minneapolis , mn . ) and a radi pressure wire ( radi medical , a st jude medical company , st . initially the atrioventricular and interventricular intervals of the implanted system were optimised to obtain the maximal lvdp / dtmax . lv pacing was performed from basal posterolateral , mid - posterolateral , lv apical and lv septal locations , and optimal ahr obtained after optimisation of the crt system for all positions . if none of the 4 endocardial positions were opposite the position of the coronary sinus lead , an additional measurement was done at this location . a rise in lvdp / dtmax 15 % from baseline was considered to be a positive haemodynamic response . in all patients with an ahr of 15 % during lv endocardial pacing , the interval between the onset of the qrs complex and the intrinsic activation at the lv electrode ( q - lv interval ) was measured and the ratio between q - lv / qrs width interval calculated . this ratio expresses the relation between q - lv interval and qrs width , which is a better indicator for late or early lv sensing than the absolute value of q - lv . notwithstanding that all patients were clinical non - responders , in 5 patients lvdp / dtmax with the implanted coronary sinus system increased 15 % after optimisation : average 30.2 % , 15.644.5 % ( table 2 ) . in 3 out of these 5 patients with the coronary sinus lead in a posterolateral position , endocardial pacing did not increase the lvdp / dtmax substantially ( ahr less than 3 % and even an adverse effect from lv endocardial pacing was observed in one patient ) . in one of the two remaining patients with an apical position of the coronary sinus lead , the ahr could be increased during lv endocardial pacing from 19.766 % and we considered the increase sufficient to justify the upgrade to lv endocardial pacing ( fig . 1 ) . table 2individual haemodynamic results of all clinical non - responders ( 24 patients)patientqrs morphologybaseline lvdp / dtmax ( mmhg / s)position cs leadahr cs lead ( % ) ahr cs - level - endo(%)lv endo optimal positionahr lvendo ( % ) pt . 07non - lbbb893pl - mid 23.4lv septal 21.5pt . 08lbbb827pl - bas44.951.0pl - bas51.0pt . 24rvp489lv apical12.717.2pl - mid24.1average891 2478.7 15.810.5 8.919.6 17.5haemodynamic measurements of 24 clinical nonresponders to crt . baseline lvdp / dtmax lvdp / dtmax with intrinsic rhythm or right ventricular pacing , ahr cs lead acute haemodynamic response from the coronary sinus ( cs ) lead expressed as percentage rise in lvdp / dtmax from baseline , ahr cs level endo acute haemodynamic response at an endocardial location opposite the cs lead , lv endo optimal position anatomic site with the highest ahr , ahr lv endo acute haemodynamic response from the optimal endocardial position . grey shaded patients have an ahr from the cs lead 15 % and are considered haemodynamic responders , pl - bas basal posterolateral , pl - mid mid - posterolateral , rvp right ventricular pacing . individual haemodynamic results of all clinical non - responders ( 24 patients ) haemodynamic measurements of 24 clinical nonresponders to crt . baseline lvdp / dtmax lvdp / dtmax with intrinsic rhythm or right ventricular pacing , ahr cs lead acute haemodynamic response from the coronary sinus ( cs ) lead expressed as percentage rise in lvdp / dtmax from baseline , ahr cs level endo acute haemodynamic response at an endocardial location opposite the cs lead , lv endo optimal position anatomic site with the highest ahr , ahr lv endo acute haemodynamic response from the optimal endocardial position . grey shaded patients have an ahr from the cs lead 15 % and are considered haemodynamic responders , pl - bas basal posterolateral , pl - mid mid - posterolateral , rvp right ventricular pacing . all views are in left anterior oblique ( lao ) showing the haemodynamic effects of stimulation from the coronary sinus lead and lv endocardial stimulation in the mid - posterolateral area ( left upper panel ) , basal posterolateral area ( left lower panel ) , lv septum ( right upper panel ) and lead positions after lv endocardial implantation of a permanent lead in the lv basal posterolateral segment . arrows indicate the position of the bipolar pacing lead angiographic pictures of the haemodynamic study in patient no . all views are in left anterior oblique ( lao ) showing the haemodynamic effects of stimulation from the coronary sinus lead and lv endocardial stimulation in the mid - posterolateral area ( left upper panel ) , basal posterolateral area ( left lower panel ) , lv septum ( right upper panel ) and lead positions after lv endocardial implantation of a permanent lead in the lv basal posterolateral segment . arrows indicate the position of the bipolar pacing lead in the 19 patients with an ahr from the implanted system < 15 % , 9 patients had an increase in ahr above the 15 % limit . the results of the total group of 24 patients showed a positive response in 11 patients with an apical lead position in 7 , anterolateral in 2 and mid - posterolateral in 2 patients . a negative response of endocardial pacing was observed in 13 patients with a basal posterolateral lead position in 3 , mid - posterolateral in 8 and apical in 2 patients . in 5 out of the 9 acute haemodynamic non - responders ( ahr < 15 % ) with lbbb , the ahr could be increased above the 15 % level by lv endocardial pacing . the average ahr with the coronary sinus system was 9.6 % ( 5.814.9 % ) and at the lv endocardial optimal position 25.8 % ( 19.034.3 % ) . the average qrs width in these patients was 169 ms ( 135198 ms ) and the average q - lv at the optimal lv endocardial pacing site 157 ms ( 128176 ms ) , resulting in a q - lv / qrs ratio of 93 % . the position of the original coronary sinus lead was apical in 3 , anterolateral in 1 and mid - posterolateral in 1 patient ( fig . 2 ) . 2recordings of the temporary study of patient no . 22 with a lbbb showing the haemodynamic effects and the timing of the lv endocardial electrogram from the different positions recordings of the temporary study of patient no . 22 with a lbbb showing the haemodynamic effects and the timing of the lv endocardial electrogram from the different positions in the remaining 4 patients , the average ahr at the optimal endocardial position increased < 15 % , with an average of 7.3 % ( 1.79.9 % ) . the average qrs width in these patients was 176 ms ( 149209 ms ) and the average q - lv 136 ms ( 100174 ms ) , resulting in a q - lv / qrs ratio of 83 % . of these , only one patient with rbbb , combined with left anterior hemiblock , showed an increase in the ahr , from 9.1 % ( apical coronary sinus position ) to 25.8 % at an endocardial basal posterolateral position . 3 ) ; one had a non - specific intraventricular conduction delay with a qrs complex of 175 ms . the coronary sinus lead position was mid - posterolateral in 2 and apical in 1 patient . 3recordings of a temporary study of patient no.18 with non - lbbb , showing the haemodynamic effects and the timing of the lv endocardial electrogram from the different positions . this recording illustrates that there is no conduction delay in the lv and a minimal haemodynamic effect recordings of a temporary study of patient no.18 with non - lbbb , showing the haemodynamic effects and the timing of the lv endocardial electrogram from the different positions . this recording illustrates that there is no conduction delay in the lv and a minimal haemodynamic effect three out of the 6 patients with rv pacing became haemodynamic responders ( lv dp / dtmax from average 9.7 % , ( 9.312.7 % ) increased to 21.8 % ( 16.624.8 % ) ) . in the other 3 patients the average ahr increased from 2.9 % ( 1.14.0 % ) to 9.7 % ( 7.811.4 % ) . in 2 patients the coronary sinus lead was in a mid - posterolateral position , one in an apical position . individual details are summarised in table 2 and a flow chart overview in fig . 4 . 4flow chart of the study showing the haemodynamic results in relation to the coronary sinus lead positions . ahr acute haemodynamic response , ant lat anterolateral , bas basal , cs coronary sinus , endo endocardial , lbbb left bundle branch block , lv left ventricular , pl posterolateral , pts patients , rv right ventricular flow chart of the study showing the haemodynamic results in relation to the coronary sinus lead positions . ahr acute haemodynamic response , ant lat anterolateral , bas basal , cs coronary sinus , endo endocardial , lbbb left bundle branch block , lv left ventricular , pl posterolateral , pts patients , rv right ventricular when pacing endocardially , opposite the epicardial coronary sinus lead , we found that lvdp / dtmax did not differ significantly for any of the patients : lvdp / dtmax from the coronary sinus averaged 1046 292 mmhg / s vs. 1068 296 mmhg / s from the corresponding lv endocardial site ( p = 0.37 ) . we found that basal or mid - posterolateral segments had the highest ahr of the tested endocardial sites in all 14 patients who showed an ahr 15 % rise in lvdp / dtmax compared with baseline . because either the basal or mid - posterolateral segment had the highest ahr , we also calculated the combined best results from basal posterolateral and mid - posterolateral ( pl - best ) . pl - best had a rise to 30.2 13.6 % in lvdp / dtmax , basal posterolateral 27.7 15.2 % , mid - posterolateral 24.6 12.6 % , lv apical 14.8 9.1 % and lv septal 17.0 11.2 % . we also found that in 12 out of the 14 patients ( 85 % ) with an ahr 15 % , the lv endocardial site with the longest time interval between the onset of the qrs complex and lv sensing ( q - lv interval ) resulted in the best haemodynamic response . the average qrs width was 133 22 ms ( range 120205 ms ) and average q - lv interval 155 26 ms ( 128205 ms ) . the average q - lv / qrs width ratio , which is q - lv expressed as a percentage of qrs width , was 90 % . table 3haemodynamic results for all 4 endocardial locations and maximum q - lv interval for 14 patients that showed a rise in lvdp / dtmax 15 % from endocardial pacingpts.ahr pl - best ( % ) ahr pl - basal ( % ) ahr pl - mid ( % ) ahr lv apical ( % ) ahr lv septal ( % ) qrs width ( ms)max q - lv interval ( ms)location longest q - lvpt.0124.224.22.71.72.1135128pl - baspt.0331.631.625.84.826.1174165pl - baspt.0424.818.724.814.219.6196205pl - midpt.0520.120.118.018.111.8165163pl - midpt.0851.051.046.022.029.2154138pl - baspt.1219.013.519.017.613.7198176pl - midpt.1316.55.316.68.89.6152128pl - midpt.1566.066.050.231.347.3200181pl - midpt.1625.825.814.1 0.612.2175147pl - baspt.2019.719.716.78.916.3156124pl - midpt.2134.928.034.916.96.9174132pl - midpt.2234.334.320.315.614.4175155pl - baspt.2331.026.231.026.718.6160138pl - midpt.2424.122.924.117.211.5210192pl - mid30.2 13.627.7 15.224.6 12.614.8 9.117.1 11.2173 22155 26 ahr acute haemodynamic response expressed as the percentage rise in lvdp / dt from baseline , pl - best best result from either basal posterolateral ( pl - bas ) or mid - posterolateral ( pl - mid ) region , max q - lv longest interval measured between onset of the qrs complex and intrinsic activation at the lv electrode . grey shaded area indicates 2 patient in whom the longest q - lv interval did not correspond with the best haemodynamic response . haemodynamic results for all 4 endocardial locations and maximum q - lv interval for 14 patients that showed a rise in lvdp / dtmax 15 % from endocardial pacing ahr acute haemodynamic response expressed as the percentage rise in lvdp / dt from baseline , pl - best best result from either basal posterolateral ( pl - bas ) or mid - posterolateral ( pl - mid ) region , max q - lv longest interval measured between onset of the qrs complex and intrinsic activation at the lv electrode . grey shaded area indicates 2 patient in whom the longest q - lv interval did not correspond with the best haemodynamic response . ten patients were considered possible candidates for lv endocardial pacing : 9 patients in whom a rise in lvdp / dtmax 15 % level was only obtained by endocardial pacing , and the patient that showed a substantial additional rise in ahr with lv pacing compared with coronary sinus pacing ( patient 15 ) . of the remaining 9 patients , and after deliberation with the patients about the possible benefits and risks , 5 of them finally agreed to lv endocardial implant at the optimal lv site as indicated from the acute study . all five patients improved clinically with a reduction of at least 1 class in the nyha score after 6 months of follow - up . two patients with a positive response in the temporary study refrained from implantation of an lv endocardial lead . one patient with a positive ahr from endocardial pacing also improved significantly after changing stimulation from the distal to the proximal electrode with a more basal position , and became a clinical responder after optimisation of the atrioventricular and interventricular interval . in one patient , the deterioration of his condition after initial improvement proved to be the result of a blunted chronotropic response after increasing beta - blocker dosage following an episode of atrial fibrillation resulting in an inappropriate icd shock . notwithstanding that all patients were clinical non - responders , in 5 patients lvdp / dtmax with the implanted coronary sinus system increased 15 % after optimisation : average 30.2 % , 15.644.5 % ( table 2 ) . in 3 out of these 5 patients with the coronary sinus lead in a posterolateral position , endocardial pacing did not increase the lvdp / dtmax substantially ( ahr less than 3 % and even an adverse effect from lv endocardial pacing was observed in one patient ) . in one of the two remaining patients with an apical position of the coronary sinus lead , the ahr could be increased during lv endocardial pacing from 19.766 % and we considered the increase sufficient to justify the upgrade to lv endocardial pacing ( fig . 1 ) . table 2individual haemodynamic results of all clinical non - responders ( 24 patients)patientqrs morphologybaseline lvdp / dtmax ( mmhg / s)position cs leadahr cs lead ( % ) ahr cs - level - endo(%)lv endo optimal positionahr lvendo ( % ) pt . 07non - lbbb893pl - mid 23.4lv septal 21.5pt . 08lbbb827pl - bas44.951.0pl - bas51.0pt . 24rvp489lv apical12.717.2pl - mid24.1average891 2478.7 15.810.5 8.919.6 17.5haemodynamic measurements of 24 clinical nonresponders to crt . baseline lvdp / dtmax lvdp / dtmax with intrinsic rhythm or right ventricular pacing , ahr cs lead acute haemodynamic response from the coronary sinus ( cs ) lead expressed as percentage rise in lvdp / dtmax from baseline , ahr cs level endo acute haemodynamic response at an endocardial location opposite the cs lead , lv endo optimal position anatomic site with the highest ahr , ahr lv endo acute haemodynamic response from the optimal endocardial position . grey shaded patients have an ahr from the cs lead 15 % and are considered haemodynamic responders , pl - bas basal posterolateral , pl - mid mid - posterolateral , rvp right ventricular pacing . individual haemodynamic results of all clinical non - responders ( 24 patients ) haemodynamic measurements of 24 clinical nonresponders to crt . baseline lvdp / dtmax lvdp / dtmax with intrinsic rhythm or right ventricular pacing , ahr cs lead acute haemodynamic response from the coronary sinus ( cs ) lead expressed as percentage rise in lvdp / dtmax from baseline , ahr cs level endo acute haemodynamic response at an endocardial location opposite the cs lead , lv endo optimal position anatomic site with the highest ahr , ahr lv endo acute haemodynamic response from the optimal endocardial position . grey shaded patients have an ahr from the cs lead 15 % and are considered haemodynamic responders , pl - bas basal posterolateral , pl - mid mid - posterolateral , rvp right ventricular pacing . all views are in left anterior oblique ( lao ) showing the haemodynamic effects of stimulation from the coronary sinus lead and lv endocardial stimulation in the mid - posterolateral area ( left upper panel ) , basal posterolateral area ( left lower panel ) , lv septum ( right upper panel ) and lead positions after lv endocardial implantation of a permanent lead in the lv basal posterolateral segment . arrows indicate the position of the bipolar pacing lead angiographic pictures of the haemodynamic study in patient no . all views are in left anterior oblique ( lao ) showing the haemodynamic effects of stimulation from the coronary sinus lead and lv endocardial stimulation in the mid - posterolateral area ( left upper panel ) , basal posterolateral area ( left lower panel ) , lv septum ( right upper panel ) and lead positions after lv endocardial implantation of a permanent lead in the lv basal posterolateral segment . arrows indicate the position of the bipolar pacing lead in the 19 patients with an ahr from the implanted system < 15 % , 9 patients had an increase in ahr above the 15 % limit . the results of the total group of 24 patients showed a positive response in 11 patients with an apical lead position in 7 , anterolateral in 2 and mid - posterolateral in 2 patients . a negative response of endocardial pacing was observed in 13 patients with a basal posterolateral lead position in 3 , mid - posterolateral in 8 and apical in 2 patients . in 5 out of the 9 acute haemodynamic non - responders ( ahr < 15 % ) with lbbb , the ahr could be increased above the 15 % level by lv endocardial pacing . the average ahr with the coronary sinus system was 9.6 % ( 5.814.9 % ) and at the lv endocardial optimal position 25.8 % ( 19.034.3 % ) . the average qrs width in these patients was 169 ms ( 135198 ms ) and the average q - lv at the optimal lv endocardial pacing site 157 ms ( 128176 ms ) , resulting in a q - lv / qrs ratio of 93 % . the position of the original coronary sinus lead was apical in 3 , anterolateral in 1 and mid - posterolateral in 1 patient ( fig . 2 ) . 2recordings of the temporary study of patient no . 22 with a lbbb showing the haemodynamic effects and the timing of the lv endocardial electrogram from the different positions recordings of the temporary study of patient no . 22 with a lbbb showing the haemodynamic effects and the timing of the lv endocardial electrogram from the different positions in the remaining 4 patients , the average ahr at the optimal endocardial position increased < 15 % , with an average of 7.3 % ( 1.79.9 % ) . the average qrs width in these patients was 176 ms ( 149209 ms ) and the average q - lv 136 ms ( 100174 ms ) , resulting in a q - lv / qrs ratio of 83 % . four patients did not have lbbb . of these , only one patient with rbbb , combined with left anterior hemiblock , showed an increase in the ahr , from 9.1 % ( apical coronary sinus position ) to 25.8 % at an endocardial basal posterolateral position . 3 ) ; one had a non - specific intraventricular conduction delay with a qrs complex of 175 ms . the coronary sinus lead position was mid - posterolateral in 2 and apical in 1 patient . 3recordings of a temporary study of patient no.18 with non - lbbb , showing the haemodynamic effects and the timing of the lv endocardial electrogram from the different positions . this recording illustrates that there is no conduction delay in the lv and a minimal haemodynamic effect recordings of a temporary study of patient no.18 with non - lbbb , showing the haemodynamic effects and the timing of the lv endocardial electrogram from the different positions . this recording illustrates that there is no conduction delay in the lv and a minimal haemodynamic effect three out of the 6 patients with rv pacing became haemodynamic responders ( lv dp / dtmax from average 9.7 % , ( 9.312.7 % ) increased to 21.8 % ( 16.624.8 % ) ) . in the other 3 patients the average ahr increased from 2.9 % ( 1.14.0 % ) to 9.7 % ( 7.811.4 % ) . in 2 patients the coronary sinus lead was in a mid - posterolateral position , one in an apical position . individual details are summarised in table 2 and a flow chart overview in fig . 4 . 4flow chart of the study showing the haemodynamic results in relation to the coronary sinus lead positions . ahr acute haemodynamic response , ant lat anterolateral , bas basal , cs coronary sinus , endo endocardial , lbbb left bundle branch block , lv left ventricular , pl posterolateral , pts patients , rv right ventricular flow chart of the study showing the haemodynamic results in relation to the coronary sinus lead positions . ahr acute haemodynamic response , ant lat anterolateral , bas basal , cs coronary sinus , endo endocardial , lbbb left bundle branch block , lv left ventricular , pl posterolateral , pts patients , rv right ventricular when pacing endocardially , opposite the epicardial coronary sinus lead , we found that lvdp / dtmax did not differ significantly for any of the patients : lvdp / dtmax from the coronary sinus averaged 1046 292 mmhg / s vs. 1068 296 mmhg / s from the corresponding lv endocardial site ( p = 0.37 ) . we found that basal or mid - posterolateral segments had the highest ahr of the tested endocardial sites in all 14 patients who showed an ahr 15 % rise in lvdp / dtmax compared with baseline . because either the basal or mid - posterolateral segment had the highest ahr , we also calculated the combined best results from basal posterolateral and mid - posterolateral ( pl - best ) . pl - best had a rise to 30.2 13.6 % in lvdp / dtmax , basal posterolateral 27.7 15.2 % , mid - posterolateral 24.6 12.6 % , lv apical 14.8 9.1 % and lv septal 17.0 11.2 % . we also found that in 12 out of the 14 patients ( 85 % ) with an ahr 15 % , the lv endocardial site with the longest time interval between the onset of the qrs complex and lv sensing ( q - lv interval ) resulted in the best haemodynamic response . the average qrs width was 133 22 ms ( range 120205 ms ) and average q - lv interval 155 26 ms ( 128205 ms ) . the average q - lv / qrs width ratio , which is q - lv expressed as a percentage of qrs width , was 90 % . table 3haemodynamic results for all 4 endocardial locations and maximum q - lv interval for 14 patients that showed a rise in lvdp / dtmax 15 % from endocardial pacingpts.ahr pl - best ( % ) ahr pl - basal ( % ) ahr pl - mid ( % ) ahr lv apical ( % ) ahr lv septal ( % ) qrs width ( ms)max q - lv interval ( ms)location longest q - lvpt.0124.224.22.71.72.1135128pl - baspt.0331.631.625.84.826.1174165pl - baspt.0424.818.724.814.219.6196205pl - midpt.0520.120.118.018.111.8165163pl - midpt.0851.051.046.022.029.2154138pl - baspt.1219.013.519.017.613.7198176pl - midpt.1316.55.316.68.89.6152128pl - midpt.1566.066.050.231.347.3200181pl - midpt.1625.825.814.1 0.612.2175147pl - baspt.2019.719.716.78.916.3156124pl - midpt.2134.928.034.916.96.9174132pl - midpt.2234.334.320.315.614.4175155pl - baspt.2331.026.231.026.718.6160138pl - midpt.2424.122.924.117.211.5210192pl - mid30.2 13.627.7 15.224.6 12.614.8 9.117.1 11.2173 22155 26 ahr acute haemodynamic response expressed as the percentage rise in lvdp / dt from baseline , pl - best best result from either basal posterolateral ( pl - bas ) or mid - posterolateral ( pl - mid ) region , max q - lv longest interval measured between onset of the qrs complex and intrinsic activation at the lv electrode . grey shaded area indicates 2 patient in whom the longest q - lv interval did not correspond with the best haemodynamic response . haemodynamic results for all 4 endocardial locations and maximum q - lv interval for 14 patients that showed a rise in lvdp / dtmax 15 % from endocardial pacing ahr acute haemodynamic response expressed as the percentage rise in lvdp / dt from baseline , pl - best best result from either basal posterolateral ( pl - bas ) or mid - posterolateral ( pl - mid ) region , max q - lv longest interval measured between onset of the qrs complex and intrinsic activation at the lv electrode . grey shaded area indicates 2 patient in whom the longest q - lv interval did not correspond with the best haemodynamic response . ten patients were considered possible candidates for lv endocardial pacing : 9 patients in whom a rise in lvdp / dtmax 15 % level was only obtained by endocardial pacing , and the patient that showed a substantial additional rise in ahr with lv pacing compared with coronary sinus pacing ( patient 15 ) . of the remaining 9 patients , and after deliberation with the patients about the possible benefits and risks , 5 of them finally agreed to lv endocardial implant at the optimal lv site as indicated from the acute study . all five patients improved clinically with a reduction of at least 1 class in the nyha score after 6 months of follow - up . two patients with a positive response in the temporary study refrained from implantation of an lv endocardial lead . one patient with a positive ahr from endocardial pacing also improved significantly after changing stimulation from the distal to the proximal electrode with a more basal position , and became a clinical responder after optimisation of the atrioventricular and interventricular interval . in one patient , the deterioration of his condition after initial improvement proved to be the result of a blunted chronotropic response after increasing beta - blocker dosage following an episode of atrial fibrillation resulting in an inappropriate icd shock . this observational study showed that endocardial lv pacing , guided by acute haemodynamic studies , could improve clinical outcome in patients not responding to conventional crt . it also demonstrated the importance of lead location , as the clinical response improved with a posterolateral position of the lead instead of the original apical or anterolateral lead location . conversely , none of the patients who already had a coronary sinus lead in a posterolateral position improved sufficiently with endocardial pacing at any site during the acute haemodynamic study to justify endocardial lead implant . this is in line with the madit - crt trial , where the apical and anterior positions of the lv lead had a lower clinical response . noteworthy , and similar to what has previously been reported , endocardial pacing opposite the coronary sinus pacing site did not improve the ahr in our study [ 1113 ] . this is in contrast with animal experiments that show significantly better haemodynamic results with endocardial vs. epicardial pacing at the same location . there is no clear single cause for this difference and it is most probably related to variance in the anatomical and electrophysiological substrate . besides this , in animal experience the lead configuration guarantees an exact position of the endocardial lead opposite to the epicardial position ; this in contrast with human studies in which the endocardial lead is placed as close as possible to its epicardial counterpart . therefore , although not examined in this and other studies , one either might speculate that epicardial pacing opposite the optimal endocardial sites , via the coronary sinus or surgically by thoracoscopy , might have resulted in similar benefits . if feasible , this might be a good alternative to avoid the uncertainty of thromboembolic complications with lv endocardial pacing . our observational study also showed some limitations in the use of acute haemodynamic studies to predict clinical outcome . first , five clinical non - responders showed a clear ahr according to the accepted criteria ( even with the higher than usual cut - off value of 15 % increase in lvdp / dtmax to exclude borderline cases ) but failed to respond clinically . still , when a substantial further increase in lvdp / dtmax from endocardial pacing at a posterolateral position compared with the apical coronary sinus lead ( 66 vs. 19.7 % ) was obtained in one patient , endocardial lead implantation resulted in a positive clinical response . this illustrates the limitation of cut - off values in crt studies of the 15 % level as surrogate for differentiating between clinical response and non - response , and that what is considered a positive haemodynamic response can still be significantly improved in the presence of an anterior or apical lead position . a similar discrepancy is found with echocardiography as the observed presence or absence of decrease in lv end - diastolic volume or end - systolic volume and clinical response does not always correlate . to err on the safe side , we therefore proceeded to a permanent lv endocardial implantation when the final increase in ahr was at least 25 % . the best ahr correlates well with the longest time interval between onset qrs and lv sensing in relation to the qrs width in the individual patient . the results of the group of patients with non - lbbb are in line with what could be expected according to the literature . the absence of response correlated well to the lack of conduction delay in any of the lv segments on the intracardiac lv electrogram . the only responder to endocardial pacing in this group had a significant lv conduction delay due to the left anterior hemiblock that accompanied rbbb . it is therefore questionable if one should proceed with endocardial mapping in this cohort of patients given the disappointing results . this observational study showed that a temporary acute haemodynamic study could be useful in non - responders to establish the chance of improvement with alternative pacing sites before embarking on complex procedures as endocardial lv pacing . it remains , however , uncertain what the optimal cut - off value for increase in lvdp / dtmax is before the improvement justifies intervention . the longest q - lv interval is a reliable indicator for the optimal electrode position in the individual patient . endocardial pacing opposite epicardial sites does not show acute haemodynamic improvement , so epicardial pacing at the optimal site may be a good alternative for endocardial lv lead placement . although all patients who had a lv endocardial system implanted after a positive ahr improved clinically , larger randomised series are necessary to justify this technique before definitive conclusions can be drawn . van gelder has received training and education on pacing and crt from st jude medical , is a consultant with radi pressure wire systems , a st jude medical company , and consultant for pacing and crt with sorin group crm sas . b.m . van gelder has received training and education on pacing and crt from st jude medical , is a consultant with radi pressure wire systems , a st jude medical company , and consultant for pacing and crt with sorin group crm sas .

introductionnon response to cardiac resynchronisation therapy ( crt ) may be related to the position of the coronary sinus lead.methodswe studied the acute haemodynamic response ( ahr ) from alternative left ventricular ( lv ) endocardial pacing sites in clinical non - responders to crt . ahr and the interval from qrs onset to lv sensing ( q - lv interval ) from four different endocardial pacing sites were evaluated in 24 clinical non - responders . a rise in lvdp / dtmax 15 % from baseline was considered a positive ahr . we also compared the ahr from endocardial with the corresponding epicardial lead position.resultsthe implanted system showed an ahr 15 % in 5 patients . in 9 of the 19 remaining patients , ahr could be elevated to 15 % by endocardial lv pacing . the optimal endocardial pacing site was posterolateral . there was no significant difference in ahr between the epicardial and the corresponding endocardial position . the longest q - lv interval corresponded with the best ahr in 12 out of the 14 patients with a positive ahr , with an average q - lv / qrs width ratio of 90 % .conclusionsacute haemodynamic testing may indicate an alternative endocardial pacing site with a positive ahr in clinical non - responders . the q - lv interval is a strongly correlated with the optimal endocardial pacing site . endocardial pacing opposite epicardial sites does not result in a better ahr .

pubmed

autosomal dominant polycystic kidney disease ( adpkd ) affects between 1 in every 500 and 1 in every 1000 individuals and is found in all racial and ethnic groups . the disease may not be clinically apparent until the third or fourth decade of life and only about half of the patients will progress to renal failure . in young patients , there is no way to predict whether or when renal failure will develop , although several risk factors such as hypertension , renal hemorrhage , multiple pregnancies , male gender , and pkd1 disease genotype have been associated with a more rapid course . complications associated with pregnant subjects with chronic renal failure undergoing dialysis include maternal hypertension , preeclampsia , anemia , infected renal and hepatic cysts , pyelonephritis , and rupture of cerebral artery aneurysm during labor with subarachnoid hemorrhage . however , with careful and effective perioperative monitoring spinal anesthesia can be safely performed for cesarean section in patients undergoing hemodialysis . an 80 kg 35-year - old full term primigravida with oligohydramnios was scheduled for emergency lower segment cesarean section ( lscs ) . she was diagnosed to have adpkd in the seventh month when she complained of flank pain , generalized swelling , and decreased urine output . she was hospitalized and hemodialyzed thrice a week for the next 2 months to maintain blood urea nitrogen ( bun ) , serum creatinine , and potassium values within acceptable limits . the nephrologists and obstetricians decided to proceed with an emergency lscs as she was full term . clinical parameters were heart rate of 102/ min regular , systemic blood pressure of 120/74 mm hg in supine position , central venous pressure ( cvp ) 1 - 2 cm h2o . preoperative investigations showed hemoglobin ( hb ) of 10.2 gm% , normal leucocyte count , and mild proteinurea and pus cells in urine . her biochemical profile revealed bun of 30 mg / dl , serum creatinine 3.7 mg% , serum sodium 140 mmol l , serum potassium 5 mmol l , and normal liver function tests . coagulation profile was bleeding time 3 min , clotting time 9 min , activated partial thromboplastin time ( aptt ) 30 s , platelet count 150 10l , prothrombin time 13 s , and international normalized ratio 1:2 . arterial blood gas on air showed ph 7.41 , pco2 30.8 mm hg , po2 93.3 mm hg , spo2 98% , and hco3 22 meq . after obtaining informed written consent , patient monitoring included pulse oximeter , cardioscope , noninvasive blood pressure ( nibp ) , cvp , and urine output measurement . a peripheral line of 18 g in the left arm was used to preload her with 500 ml of normal saline till cvp was 5 cm h2o . spinal anesthesia was successfully given in the l3 - 4 intervertebral space using 1 ml of 0.5% hyperbaric bupivacaine ( 5 mg ) with 25 mcg fentanyl in the left - lateral position with a 25 g spinal needle . intraoperatively , bp was in the range of 100 - 120 mm hg systolic , hr 70 - 100/ min , cvp 5 - 6 cm h2o and urine output of approximately 1 a 2.0 kg live male child was delivered ( apgar score of 9 at 5 min ) . patient did not complain of pain for about 4 h ; thereafter , she was given intravenous tramadol 8 hourly . postoperatively , she maintained normal hemodynamic and biochemical parameters and did not require any hemodialysis . there was no neurological deficit ; she was shifted to the nephrology unit after 72 h and discharged on day 7 . women with moderate or severe antenatal renal insufficiency often experience deterioration of their renal functions during pregnancy . the pathophysiology by which pregnancy exacerbates renal disease is unknown . one hypothesis is that increased glomerular perfusion which normally accompanies pregnancy paradoxically causes further injury to the kidneys in patients with preexisting impairment of function . however , this hypothesis is not supported by published data , which reveal no evidence of hyperfiltration . epstein outlined an alternative hypothesis in which preexisting renal disease may induce a cascade of platelet aggregation , microvascular fibrin thrombus formation , and endothelial dysfunction that leads to microvascular injury in the already tenuous kidneys . increased cardiac output and decreased intrarenal vascular resistance in pregnancy cause an 80% increase in renal blood flow and a 50% increase in glomerular filtration rate ( gfr ) . because of the increased gfr , a serum creatinine concentration greater than 0.8 mg / dl and a bun concentration greater than 13 mg/ dl ( which are normal values for the nonpregnant patient ) suggest renal insufficiency . tubular sodium reabsorption and osmoregulation are reset , allowing a physiologic hypervolemia during gestation . modest proteinuria also occurs during pregnancy ( up to 300 mg in 24 h ) . when serum creatinine concentration exceeds 2.0 mg / dl there is a one in three chance of developing dialysis - dependent end stage renal disease during or shortly after pregnancy . our patient presented with symptoms of renal insufficiency only in the seventh month of gestation , it is difficult to opine whether her disease progressed during pregnancy or the diagnosis was missed earlier . on presentation , the blood erythrocyte count and hematocrit may be increased above normal possibly because of abnormal erythropoietin production by the cysts ; hence , they do not have anemia as profound as that occurring in other types of terminal renal disease . for ambulatory chronic renal failure patients , hb of 11 - 12 g / dl blood coagulation studies and platelet counts are usually normal in patients of adpkd , as was the case in our patient . hypertension an early symptom , occurring in approximately 60% of patients secondary to stretching of arterioles across expanding cysts leading to increased secretion of renin and angiotensin . although the patient was not anuric and did not have ecg changes due to hyperkalemia , it was decided to dialyze her at least thrice a week . during pregnancy early dialysis is recommended ( when the gfr is approximately 10 ml / min , rather than waiting until it falls further ) and treating with more frequent and longer hemodialysis sessions . problems occurring in a pregnant patient on hemodialysis are need for vascular access , cardiovascular instability , large fluid , and electrolyte shifts , need for anticoagulation of extracorporeal circuit and risk of hepatitis . fetal heart rate monitoring is recommended , even when hypotension and major fluid shifts are avoided , as doppler examination of uterine and umbilical artery flow during hemodialysis suggest redistribution of arterial flow away from the uteroplacental vascular bed . bun levels are kept below 80 mg / dl predialysis and 30 mg / dl postdialysis . at birth , the increase in concentration of most coagulation factors is associated with a shortening of the prothrombin and aptt during pregnancy . there are reports of safe administration of epidural anesthesia , without any neurologic complications , in thrombocytopenic ( platelet count < 100 000/cumm ) healthy pregnant women , preeclampsia , and women with autoimmune thrombocytopenic purpura . they state that in the absence of clinical evidence of bleeding , regional anesthesia should not necessarily be withheld in pregnant women with a platelet count of less than 1 00 000/cu mm . no hemorrhagic complications were observed in 24 women with unsuspected thrombocytopenia ( platelet counts of 15 - 99 10/l ) who received spinal anesthesia or in 24 children with acute lymphoblastic leukemia and platelet counts below 20 10/l . intravenous unfractionated heparin should be stopped 4 - 6 hrs before neuroaxial blockade and a normal aptt should be confirmed . since our patient had been dialyzed the previous day and her electrolytes , aptt , platelet counts , bleeding time and hemoglobin values were within acceptable limits , we decided to proceed with spinal anesthesia . high degree of suspicion for any neurological complications should be maintained in the postoperative period . preservation of renal function intraoperatively depends on maintaining an adequate intravascular fluid volume and minimizing drug - induced cardiovascular depression . we used low dose of local anesthetic with intrathecal opioid in an effort to preserve renal and uterine blood flow . low dose of spinal anesthesia has the advantage of providing cardiovascular stability and it is advocated that 8 mg of 0.5% hyperbaric bupivacaine is the optimal dose for cesarean section . we were able to achieve a sensory level of t6 with 5 mg of 0.5% bupivacaine combined with fentanyl 25 mcg . bogra j et al . studied the synergistic , potentiating effect of fentanyl on bupivacaine in spinal anesthesia for cesarean section , on comparing the hemodynamic stability of equipotent doses of bupivacaine and bupivacaine fentanyl , they found the latter to be more stable .

renal disease , either preexisting or occurring during gestation may impair maternal and fetal health . a 35-year - old primigravida with autosomal dominant polycystic kidney disease on hemodialysis was scheduled for emergency cesarean section . she was managed successfully with low - dose intrathecal bupivacaine and fentanyl . in the case of pregnancy in such a patient , early involvement of the nephrologists along with the obstetrician can improve maternal and fetal outcome .

pubmed

it also depicts summary of the pattern of milk yield determined by the biological efficiency of the cow . the cost of milk production depends to a large extent on the persistency of lactation , that is , the rate of decline in production after peak milk yield . high persistency is associated with a slow rate of decline in milk production , whereas low persistency is associated with a rapid rate of decline in milk yield . in general declining rate of milk production estimates of heritabilities for milk yield and persistency traits in hf cows have been reported [ 3 , 4 ] . the lactation curve models have been used to predict the milk yield at any point of the lactation [ 5 , 6 ] . various models have been tried by different researchers to fit the lactation curve in indigenous as well as exotic cattle [ 710 ] . further getting the test - day milk yield information from the field conditions is not easy and there is every chance of missing the data due to certain inevitable circumstances . in such case mathematical models may prove beneficial for prediction of milk production performance using . in view of this the lactation curve was composed for the normal vis - - vis mastitis cows using gamma type function during different parity to find out the accuracy of model in predicting the milk yield with minimum error in normal and mastitis crossbred cows . the data on 11728 fortnightly test - day milk yields ( ftdmys ) of 733 karan fries cows for a period of 12 years ( 20002011 ) was collected from livestock farm of national dairy research institute , karnal , india . the climate of the farm is subtropical in nature with the lowest temperature reaching 2c during winter and the highest temperature up to 45c in the summer months . the annual rainfall is about 760 to 960 mm , out of which most of the rainfall is received during the months of july and august . the amount of milk recorded in 24-hour duration on any particular day is called the test - day ( td ) milk yield and is expressed in kilograms . the fortnightly test - day intervals of 15 days were considered starting from days 5 , 20 , 35 , 50 , 65 , 80 , 95 , 110 , 125 , 140 , 155 , 170 , 185 , 200 , 215 , 230 , 245 , 260 , 275 , 290 , and 305 of lactation . twenty - one ( 21 ) fortnightly test - day milk yield ( ftdmy ) records were considered per lactation ( from the 6th to the 305th day of lactation ) . the data was grouped as mastitis ( cows suffering from clinical mastitis ) and nonmastitis ( normal cows ) . statistical analysis of data was carried out using gamma type function with the following equation : ( 1)yt = atbect , where yt is the average daily yield in the tth fortnight , a is the initial milk yield after calving , b is the ascending slope parameter up to the peak yield , and c is the descending slope parameter . the constants were derived by solving the above equation after transformation on the log scale : ( 2)ln(yt)=ln(a)+bln(t)ct . the milk yield up to week t the fortnightly test - day milk yield was less in td-1 ( 9.78 0.29 kg / d ) and increased ( p < 0.01 ) gradually to attain peak in td-3 and td-4 ( 14.34 0.38 and 14.48 0.37 kg / d ) in normal cows ; however , in mastitis cows , the peak milk yield was attained in td-3 ( 13.15 0.37 kg / d ) and was maintained only for this period ( figure 1 ) . the cows of both of the groups exhibited a steady decline ( p < 0.01 ) in milk yield with increase in fortnights of lactation . in general the fortnightly test - day milk yield increased till td-3 and declined steadily with advancement of lactation . in the second parity the ftdmy showed an increasing trend from td-1 to td-4 and decreased thereafter gradually in both of the groups . a minimum ftdmy of 11.31 0.39 kg / d and 9.18 0.44 kg / d and a maximum ftdmy of 17.19 0.44 kg / d and 15.53 0.46 kg / d were observed on td-4 in both of the groups ( figure 2 ) . kg / d to 9.55 0.47 kg / d in td-21 and the maximum ftdmy of 19.08 0.53 kg / d and 16.44 0.47 an increase in ftdmy was observed from 14.36 kg and 12.34 kg in td-1 to a peak yield of 20.50 0.59 kg / d in td-4 and 17.63 kg / d in td-3 which subsequently declined ( p < 0.05 ) to 10.50 0.54 kg / d and 9.54 0.54 kg / d in td-21 in normal and mastitis kf cows ( figure 3 ) . the ftdmy milk yield in the 4th parity was maximum in comparison to the first , second , and third parity in both normal and mastitis cows ( figure 4 , table 2 ) . an increase in milk production was observed up to the 4th fortnight and milk productions decreased ( p < 0.01 ) during the 5th parity in normal cows . in mastitis cows increase in milk yield was noticed during the first three fortnights and milk production decreased subsequently ( p < 0.05 ) until the end of lactation ( figure 5 ) . the parity - wise estimated lactation curve parameters for ftdmy indicated higher average initial milk yield ( a ) in normal cows as compared to mastitis cows in different parities ( table 1 ) . the peak milk yield was observed in the 4th parity ( 16.45 kg and 14.42 kg ) while minimum yield was observed in the 1st parity in both normal and mastitis cows ( 11.28 versus 10.35 kg / d ) . lactation curve parameter of ascending slope parameter up to peak yield ( b ) was similar in both the groups in the 1st parity , declined in the 2nd parity , and increased in the subsequent parities . the descending slope parameter ( c ) was lower in the 1st parity ( 0.041 ) and higher in the 4th parity ( 0.072 ) in normal cows , but , in mastitis cows , it was minimum in the 1st parity ( 0.043 ) and maximum in the 5th and above parity ( 0.070 ) . further higher persistency of lactation was observed in the 1st parity ( 54.34 and 51.40 ) which declined till the 5th and above parity in all of the cows . the fortnights of peak milk yield ranged from averagely 4.53 to 6.15 fortnights in normal cows and from 4.49 to 5.88 fortnights in mastitis cows . the cows in the first parity took more time ( fortnight ) to attain peak yield in comparison to subsequent parity ; however , peak milk yield increased ( p < 0.05 ) with increase in number of parity in both of the groups . the peak yield was minimum in the 1st parity ( 13.89 kg / d and 12.58 kg / d ) and maximum in the 4th parity ( 19.67 kg / d and 16.61 the coefficient of determination of variation ( r ) ranged from 84.88% to 98.18% in normal cows and from 83.93% to 97.68% in mastitis cows during different parities . the rmse value was minimum in the 3rd parity ( 0.09 kg ) and maximum in the 5th and above parity ( 0.16 kg ) in normal cows ; however , in mastitis cows rmse was minimum in the 5th and above parity ( 0.09 ) and maximum in the 4th parity ( 0.13 kg ) . the pattern of change in the ftdmy observed in this study was in agreement with the earlier reports in normal cattle and buffaloes [ 1217 ] . however comparable literature on fortnightly test - day milk yield ( ftdmy ) and fortnightly average daily milk yield ( fadmy ) in mastitis crossbred kf cows is scanty . the peak milk yield attained during the 3rd and 4th fortnights in both of the groups corroborates with the values reported earlier in cows [ 10 , 18 , 19 ] . the trend in ftdmy in both of the groups suggested that peak yield duration is not affected by incidence of mastitis in spite of significant decline in milk yield ( p < 0.01 ) . the lower initial milk yield ( a ) in the first parity and an increasing trend in subsequent parity have also been reported in normal kf cows and butana dairy cows . many research findings indicated an increase in initial milk yield value with increase of parity order and the highest initial milk yield reaches the 5th lactation in friesian and ayrshire crossbred and kf cows [ 2125 ] ; however , such trend was evident up to the 4th parity in this study . the lower initial yield , peak milk yield , and greater persistency in the first parity than the subsequent parity in kf cows as observed in this study corroborate earlier reports [ 6 , 2631 ] . this is expected because lactation curves of the first parity are characterized by a less peak milk production and a greater persistency in ruminants [ 11 , 20 , 24 ] . the decline in persistency of lactation with increase in parity order is attributed to age factor as older animals start their lactation at a higher level and had a rapid rate of decline in milk production due to regression of alveolar cells with advancement in age . further , first - calving animals have less body weight and maturation of mammary tissue is still active , which counterbalances decline in milk production . during the first lactation , animal encounters unfamiliar situations , including the atmosphere of the milking parlor , presence of dairy farmer , and the milking procedures . thus variation in the initial milk yield in different parities could be due to addition of more number of alveolar cells ( secretory cells ) at each successive pregnancy which reach their maximum numbers at about the 5th calving and diminish gradually thereafter . the value of ascending slope parameter up to peak yield ( b ) in the present study was lower than reported earlier in different parity of friesian and ayrshire crossbred cows due to difference in milk yield of cows , management practices followed in a farm , and breed difference . bwith advancement of parity in both of the groups was also reported in butana dairy cows , hf cows , and crossbred cows b was lower than the reported values in hf , kf , and sahiwal cows [ 10 , 21 , 36 ] than the value of parameter the higher descending slope parameter ( c ) observed in this study was in agreement with the findings of previous reports [ 33 , 35 ] in crossbred cows and was lower than reported values in sahiwal cows and holstein cows . c in the first parity than the rest of the parities indicated better utilization of feeds and less susceptibility of cows to metabolic and reproductive disorders . the marginal differences in parameters a , b , and c in mastitis cows rather than the normal ones were due to disturbed milk secretion and apoptosis of mammary secretory cells that is solely responsible for the decline in milk yield after peak lactation [ 38 , 39 ] . it has been found that turn of lactation curve was unimportant on the level of persistency of lactation in brown swiss cows . further occurrence of atypical shapes of lactation curve characterized by the absence of the lactation peak varied from 25 to 42% . previous report also supports the fact that peak yield is lower in the first parity and increases in subsequent parity . the lower initial and peak milk yield in the first parity and more time to attain peak yield in the first parity than the remaining parities were in agreement with previous reports in norwegian mastitis cows [ 25 , 33 ] . it has been found that holstein x zebu cows require 71 days to reach peak yield , which was less than the predicted 6.15 fortnights observed in crossbred cows . however , predicted fortnightly peak milk yield was nonsignificantly different in both mastitis and normal cows ( 5.88 versus 6.15 kg / d ) . further moderate to high heritability estimates for different lactation curve shape parameters suggest that these traits can be included in selection schemes . it has been reported that the milk secretory tissue requires more time for peak activity in primiparous cows than multiparous cows due to reduced udder size , less digestive capacity , and directs partitioning of nutrients [ 24 , 41 ] . the high persistency is associated with a slow rate of decline in milk yield after peak production , while low persistency is associated with a rapid rate of decline in milk yield due to less feed intake . further persistent lactations are characterized by lower peak yield and reduced metabolic stress in early lactation . the high score of goodness of fit ( r value ) of gamma type function corroborates the earlier reports in cows [ 10 , 19 , 21 , 22 , 36 ] . the gamma function has been reported as the best model because of the small error variance and high determination coefficient . from gamma function , approximately one third ( 31.28% percent ) of lactation curves were named atypical . atypical curve percentages were found as follows : 44.19% downhill , 45.08% concave , 7.32% lna negative , and 3.39% c negative . further first lactation goodness of fit in normal and mastitis cows indicated more fitness of the model in kf crossbred cows as evident from low rmse value . the latter confirms accuracy of gamma function model to predict milk yield of mastitis cows also [ 11 , 46 ] . the similar value of r in normal and mastitis cows further indicated accuracy of model level . the similar rmse values in mastitis and normal kf cows during different parities also support this fact . the lactation curve parameters for fortnightly test - day milk yield exhibited similar trend in normal and mastitis infected cows by attaining peak milk yield in the 3rd and 4th td followed by a gradual decline in milk yield till the end of lactation . the steeper decline in descending slope ( c ) and increase in initial ftdmy due to steeper rise in ascending slope from the 1st parity onwards could be used as a marker of persistency during different parities in cows . further , higher r and lower rmse confirm the validity of gamma type function in predicting the milk yields in cows suffering from mastitis .

data pertaining to 11728 test - day daily milk yields of normal and mastitis karan fries cows were collected from the institute herd and divided as mastitis and nonmastitis and parity - wise . the data of lactation curves of the normal and mastitis crossbred cows was analyzed using gamma type function . ftdmy in normal and mastitis cows showed an increasing trend from td-1 to td-4 and a gradual decrease ( p < 0.01 ) thereafter until the end of lactation ( td-21 ) in different parities . the ftdmy was maximum ( peak yield ) in the fourth parity . parity - wise lactation curve revealed a decrease in persistency , steeper decline in descending slope ( c ) , and steeper increase in ascending slope ( b ) from 1st to 5th and above parity . the higher coefficient of determination ( r2 ) and lower root mean square error ( rmse ) indicated goodness and accuracy of the model for the prediction of milk prediction performance under field conditions . clinical mastitis resulted in a significantly higher loss of milk yield ( p < 0.05 ) . the ftdmy was maximum ( p < 0.05 ) in the fourth parity in comparison to the rest of parity . it is demonstrated that gamma type function can give the best fit lactation curve in normal and mastitis infected crossbred cows .

pubmed

mouse lifespan , weight , and food consumption were monitored as described in detail previously ( 15 ) . briefly , male b6c3f1 mice ( harlan breeders ; indianapolis ) were randomly assigned to treatment groups at 12 months of age . two hundred ninety - seven control mice were shifted from ad libitum chow feeding ( diet # 5001 , purina mills , richmond , in ) to daily feeding with 13.3 kcal / day / mouse of control diet [ american institute of nutrition ( ain)-93 m diet ; diet no . four groups of 18 mice each were shifted to daily feeding with an identical quantity of control diet supplemented with ndga at either 1.5 , 2.5 , 3.5 , or 4.5 g per kg diet ( approximately 1.59 , 2.65 , 3.71 , 4.77mg / kg body weight / day , respectively ) . food consumption and health were monitored at the time of feeding , and any uneaten food noted . with few exceptions , ndga was mixed with the powdered diet and cold - pressed into 1 g pellets by bio - serv . the mice had ad libitum access to tap water , which was acidified ( ph 4.0 ) to reduce colonization by pseudomonas ( 16,17 ) . the health of the mice was examined twice daily by laboratory staff and weekly by a veterinarian . this study was approved by the institutional animal care and use committee at the university of california , riverside . these lifespan studies utilized an unbalanced statistical design to minimize the number of mice per test group while maintaining statistical power ( 18 ) . unbalanced designs have economic and logistic advantages for comparing multiple treatments to a common control ( 18 ) . the group sizes in this study are similar to those required for a weibull survival analyses with a 75% probability of detecting an 10% increase in mean lifespan with a 1% probability of a false positive ( .01 ) . the weibull analysis is more stringent than the comparison of kaplan meier survival curves using the mantel cox or gehan breslow wilcoxon tests , implemented in graphpad prism 5.01 , which are used here . the significance of the differences in body weights between the treated and control groups was judged using a linear mixed effects model ( 19,20 ) as described previously ( 15 ) . in brief , we modeled the mean response by a set of fixed effects assumed to be shared by mice and a set of random effects that are unique to a particular mouse . additionally , our model imposed a common intercept since all mice were on the same diet at the time of the first measurement . to determine which group weights were significantly different than those of the controls , a bayesian information criterion ( bic ) model selection criteria , a likelihood ratio test ( lrt ) , and an akaike s information criterion ( aic ) model selection criteria were used ( table 1 and results ) . food consumption was determined by totaling the amount eaten by each treatment group during each time period , adjusted for the number of mice . summary of the statistical analysis of mouse group weights ( figure 2 ) using bic model selection removing each diet individually akaike s information criterion . drosophila ( wild - type oregon - r - c ; bloomington drosophila stock center , department of biology , indiana university , bloomington , in ) lifespan determinations were performed as described in detail previously ( 21,22 ) , using 0 , 1 , or 3mg / ml ndga . capillary feeder ( cafe ) assays ( 23 ) and fecal plaque assays ( fpas ( 24,25 ) ) were performed as described ( 26 ) . the effect of ndga on fpa plaque size was determined as described ( 21,22 ) . the effects of ndga on locomotor activity were determined as described previously ( 26 ) . briefly , one day old male flies were exposed to 3 mm ndga in dmso or vehicle treatments in 9525 mm glass vials for 3 days ( at 25 c with 12 hour light / dark cycle ) . after 3 days , movement of the flies was monitored for 72 hours using a trikinetics locomotor activity monitor ( lam 32 ) . means sem were calculated for 30 minutes time increments over the entire 72 hour trial ( 144 recordings ) . an unpaired , two - sample t test was used to determine the significance of differences between experimental and control treatments using graphpad prism 5 . mouse lifespan , weight , and food consumption were monitored as described in detail previously ( 15 ) . briefly , male b6c3f1 mice ( harlan breeders ; indianapolis ) were randomly assigned to treatment groups at 12 months of age . two hundred ninety - seven control mice were shifted from ad libitum chow feeding ( diet # 5001 , purina mills , richmond , in ) to daily feeding with 13.3 kcal / day / mouse of control diet [ american institute of nutrition ( ain)-93 m diet ; diet no . four groups of 18 mice each were shifted to daily feeding with an identical quantity of control diet supplemented with ndga at either 1.5 , 2.5 , 3.5 , or 4.5 g per kg diet ( approximately 1.59 , 2.65 , 3.71 , 4.77mg / kg body weight / day , respectively ) . food consumption and health were monitored at the time of feeding , and any uneaten food noted . with few exceptions , ndga was mixed with the powdered diet and cold - pressed into 1 g pellets by bio - serv . the mice had ad libitum access to tap water , which was acidified ( ph 4.0 ) to reduce colonization by pseudomonas ( 16,17 ) . the health of the mice was examined twice daily by laboratory staff and weekly by a veterinarian . this study was approved by the institutional animal care and use committee at the university of california , riverside . these lifespan studies utilized an unbalanced statistical design to minimize the number of mice per test group while maintaining statistical power ( 18 ) . unbalanced designs have economic and logistic advantages for comparing multiple treatments to a common control ( 18 ) . the group sizes in this study are similar to those required for a weibull survival analyses with a 75% probability of detecting an 10% increase in mean lifespan with a 1% probability of a false positive ( .01 ) . the weibull analysis is more stringent than the comparison of kaplan meier survival curves using the mantel cox or gehan breslow wilcoxon tests , implemented in graphpad prism 5.01 , which are used here . the significance of the differences in body weights between the treated and control groups was judged using a linear mixed effects model ( 19,20 ) as described previously ( 15 ) . in brief , we modeled the mean response by a set of fixed effects assumed to be shared by mice and a set of random effects that are unique to a particular mouse . additionally , our model imposed a common intercept since all mice were on the same diet at the time of the first measurement . to determine which group weights were significantly different than those of the controls , a bayesian information criterion ( bic ) model selection criteria , a likelihood ratio test ( lrt ) , and an akaike s information criterion ( aic ) model selection criteria were used ( table 1 and results ) . food consumption was determined by totaling the amount eaten by each treatment group during each time period , adjusted for the number of mice . summary of the statistical analysis of mouse group weights ( figure 2 ) using bic model selection removing each diet individually akaike s information criterion . drosophila ( wild - type oregon - r - c ; bloomington drosophila stock center , department of biology , indiana university , bloomington , in ) lifespan determinations were performed as described in detail previously ( 21,22 ) , using 0 , 1 , or 3mg / ml ndga . capillary feeder ( cafe ) assays ( 23 ) and fecal plaque assays ( fpas ( 24,25 ) ) were performed as described ( 26 ) . the effect of ndga on fpa plaque size was determined as described ( 21,22 ) . the effects of ndga on locomotor activity were determined as described previously ( 26 ) . briefly , one day old male flies were exposed to 3 mm ndga in dmso or vehicle treatments in 9525 mm glass vials for 3 days ( at 25 c with 12 hour light / dark cycle ) . after 3 days , movement of the flies was monitored for 72 hours using a trikinetics locomotor activity monitor ( lam 32 ) . means sem were calculated for 30 minutes time increments over the entire 72 hour trial ( 144 recordings ) . an unpaired , two - sample t test was used to determine the significance of differences between experimental and control treatments using graphpad prism 5 . male mice were fed ndga in their food at 1.5 , 2.5 , 3.5 , or 4.5g / kg diet ( 160 , 267 , 373 , 480mg / kg bw / d ) , beginning at 12 months of age . median lifespan was extended by 12% at 373mg / kg bw / d ( ~13.5mg / mouse / day ; mantel cox p = .008 ; gehan breslow wilcoxon p = .009 ; figure 1 ) . wilcoxon test gives more weight to deaths at early time points , while the mantel - cox test gives equal weight to all time points . despite its effects on median lifespan , we found no effect of 373 mg of ndga / kg bw / d ( 3.5g / kg diet ) on maximum lifespan using the method of gao et al . maximum lifespan is variously defined as the lifespan of the longest lived 10% or 20% of a cohort . here , we eliminated the lifespan data below a pooled quantile of 0.8 . the resulting mann shown are the lifespans of mice consuming ain-93 m diet without additions ( filled circles ; median survival 983 days ) ; mice consuming 1.5 g of ndga / kg ain-93 m diet ( open circles ; median survival 969 days ; mantel cox p = .769 ; gehan breslow wilcoxon p = .991 ) ; mice consuming 2.5 g of ndga / kg diet ( open squares ; median survival 1059 days ; mantel - cox p = .528 ; gehan breslow wilcoxon p = .490 ) ; mice consuming 3.5 g of ndga / kg diet ( open upward pointing triangles ; median survival 1099 days ; mantel - cox p = .008 ; gehan breslow wilcoxon p = .009 ) ; and mice consuming 4.5 g of ndga / kg diet ( open downward pointing triangles ; median survival 995 days ; mantel cox p = .963 ; gehan breslow wilcoxon p = .647 ) . the dose of ndga which extended median lifespan is within 12% of that initially reported to extend the lifespan of the nih - itp mice ( ~417mg / kg bw / d ( 12 ) ) . in our study , only this dose extended lifespan ( figure 1 ) . recently , the nih - itp reported lifespan extension using censored data at higher and lower dosages . further study will be needed to resolve these differences . the statistical design and animal husbandry used in these studies have been described in detail elsewhere ( 15,18,26,28 ) . food consumption and body weight food consumption and body mass were monitored throughout the study ( figure 2 ) . the mice ate essentially all their food each day , with the exception of a single excursion by the 1.5g / kg diet group late in life . this is consistent with the instability of the weights in late life due to the smaller number of mice and the presence of more age - related pathologies . shown is the body weight of the control ( closed black circles ) ; 40% cr fed ( closed orange circles ) ; 20% cr fed ( closed fuchsia circles ) ; 1.5 g ndga / kg diet ( closed gold circles ) ; 2.5 g ndga / kg diet ( closed green circles ) ; 3.5 g ndga / kg diet ( closed purple circles ) ; and 4.5 g ndga / kg diet ( closed blue circles ) . food consumption as a percent of the kcal offered with respect to the kcals consumed during the preceding month is shown for control , ( open black circles ) ; 40% cr fed ( open orange circles ) ; 20% cr fed ( open fuchsia circles ) ; 1.5 g ndga / kg diet ( open gold circles ) ; 2.5 g ndga / kg diet ( open green circles ) ; 3.5 g ndga / kg diet ( open purple circles ) ; and 4.5 g ndga / kg diet ( open blue circles ) . a preliminary report of these data from 365 to 1,063 days of age was published previously ( 14 ) . the weights of the mice were analyzed using a mixed effects model with a bic model selection criterion , as described previously [ table 1 ; reference ( 15 ) ] . these results suggest that the weights of the 2.5 and 4.5g / kg diet - treated mice , as well as the 20% and 40% cr mice were reduced significantly relative to those of the control group ( figure 2 ; table 1 ) . the differences between the weights of the control and the 1.5 and 3.5g / kg diet - treated groups were close to significance by this test ( table 1 ) . a lrt suggests that the weights of all the groups except the 1.5g / kg diet group differed significantly from those of the control group . an aic model selection criteria suggests that all the diets groups differed significantly from the control group ( table 1 ) . the group consuming the highest dose of ndga weighed less than the 20% calorically restricted ( 20% cr ) group at younger ages , when the data were more accurate due to sample sizes . pathologies associated with mortality death - related pathology has not been reported previously ( 1012 ) . the results of the necropsies from our study are summarized in tables 2 and 3 . focusing on the aggregated results for the pathologies for all the groups , there was a highly or very highly significant increase in liver and lung tumors , enlarged thymuses ( indicative of thymomas or lymphomas ) , and hemorrhage into the peritoneum ( hemorrhagic diathesis ; table 2 ) . response relationship for these pathologies , likely due to the relatively small group sizes . however , even the lowest dose of ndga produced significant pathologies ( table 2 ) . ndga decreased the size of liver tumors by 24% relative to those found in the control mice , suggesting that it may decrease the rate of tumor growth . however , the total mass of the tumors per mouse was unchanged , suggesting it may increase hepatic tumorigenesis ( tables 2 and 3 ; see discussion below ) . necropsy results from the mouse longevity studies shown in figure 1 * number of necropsied mice in each treatment group . significance of the differences from the control values were determined using fisher s exact test . for convenience , liver tumor mass of the mice shown in table 2 and figure 1 * tumor mass was calculated as ( /6 ) l w h 1.0 g , where l is length , w is width , and h is the height of each tumor . significant changes are in bold . in view of the clinical evidence of hepatotoxicity and nephrotoxicity in humans and rodents consuming ndga , we investigated whether ndga produced signs of toxicity at the concentrations used in these lifespan studies . male b6c3f1 mice were treated with ndga or control diet for 9 weeks , and clinical chemistries performed . alanine transaminase ( alt ) was significantly decreased by the higher doses of ndga , which is the opposite of what would be expected if the drug was toxic to the liver or kidneys . aspartate transaminase ( ast ) , alkaline phosphatase , bilirubin , and creatinine levels , which can also be signs of toxicity if elevated , were unchanged . con = control ; p = p - value . * eight male b6c3f1 mice were treated with ndga at each dosage or with control diet for 8 weeks . at 21 months of age clinical chemistries were performed on the 68 mice in each group with no obvious signs of pathology at necropsy . blood glucose levels were measured with the freestyle lite blood glucose monitoring system ( abbot laboratories ) . the other blood tests were performed by the comparative pathology laboratory , university of california , davis . all , results and p - values for all ndga treated groups considered together ; two lowest , the number of pathologies and the p - values for the 1.5 and 2.5g / kg diet fed mice considered together ; two highest , the number of pathologies and p - values for the 3.5 and 4.5g / kg diet fed mice considered together . calculated using unpaired two - sample t tests for convenience , significant changes are indicated in bold . however , the elevated triglyceride and glucose levels seem more likely to have resulted from the alterations in energy homeostasis evinced by the decrease in body weight in the absence of a change in caloric consumption ( figure 2 ; table 1 ) . conservation of the lifespan response to further characterize the effects of the drug , we investigated its effects on the lifespan of drosophila . optimum oral treatment with ndga at 3.0mg / ml medium increased the median lifespan of the flies by approximately 23% ( figure 3 ) . these results suggest that at least some of the target(s ) of ndga action are phylogenically conserved between mice and flies . the relatively short lifespans of the control flies are the result of using large flybottles , which induce more flight time ( 29 ) , male flies only ( 30 ) , mildly elevated incubation temperatures ( 31 ) , and food with a high protein concentration ( 3234 ) . each of these parameters somewhat shortens lifespan , although the flies remain responsive to compounds that lengthen lifespan [ e.g. references ( 21,22,26 ) ] . shown is the survival of drosophila fed diets containing either vehicle alone ( closed circles ; median survival 20 days ) , vehicle containing 1.0mg / ml ndga ( upward pointing triangles ) , or vehicle containing 3.0mg / ml ndga ( downward pointing triangles ; median survival 26 days ; both mantel cox and gehan breslow because drug induced cr or reduced locomotor activity might increase drosophila lifespan ( 29,35 ) , we investigated these parameters in ndga - treated flies . we found no effect of ndga on food consumption ( tables 5 and 6 ) , body weight ( table 7 ) , or locomotor activity ( table 8) . food consumption was quantified in two ways . we used our modifications of the cafe assay ( table 5 ) and the fecal plaque assay [ fpa ; table 6 ; references ( 21,22 ) ] . no effect on food consumption long - term ndga treatment also did not change body weight ( table 7 ) . locomotor activity was quantified using a lam 32 activity monitor ( trikinetics ) as described previously [ table 8 ; reference ( 26 ) ] . there was no significant difference between the activity of flies consuming food containing ndga or an equivalent volume of vehicle . together , these results indicate that the effects of ndga on drosophila lifespan were not due to cr or altered locomotor activity . further , we did not detect a disequilibrium between food intake and weight in this species as we did in the mice . ndga does not alter food consumption in drosophila as measured using cafe assays * one day old male flies were starved for 2 hours and exposed to capillary tubes containing a solution of 5% sucrose and 5% yeast extract with the addition 1% by volume of either 3 mm ndga dissolved in dmso , or dmso alone . a small volume of red food coloring was added to the food to aid in the visualization of the liquid . for a complete description see methods . consumption per fly is the mean sem of the difference between the initial volume in the capillary pipets at the start and completion of assay conducted with flies present less the average amount of liquid loss to evaporation in control assays conducted in the absence of flies , divided by the number of flies in the assays . four bottles per condition ( 50 flies per bottle ) . each bottle had four capillary tubes inserted into its sides . nul control bottles were identical except for the absence of flies . the significance of the difference in food consumption between the treated and control flies was determined using an unpaired , two - sample t test . two day old flies were starved for 2 hours and exposed to capillary tubes containing a solution of 5% sucrose and 5% yeast extract with the addition 1% by volume of either 3 mm ndga dissolved in dmso , or dmso alone . a small volume of red food coloring was added to the food to aid in the visualization of the fecal plaques . the significance of the differences between the treated and control groups was determined using an unpaired two - sample t test . * one day old flies were fed for 7 days with food containing either 3 mm ndga or an equal volume of vehicle ( dmso ) . a total of 7 groups ( n ) of 25 flies were weighed for each treatment condition . mean weight sem was calculated by dividing the total weight in milligrams of each group of flies by the number of individuals in the group . the significance of the differences in weight was determined using unpaired two - sample t tests . * flies were treated with either 3 mm ndga , dissolved in dmso , or medium containing an equal volume of dmso alone for 3 days , followed by 72 hours of monitoring at 25 c in a lam 32 activity monitor ( trikinetics ) . ten flies in each of 10 vials were monitored simultaneously for each condition . the average number of infrared beam disruptions per 72-hour period by the groups of 10 flies . the significance of the differences in the mean number of beam breaks for treated and control flies determined using unpaired two - sample t tests . the results presented here show that ndga can extend murine and drosophila lifespan . in our study , ndga consumption , even at doses below its therapeutic dose , was associated with an increase in multiple tumor types and hemorrhagic diathesis . the drug also increased the lifespan of drosophila , suggesting its target(s ) and mechanism(s ) of action may be phylogenetically conserved . however , aspects of the response in flies were unlike those in mice . in flies , ndga did not appear to affect energy uptake or utilization . in our studies , 3.5g / kg diet ( 373 mg ndga / kg bw / d ) extended lifespan , but higher and lower dosages were ineffective . the nih - itp found that ndga extended male mouse lifespan at 2.5 g ndga / kg diet , which they estimated supplied approximately 417mg / kg bw / d ( 10,11 ) . however , the actual dose was uncertain because food intake was not continuously measured in this study . more recently , they report that ndga at 0.8 or 5.0g / kg food produced lifespan effects similar to that of 2.5g / kg diet . these data were censored at 30% survival , and thus further study will be required to determine the dose response range of the lifespan effect in mice ( 12 ) . the nih - itp did not report necropsy or toxicity studies for their ndga - treated mice ( 10,12 ) . based on the mortality - related pathologies we found , there was an association between ndga consumption and an increased incidence of liver , lung , and thymus tumors , and increased hemorrhagic diathesis . there was little evidence for acute liver or kidney toxicity as measured by serum chemistries . however , others reported that treatment of mice with 5.0 g ndga / kg diet , a concentration reported to extend lifespan by the nih - itp , stunted the growth of male and female mice , and induced inflammatory cecal lesions , hemorrhages , and cysts in rats [ reviewed in ( 6 ) ] . thus , the doses of ndga which extend murine lifespan appear to overlap the dosages which produce serious pathologies . in humans , the consumption of dietary supplements containing creosote leaf and stem extracts resulted in toxic hepatitis , cirrhosis , and fulminant liver failure ( 4,7,36,37 ) . however , ndga also was found to suppress signaling through the igfir and to suppress the androgen - stimulated growth of human prostate cancer cells in culture ( 3840 ) . these studies led to clinical investigation of its safety and efficacy for the treatment of prostate cancer ( 41,42 ) . in humans , continuous oral dosing at up to 2.5g / d ( ~31mg / kg bw / d ) , one - tenth of the dose which extended lifespan in this study in mice , was tolerated , except for the development of elevated ast or alt ( transaminitis ) in some patients ( 42 ) . drug dosages in rodents are often scaled down approximately 10-fold in mg per kg body weight for investigational studies in humans ( 14 ) . in a phase ii study of men with non - metastatic hormone - sensitive prostate cancer , oral doses of 2.0 g ndga / d in 28 day cycles produced transaminitis in 8 patients ( 67% ) . the development of less toxic derivatives of ndga , such as tetra - o - methyl nordihydroguaiaretic acid , are underway for potential use as cancer therapeutics ( 43,44 ) . possible mechanisms for the longevity effects of ndga a large number of molecular mechanisms have been proposed to explain the presumed , but as yet largely undocumented , health benefits of ndga [ reviewed in ( 4 ) ] . perhaps the most probable mechanism for the longevity effects of ndga is its inhibitory effects on the activity of mammalian target of rapamycin complex 1 ( mtorc1 ( 45 ) ) . mtorc1 inhibition is a well - established mechanism for the extension of mouse and drosophila lifespan ( 22,4649 ) . a recent review of the literature argues that rapamycin inhibition of mtorc1 extends mouse lifespan by suppressing cancer formation and growth ( 50 ) . however , it will be important to discover whether the dosages of ndga that extend lifespan actually inhibit mtorc1 activity in tissues shown to be important to its longevity effects . in our studies , ndga decreased the mass of liver tumors by 24% relative to those found in control mice , suggesting that it decreased the rate of liver tumor growth ( table 3 ) . these observations are consistent with in vitro and in vivo studies showing that ndga inhibits the growth and proliferation , and induces the apoptosis of multiple cancer cell types ( 5154 ) . these effects likely result from the inhibitory effects of ndga on growth factor signaling ( 3840 ) . in mammals , ndga inhibits igfir activation , igfir and her2 tyrosine kinase activity , androgen dependent growth of cultured prostate tumor cells , and growth of cultured her2-overexpressing human breast cancer cells ( 3840 ) . in our studies , ndga appeared to increased hepatic tumor number ( tables 2 and 3 ) , and to increase the prevalence of lung and thymus tumors ( table 2 ) . taken together , the data suggest that while ndga reduces tumor growth rates , it increases tumor formation . inhibition of igfi receptor signaling can enhance rodent lifespan , although these effects are sex and background dependent ( 55,56 ) . there is less of an effect on lifespan on the c57bl/6j background than on the 129/svpas background ( 56 ) . female mice heterozygous null for the igf1r exhibit extended longevity , while their male counterparts do not ( 56,57 ) . in other studies , neither male nor female mice with reduced igfi levels experience an increase in mean lifespan ( 55 ) . together , these results suggest that if a lifespan effect is observed when igfr activity is inhibited , females , rather than males respond . thus , this response is the opposite of that found with ndga , where males rather than females respond . thus , reduced igfir activity alone is unlikely to be the major mechanism by which ndga extends murine lifespan . hormetic stimuli are thought to produce molecular damage at low doses , which stimulate increased maintenance and repair , and thereby increased longevity ( 58 ) . at high doses masoro and others have proposed that cr is a hormetic agent ( 35,59,60 ) . in one example , the genic profiles produced by a group of progeroid mutations recapitulate the profiles produced by the prolongevity treatments of cr and gh deficiency in mice ( 60 ) . the data regarding ndga are not sufficiently developed to indicate whether it is a hormetic longevity agent . it appears to produce molecular damage at its optimum dosage for increasing lifespan , as evinced by the induction of cancers ( table 2 ) and modest liver and kidney toxicity in humans ( 41,42 ) and mice ( table 4 ) . an unanswered question is whether the less toxic derivatives of ndga such as tetra - o - methyl nordihydroguaiaretic acid will also be capable of extending murine and drosophila lifespan ( 43,44 ) . one theory proposes that ndga exerts positive effects on health because it is a potent scavenger of activated oxygen and nitrogen species in vitro and in vivo ( 4 ) . however , exogenous antioxidants have not been shown to reproducibly extend mammalian lifespan , and they can shorten it ( 15,28,6164 ) . further , the consumption of agents that increase oxidative stress , rather than reduce it , appear to be associated with enhanced longevity in mammals and other species ( 65 ) . ndga reportedly suppresses proinflammatory gene expression and prostaglandin e 2 production and thereby inhibits arachidonic acid 5-lipoxygenase and cytokine - stimulated activation of microglia and macrophages ( 66,67 ) . some data support an inverse correlation between inflammation and lifespan ( 68 ) . however , correlative evidence has been misleading in the context of oxidative stress ( 61 ) . in the absence of direct evidence for an association between reduced inflammation and extended lifespan , inhibition of oxidative phosphorylation can increase rodent and fly lifespan ( 69,70 ) . however , decreased rates of oxidative phosphorylation are not consistent with the dose responsive loss of body weight observed in our mice ( figure 2 ; table 1 ) . inhibition of oxidative phosphorylation should preserve body weight , since it reduces the utilization of calories . in flies , ndga had no effect on body weight , physical activity , or food consumption ( tables 58 ) . thus , our data are inconsistent with the extension of lifespan through inhibition of oxidative phosphorylation . the funders had no role in study design , data collection or analysis , decision to publish , or preparation of the manuscript .

mesonordihydroguaiaretic acid ( ndga ) extends murine lifespan . the studies reported here describe its dose dependence , effects on body weight , toxicity - related clinical chemistries , and mortality - related pathologies . in flies , we characterized its effects on lifespan , food consumption , body weight , and locomotion . b6c3f1 mice were fed ain-93 m diet supplemented with 1.5 , 2.5 , 3.5 , or 4.5 g ndga / kg diet ( 1.59 , 2.65 , 3.71 and 4.77mg / kg body weight / day ) beginning at 12 months of age . only the 3.5mg / kg diet produced a highly significant increase in lifespan , as judged by either the mantel cox log - rank test ( p = .008 ) or the gehan breslow wilcoxon test ( p = .009 ) . ndga did not alter food intake , but dose - responsively reduced weight , suggesting it decreased the absorption or increased the utilization of calories . ndga significantly increased the incidence of liver , lung , and thymus tumors , and peritoneal hemorrhagic diathesis found at necropsy . however , clinical chemistries found little evidence for overt toxicity . while ndga was not overtly toxic at its therapeutic dosage , its association with severe end of life pathologies does not support the idea that ndga consumption will increase human lifespan or health - span . the less toxic derivatives of ndga which are under development should be explored as anti - aging therapeutics .

pubmed

genetic variation contributes to both disease susceptibility and treatment response . genome - wide association studies ( gwas ) have enabled rapid discovery of genetic variants contributing to the pathogenesis of complex genetic diseases ( manolio 2010 ) , as well as detection of many pharmacogenetic markers ( link et al . the driving hope of these major advances in genetic epidemiology is that promotion of personalized medicine will improve medical decision - making . although use of the term personalized medicine is often limited to the identification of the optimal drug and the optimal dosage for a subgroup of patients , current personalized medicine applications are far more broad , and might include situations of withholding treatment , preventive interventions , or targeted treatment options for individual patients . in prostate cancer , for example , dna biomarker tests may be used to determine whether treatment may be safely delayed for a period of watchful waiting . if the tumor is demonstrated due to lack of genes causing an aggressive form of the cancer , it may remain stable for decades , and the need for radical surgical resection with subsequent radio- or chemotherapy may be obviated ( kroll 2008 ) . in contrast , in other instances , genetic profiles may be used to determine preventive interventions . this approach is already used for some forms of hereditary cancer , in which individual genetic testing is the basis for deciding upon specific , sometimes very radical interventions such as preventive surgery ( kroll 2008 ) . beyond treatment schemes that are applied identically across large subgroups of patients to which some authors have applied the distinct term stratified medicine ( trusheim et al . 2007)other personalized medicine applications offer targeted treatment options for individual patients . anti - inflammatory therapies , for example , such as anti tnf , anti il-6 , or anti il-1 , are thought to be effective in inflammatory diseases , such as crohn s disease ( buchner et al . other molecules seem suitable for use as anti - inflammatory therapy as well . to choose one or a combination of different anti - inflammatory therapies , the physician might first obtain the genetic profile of a patient by sequencing . depending on the individual dna profile , the physician might select the anti - inflammatory combination therapy , of which there are many . though foundational to personalized medicine , biological markers , biomarkers for short , are diversely defined in the literature . gallo et al . ( 2011 ) summarize some of the definitions , and observe that the most commonly adopted definition states that a biomarker is any substance or biological structure that can be measured in the human body and may influence , explain or predict the incidence or outcome of disease . however , it is a matter of debate whether the qualification that biomarkers must be measured in the human body is a reasonable limitation . ( 2011 ) who define biomarkers as any substance , structure or process that can be measured in biospecimen and which may be associated with health - related outcomes . from our perspective , this definition is too general and should include a specific association with a health or clinical outcome . our preference , therefore , is for the definition developed by the biomarkers definitions working group ( 2001 ) : a biomarker is a characteristic that is objectively measured and evaluated as an indicator of normal biological processes , pathogenic processes or pharmacologic responses to a therapeutic intervention . although more than a decade old , this definition is comprehensive and sufficiently broad to capture the full range of current biomarker applications , described in detail in the table 1.table 1important terms in biomarker studiestermexplanationbiomarkera characteristic that is objectively measured and evaluated as an indicator of normal biological processes , pathogenic processes , or pharmacologic responses to a therapeutic intervention ( biomarkers definitions working group 2001).cancer biomarkera biomarker that is present in tumor tissue or serum and includes many different molecules , such as dna , mrna , or proteins . tumor biomarkers are measured in tumor tissue , and tumor dna biomarkers are measured from tumor tissue.clinical endpointa characteristic or variable that reflects how a patient feels , functions , or survives ( biomarkers definitions working group 2001).companion endpointa biomarker that is essential to the efficacy and safety of a corresponding therapeutic product ( food and drug administration 2011).copy number variant ( cnv ) biomarkera biomarker of genomic variation in which blocks of dna are missing or for which multiple copies exist.diagnostic biomarkera biomarker that relates to the diagnosis or severity of disease . the most important diagnostic biomarkers are screening biomarkers.disease biomarkera biomarker that relates to a clinical endpoint or measure of disease ( kroll 2008).dna biomarkera germline biomarker , such as snps , strs , deletions , insertions , or other variation on the dna sequence level.efficacy biomarkera biomarker that predicts a beneficial effect of a given treatment ( kroll 2008).epigenetic biomarkera biomarker that measures epigenetic alterations , such as dna methylation , histone methylation , histone acetylation , micrornas , or other non - coding rna ( bock 2009).monitoring biomarkera biomarker to monitor efficacy or side effects of a drug treatment.prognostic biomarkera biomarker that predicts the likely course of disease in a defined clinical population under standard treatment conditions.prediction modela predictive test including multiple markers.predictive biomarkera biomarker that forecasts the likely response to treatment ( buyse et al . 2011 ) . treatment response may be measured either as efficacy or as safety.predictive testtwo definitions exist in the literature a test of test of probability for an individual to develop a disease ; alternatively , a test which discriminates between individuals who will develop a disease and those who will not ( janssens and van duijn 2008).risk predictionthe generation or validation of models which make a prognosis for developing a disease or the prognosis for attaining a clinical endpoint.safety biomarkera biomarker that indicates adverse response to a treatment ( sistare et al . toxicity biomarkers are special cases of safety biomarkers.screening biomarkera biomarker to discriminate between healthy individuals and those in an early stage of the disease ( kroll 2008 ) , ideally while subjects are asymptomatic.staging biomarkera biomarker that distinguishes between different stages of chronic disease ( kroll 2008).stratification biomarkersee predictive biomarker.surrogate biomarkera biomarker that is regarded as a valid substitute for a clinical endpoint . a surrogate endpoint is expected to predict clinical benefit ( or harm or lack of benefit or harm ) ( biomarkers definitions working group 2001 ; kroll 2008).target biomarkera biomarker that reports interaction of the drug with its target ( kroll 2008).toxicity biomarkera biomarker that reports to the toxic effect of a drug on an in vitro or in vivo system ( kroll 2008 ) . important terms in biomarker studies in personalized medicine , it is necessary to distinguish between prognostic and predictive biomarkers . following buyse et al . ( 2011 ) , the difference is that prognostic biomarkers help in predicting the progress of the disease , while predictive biomarkers are connected with the response to a treatment . having clarified fundamental terms , we will proceed to a discussion of similarities and differences of biomarkers , illustrate current uses of some of these biomarkers , and outline specific aspects of diagnostic , prognostic , and predictive biomarker studies . finally , we will discuss various study designs for the validation of predictive biomarkers in detail . genetic information , coded within dna , requires stability because dna directs the production of proteins required for the cell structure and function of cells over a lifetime . some authors state that dna is stable over an individual s lifetime ( hicks and coquoz 2009 ) , and biomarkers explicitly representing this stability are termed dna biomarkers in the discussion which follows . single nucleotide polymorphisms ( snps ) , short tandem repeats ( strs ) , deletions , insertions , or other variation on the dna sequence level are among this group . due to the availability of high - throughput molecular biological facilities , snps are the most commonly used type of dna variation . in most applications snps cancer is a disease that involves changes to the dna at the cellular level , and these changes can be measured in the tumor . distinct from dna biomarkers outlined above , we will use the term dna tumor biomarkers to indicate biomarkers specific to cancerous tumors . typically , only the presence or absence of a mutation in a gene is determined . finally , we use the term general biomarkers for all other forms of biomarkers , such as rna , protein , or metabolite measurements which can be measured in biofluid , tissue , or even cell lines . while most general biomarkers share the property of being quantitative with positive measurement values , both dna biomarkers and dna tumor markers are discrete in nature ( table 2 ) . nevertheless , when used in the diagnostic process , thresholds need to be introduced for all types of biomarkers to relate biomarker measurements to clinical decision-making.table 2differences between dna biomarkers , dna tumor biomarkers , and general biomarkerscharacteristicdna biomarkerdna tumor biomarkergeneral biomarkerlevel of measurementdiscrete . in snps , one of three different genotypes is observed per subject , in generaldiscrete . in general , the measurement is whether a specific gene is mutated or notcontinuous . rna , protein , and metabolite concentrations may take almost any continuous positive valuestability , reproducibilityyesnot necessarily as different mutations may be present in different parts of the tumoronly at one specific time pointsuitable for therapy monitoringnoyesyessuitable for pharmacodynamicsnoyesyessuitable as surrogate markernono , in generalyescomplexity of measurementlowhighhightime required for measurement ( includes drawing and preparation of sample)lowhighhightime of measurementdoes not have to be specifiedneeds to be specified in advanceneeds to be specified in advanceretrospective validation of biomarkeryesnonodurability of the final biomarker testshort- to long - term ; multimarker sets may be already obsolete at start of prospective studymid - term to long - termmid - term to long - termstudy designretrospective or prospectiveprospective onlyprospective onlydna biomarkers are generally measured in the blood , tumor dna biomarkers are measured in tumor tissue , general biomarkers may be measured in biofluid , tissue , or cell lines differences between dna biomarkers , dna tumor biomarkers , and general biomarkers dna biomarkers are generally measured in the blood , tumor dna biomarkers are measured in tumor tissue , general biomarkers may be measured in biofluid , tissue , or cell lines important differences between dna biomarkers and dna tumor or general biomarkers stem from the fact that dna is stable over the entire lifetime ( table 2 ) . dna biomarkers are reproducible , can be measured at any point in time , and may be used in both prospective and retrospective studies . specifically , dna biomarkers can be prospectively validated in biobanks , i.e. , in studies , where clinical information has already been collected . the authors stress that they would not call such kind of study a prospective one . in general , sample drawing , handling and storage protocols are generally also simpler for dna biomarkers , and laboratory time and cost for measurement is lower . first , as they do not vary over lifetime , they can not be used for therapy monitoring , pharmacodynamics , or as surrogate markers . a second general problem is durability , as the rate of discovery of new dna biomarkers is frequently more rapid than their product cycles . by the time , a dna multimarker test has successfully passed all steps for marketing approval , including refunding by health insurance companies , it may already be rendered scientifically obsolete by newly discovered dna biomarkers with seemingly better performance . pertinent differences between dna tumor biomarkers and general biomarkers ( table 2 ) include the fact that dna tumor biomarkers generally can not serve as surrogate markers while general biomarkers often can and the fact that dna tumor biomarkers can show greater variation , depending on how they are measured . if a dna tumor biomarker is measured through biopsy , for example , it is possible for one biopsy probe to be tumor free , while another contains tumor cells , and this difference may result in different dna tumor biomarker results . in contrast , if a general biomarker is measured , for example , from plasma , it is generally stable for the time point of measurement . in this section , we illustrate current applications of biomarkers for diagnosis , prognosis , and prediction in personalized medicine . details of the examples outlined in this section are provided in table 3 , together with additional examples of general biomarkers , and in particular , dna biomarkers and dna tumor biomarkers , in current use.table 3examples for biomarkers in current usenametyperange of applicationcommercial useindicationtime of measureoutcomereferenceblueprintdna tumorpredictiveyesbreast cancerknown diagnosis , after surgeryreaction of individual therapieskrijgsman et al . ( 2012)egfrdna tumorpredictivepcoadvanced non - small - cell lung cancerknown diagnosis , before first - line therapyegfr tki or chemotherapykeedy et al . ( 2011)il28bdnapredictive hepatitis c virus 1 ( hcv-1)known diagnosis , before treatmentresponse to treatment with pegylated interferon ( peg - ifn ) combined with ribavirin ( rbv)holmes et al . ( 2011)k - rasdna tumorprognosticpcoadvanced colorectal cancerknown diagnosis , before chemotherapytreatment with cetuximab yes or nokarapetis et al . ( 2008)mammaprintdna tumorprognosticyesbreast cancerknown diagnosis , after operationprecise stage of tumor , aggressivity of tumorbuyse et al . ( 2011)oncotypdxdna tumorpredictive / prognosticyeser - positive , her2-negative breast cancer , colon cancerknown diagnosis , after operationchemotherapy recommended yes / nobuyse et al . ( 2011)point - of - care tests : rheumachecccpoint assaygeneraldiagnostic ( screening)yesrheumatoid arthritis ( ra)before first symptomaticearlier therapy for raegerer et al . ( 2009)slco1b1dnapredictive myocardial infarctionknown diagnosis , before treatmentreduction of statin doses cause of statin - induced myopathy , security monitoringlink et al . ( 2008)egfr epidermal growth factor receptor , egfr tki egfr tyrosine kinase inhibitor , er estrogen receptor , her2 hormone receptor , k - ras kirsten rat sarcoma viral oncogene homolog , pco provisional clinical opinion of the american society of clinical oncology examples for biomarkers in current use egfr epidermal growth factor receptor , egfr tki egfr tyrosine kinase inhibitor , er estrogen receptor , her2 hormone receptor , k - ras kirsten rat sarcoma viral oncogene homolog , pco provisional clinical opinion of the american society of clinical oncology diagnostic biomarkers are biomarkers used to determine the diagnosis or severity of a disease . the most important within this group are screening biomarkers ( table 1 ) , which are used to discriminate between healthy individuals and those in an early stage of a disease . for example , the commercially available point - of - care tests ( poct ) rheuma - chec and ccpoint , test serum for antibodies to mutated citrullinated vitmentin ( mcv ) or citrullinated peptides / proteins ( anti - ccp antibodies ) , in order to screen for rheumatoid arthritis in non - symptomatic , healthy persons ( egerer et al . 2009 ) . if a diagnosis is known , prognostic biomarkers help to predict the likely course of disease in a defined clinical population under standard treatment conditions . for example , mammaprint , a dna tumor biomarker for breast cancer prognosis is used following surgery to indicate whether risk of metastasis is low or high , and guide physicians to determine the best kind of treatment for the individual patient . in fact , in the united states , mammaprint is cleared by the food and drug administration ( fda ) as an in vitro diagnostic multivariate index assay ( ivdmia ) ( slodkowska and ross 2009 ) . in germany , as of 2012 , some health insurance companies will pay the cost of the test for particular cases . predictive biomarkers predict the likely response of a patient to a special treatment in terms of efficacy and/or safety and thus support clinical decision - making ( table 1 ) . for example , gwas conducted by three independent groups from north america , australia / northern europe , and japan ( holmes et al . 2011 ) demonstrated that the il28b gene is a strong indicator for response to standard treatment in patients with hepatitis c virus-1 ( hcv-1 ) infection . further , there is evidence for population stratification in the il28b gene , such that treatment response varied across different ethnic groups . specifically , caucasians with the good response genotype were more likely to benefit from treatment . this fact is also mentioned in a provisional guidance for the treatment of hcv ( thomas et al . 2012 ) , which notes that treatment with special inhibitors is expected to be less efficacious in persons with the non - response genotype , or african ancestry . while some biomarkers have already been approved by the fda , the use of others has been recommended in clinical guidelines such as the provisional clinical opinion from the american society of clinical oncology ( asco ) , which publishes clinical direction based on potentially practice - changing data from major studies . a recent example of this is a test for epidermal growth factor receptor ( egfr ) mutation in patients with advanced non - small - cell lung cancer , which determines whether or not the first - line egr tyrosine kinase inhibitor therapy is indicated ( keedy et al . regulatory approval is required before a new drug can enter the market . in the preclinical program , safety , pharmacology and pharmacodynamics the first three clinical phases establish pharmacokinetics , pharmacodynamics , the required dose , clinical efficacy and benefit / risk . following marketing approval , effects on morbidity and mortality endpoints , or relative effectiveness are investigated in the fourth phase ( european medicines agency 1998b ) . development programs of diagnostic tests undergo a similar process ( european medicines agency 2009 ) . in phase i , it must be demonstrated that the diagnostic test may be safely applied to humans , and that the technical properties are well understood , such as mode of application , reproducibility , etc . in phase ii phase iii studies are the so - called validation studies , in which the diagnostic test and subsequent diagnostic workup is carried out in the same manner and setting as it would be in later clinical and diagnostic practice . finally , phase iv diagnostic studies are performed to investigate whether the application of the test leads to a measurable improvement of the clinical outcome in a broader population . this phase concept is oriented towards marketing approval of diagnostic agents that are intended for in vivo administration , such as radiopharmaceuticals or contrast agents for use in imaging techniques , such as magnetic resonance imaging . in our own projects , we have found an extended phase approach , summarized in table 4 , to be more helpful from a developmental perspective for diagnostic or prognostic biomarkers ( compare the phase models of pepe 2003 , p. 215 2009).table 4phases of diagnostic or prognostic biomarker studiesphasediagnostic and prognostic biomarkerstypical sample sizesdescriptionaim of studyiadiscoveryidentification of promising biomarkers10100ibassay development , assay validationdefine and optimize analytical process into robust , reproducible , and valid device10100icretrospective validationclinical assay detects disease ; development of first algorithm for combination test50500iiretrospective refinementvalidation of early detection properties of biomarker ( set ) ; development and/or refinement of algorithm(s ) for combination tests1001,000iiiprospective investigationdetermination of diagnostic accuracy ( sensitivity , specificity ) in the situation of clinical routine2001,000ivarandomized controlled trialquantification of effect of making the biomarker information available to the doctor to reduce disease burden2001,000ivbhealth economics studyquantification of cost - effectivenessstrongly depends on clinical consideration of clinical risk phases of diagnostic or prognostic biomarker studies in phase i of this extended approach , we delineate three distinct sub - phases : discovery ( ia ) , assay development and validation ( ib ) , and retrospective validation ( ic ) . the discovery study ( phase ia ) the discovery phase might be a gwas , a genome - wide meta - analysis or even a whole genome sequencing study . in proteomics , this discovery phase might consist of expression clone arrays containing tens of thousands of recombinant human proteins ( for a recent application to prostate cancer , see massoner et al . 2012 ) or multiplex protein antigen analysis on a luminex platform ( linkov et al . the high - throughput arrays or whole genome sequences will not be the final product for the diagnostic test to be released on the market . therefore , in phase ib the identified biomarkers are adapted to a laboratory setting which might be integrated into clinical routine . the complementary technology introduced in this phase is generally more reliable and precise , i.e. , its coefficient of variation is lower and typically has reduced bias . for dna biomarkers , this phase combines assay transfer with a better - suited laboratory platform and the choice of the dna marker(s ) to be typed on this platform . in proteomics , this might represent the development of an elisa , such as the elisa for agr2 in voided urine for detecting secreted prostate cancer ( wayner et al . 2011 ) . following the discovery phase and the change in the specific laboratory technology , a first retrospective validation is generally performed ( phase ic ) to determine whether the results from the imprecise high - throughput technology still hold true . it is important to note that the sample size in this first retrospective validation is higher than in the initial high - throughput search ( table 4 , last column ) . subsequent to retrospective validation , multimarker models are developed , either as part of phase ic , with the initial cohort of patients , or as part of phase ii , in a retrospective study using patients different from those of phase i. whether initially developed in phase i or ii , this model will always undergo refinement with the second group of patients . nevertheless , a prospective investigation in phase iii may also often be necessary to obtain reliable estimators for sensitivity and specificity of dna biomarker tests , or , for prognostic biomarkers , to attain reliable estimates of the prognosis , e.g. , through the use of brier scores . to assess the impact of the diagnostic dna biomarker on patient management , a randomized controlled trial is conducted ( in phase iva ) , following marketing approval demonstrating that treating doctors knowledge of the test result improves patient outcome . finally , health economics studies may be carried out in order to assess cost - effectiveness of the diagnostic test ( phase ivb ) . a challenging element of this extended phase model for diagnostic and prognostic biomarkers is the term prospective validation for phase iii . because germline dna is assumed to be stable , the prospective investigation of an assay in an already ascertained biobank may be considered to be prospective . however , it must be definitively demonstrated that the biobank has not been used before , because multiple testing will arise if a biobank is used for biomarker validation more than once . proper definition of standards as to when biobanks may be used for prospective validation of dna biomarkers is complex . the situation becomes even more complex when considering dna tumor biomarkers or general biomarkers , as these biomarkers may change over time such that the time point of sample drawing may also be crucial . furthermore , different sample handling and storage protocols as well as age of samples may have an effect not only on biomarker quality but also on biomarker levels ( table 2 ) . irrespective of the phase model used ( for other alternatives , see , e.g. , zhou et al . 2001 , p. 61 ) , assay sensitivity the technical and analytical validity of the biomarkers is a mandatory requirement . given high assay sensitivity , the problem of missing values can be ignored because they are assumed to occur only due to random technical failure and not any systematic process . examples for current use of biomarkers , above , and detailed in table 3 , a number of biomarkers have recently been identified to predict treatment outcome , and some have proven particularly effective in resolving unconvincing or ambiguous clinical trial results . for example , in the multicenter ipass ( iressa pan - asia study ) trial of advanced non - small cell lung cancer ( nsclc ) , patients treated with either gefitinib or carboplatin plus paclitaxel , progression - free survival curves of both treatment groups crossed in the general population of patients ( 1,200 previously untreated east - asian non - smokers or former light smokers ) . when treatment groups were stratified for tumors bearing epidermal growth factor receptor gene ( efgr ) mutation , however , important differences emerged : egfr - positive patients progression - free survival was consistently longer in the gefinitab treatment group , while egfr - negative patients progression - free survival was longer in the carboplatin paclitaxel group ( mok et al . 2009 ) . as noted in sect . examples for current use of biomarkers , the strength of the relationship was sufficiently compelling that the american society for clinical oncology has since recommended that patients with nsclc should have their tumors tested for egfr mutations to determine the most appropriate first - line therapy ( keedy et al . 2011 ) . in most instances these biomarkers have been identified after the conduct of phase iii trials that were intended to provide the pivotal evidence about efficacy and positive benefit / risk of an experimental treatment required to justify market approval . an important consequence of the retrospective nature of these investigations was that the appropriateness of the biomarker validation had to be assessed on a case - by - case basis . in july 2011 , the fda issued a draft guidance for industry for in vitro companion diagnostic devices , which are predictive biomarkers essential for safe and effective use of a corresponding therapeutic product ( food and drug administration 2011 ) . the draft guidance anticipated three uses of companion diagnostics : identification of patients most likely to benefit from a particular therapeutic product;identification of patients likely to be at increased risk for adverse reactions to a therapeutic product , and;monitoring treatment response to adjust treatment in order to improve efficacy and safety . identification of patients most likely to benefit from a particular therapeutic product ; identification of patients likely to be at increased risk for adverse reactions to a therapeutic product , and ; monitoring treatment response to adjust treatment in order to improve efficacy and safety . if either treatment effect or tolerability differs with respect to the companion diagnostic , the diagnostic test can be used to refine the patient population . dna biomarkers and dna tumor biomarkers can not be used for treatment monitoring for obvious reasons . the fda guideline suggests that because companion diagnostics provide critical information for the appropriate use of drugs , they require validation as part of the evaluation of efficacy of the experimental treatments , and information about the diagnostic is reflected in the drugs labeling . the use of biobanks for prospective validation , therefore , might play a role for already approved drugs , if biomaterial allows systematic investigations into improved efficacy or safety in biomarker - defined subgroups . within the pharmaceutical industry , such research will become increasingly important to pharmacovigilance and post - marketing surveillance of drug use . valid conclusions are , however , not possible if material for genotyping is available for only so - called convenience samples ( wang et al . , clinical data should substantiate that efficacy and/or benefit / risk of the experimental treatment as compared to control is substantially larger in patients who test positive with the companion diagnostic test than in those who test negative . if the experimental treatment shows comparable positive effects in test - negative patients and in test - positive patients , the diagnostic test would not be required , and the new treatment might simply be provided to all patients . thus , diagnostic tests are only of value if clinical data or ethical reasons support that treatment be withheld in test - negative patients . in the context of clarifying specific circumstances in which a new drug might be used appropriately , information from test - negative patients must be available in order to justify population refinement , and the costs that use of the biomarker prior to treatment will incur on the health insurance system . it may be a matter of debate , or even discriminative power of the companion biomarker , whether this information might be obtained from a phase ii clinical trial , or whether it should be obtained from the same trial used to demonstrate efficacy of the experimental treatment . obviously , there is no need to demonstrate in statistical terms that the experimental treatment does not have advantages over control in test - negative patients . however , if in the same trial there is no clear trend towards superior efficacy or improved benefit / risk , especially in a phase ii trial of limited size , the usefulness of the companion biomarker might be questioned . official regulatory guidance about appropriate study designs to validate companion diagnostics together with new drugs is still sparse , although both the fda and european medicines agency ( ema ) have established procedures , in which applicants may negotiate the amount of evidence required for marketing authorization in this setting ( european medicines agency 2012 ) . pioneering work on clinical trial designs for predictive biomarker validation has been performed by sargent et al . 2011 ) , in their seminal review , discuss 10 different study designs for co - development of an experimental drug and a companion diagnostic test . in selection designs , patients are first tested with the companion test . only test - positive patients are then randomized to treatment or control ( fig . ( 2011 ) observe , although these selection designs provide clear information about treatment efficacy , they fall short in substantiating the usefulness of the companion test , as they do not demonstrate lack of benefit in marker - negative patients . as soon as a test incurs costs , its usefulness for application should be quantified , and for this a selection design is only of limited value.fig . a traditional randomized - control design where the randomization ( r ) to standard ( std ) or experimental ( exp ) therapy is independent from the results of the biomarker test . randomization to std or exp is performed in the total patient population stratified by the result of the biomarker results . c restricted design . to reduce effort , only biomarker - positive patients are randomized to std or exp . claims about the utility of a biomarker can not be made from this trial alone . d patients are randomized according to treatment based on biomarker or non - biomarker - dependent strategy . this is the most flexible design that provides information about specific individualized treatment rules according to , e.g. , a dna profile common study designs for biomarker studies . a traditional randomized - control design where the randomization ( r ) to standard ( std ) or experimental ( exp ) therapy is independent from the results of the biomarker test . randomization to std or exp is performed in the total patient population stratified by the result of the biomarker results . c restricted design . to reduce effort , only biomarker - positive patients are randomized to std or exp . claims about the utility of a biomarker can not be made from this trial alone . d patients are randomized according to treatment based on biomarker or non - biomarker - dependent strategy . this is the most flexible design that provides information about specific individualized treatment rules according to , e.g. , a dna profile in the interaction or biomarker - stratified design ( fig . 1b ) , patients are first tested for the biomarker , and then randomized to treatment or control with stratification by the companion biomarker s test result . this study design might be considered the gold - standard design for providing a sound basis for decision - making about the efficacy and benefit / risk of the experimental drug and the ability of the companion diagnostic to identify the appropriate patient population to be treated . the significance of the interaction test for the treatment effect in the test - positive and test - negative stratum may serve as a yardstick for the usefulness of the companion biomarker . if the interaction effect is significant , treatment effects in test - positive and test - negative patients differ ; this difference alone does , however , not in itself assure the utility of a companion diagnostic . a small but significant treatment effect in test negatives , even if less marked than the effect in the biomarker - positive population , might still be of clinical importance . similarly , if the treatment effect in the larger population is small and irrelevant , the subgroup of biomarker - positive patients might be too small to achieve sufficient power in the interaction test . evaluation of efficacy and benefit / risk should always consider the size of the estimated treatment effect as well . adaptive study designs offer a promising means to stop recruitment for futility in test - negative patients as soon as sufficient evidence about lack of efficacy in this subgroup is available . strategies based on conditional power may be used to formalize adaptive study designs to some extent ( lachin 2005 ; proschan and hunsberger 1995 ; schfer et al . however , in the end , the assessment of the treatment effect size and the extent of benefit / risk must be set into perspective . in instances where no gold standard are available , the utility of a diagnostic test may be evaluated by comparing biomarker - guided treatment with the standard , non - biomarker - based treatment selection . a highly simplified version of this is depicted in fig . 1c with only two profiles and two treatments in the biomarker - guided treatment group . again , significant differences between the outcome in the biomarker - guided and the conventional treatment group may be difficult to achieve and may fall short in demonstrating the efficacy of the experimental biomarker . the individual profile design , which includes a large number of different profiles leading to the selection of one out of a large number of different treatments , is easily validated if it is planned and understood as a strategic trial comparing conventional treatment selection to an individualized decision rule ( fig . for example , an individualized therapy might combine several monotherapies , each selected based on the presence or absence of a specific dna variant . however , they pose new challenges to the regulatory system , requiring that the precise particularities of the application of drugs as mono - therapy , or in combination are well understood and substantiated with clinical trials data . obviously , there is a gap between the validation of the biomarker - based treatment selection rule and the efficacy of the individual treatments to be applied based on a complex decision rule . consideration needs to be given to the question of how this gap can be filled . for example , the global statistics may demonstrate superiority of the individualized treatment selection when compared to the current standard . at the same time , some of the individual treatment combinations may seem to be inferior to the current standard . here , it is clear that an upfront discussion is required to formulate the circumstances under which such inconsistencies can be ignored and under which the overall validity of conclusions has to be questioned . similarly , none of the many investigated treatments or treatment combinations may provide sufficient clinical information enabling the assessment of the safety of the suggested combination therapy in the way this is done in the standard drug development process , and we acknowledge that this standard process is justified by historical fallacies . unless clear evidence is available that all drugs under investigation are safe and can be combined freely , a substantial amount of clinical data needs to be provided for each of the recommended treatments and treatment combinations . at least a basic assessment of safety is required because safety or benefit / risk may be different for different subpopulations identified with the biomarker rule . along the line of the fda definition of a companion test such information is a pre - requisite to use a complex decision rule in patient management . of course , sample - size requirements will substantially increase with the number of treatments and treatment combinations under investigation , and the ipass trial can be considered as a model for this . in some instances , biomarkers may be investigated that are closely related to the mechanism of action of the companion drug , and in these instances it may be possible to provide the required information about the usefulness of the companion test as early as phase ii drug development . in most instances , however , the traditional design randomizing patients to treatment and control irrespective of biomarker outcome may still be the optimal approach , such that overall superiority of a drug should first be demonstrated , subgroups defined by the companion test excluded from the labeling in retrospect , following the precautionary principle that harm does not need to be proven ( fig . the credibility of this concept depends on the degree of independent validation that can be found in the different studies during the development program and the completeness of the sampling in every study . convenience sampling in subpopulations of poorly defined origin should be avoided ( wang et al . 2010 ) . the three pillars of a physician s work are diagnosis , therapy , and prognosis . biomarkers form the basis for all aspects of personalized medicine , from what does the patient have ? ( diagnosis ) to what can the patient do about it ? ( therapy ) to how bad is it ? ( prognosis ) . although the characteristics and applications of dna biomarkers , dna tumor biomarkers and general biomarkers differ substantially , the underlying methodological principles to validate each for use in clinical routine are identical . dna biomarkers are distinct from general biomarkers in several ways , such as protein expression levels , gene expression levels or even epigenetic biomarkers . further , since germline dna does not change over time , some studies utilizing biobanks may be interpreted as prospective biomarker studies under very special conditions . however , the critical aspect prior to marketing approval of a biomarker is how convincing evidence may be achieved and when it has been achieved . novel strategies must be developed so that a biomarker assessed in biobanks can be considered validated , in particular if biobanks are to be used multiple times . an important aspect of biobank biomarker studies is the transparency of the approach , which directly relates to the quality of reporting . in general , to date , reporting of biomarker studies has been weak . in a systematic review of studies published between 2004 and 2006 , fontela et al . ( 2009 ) evaluated diagnostic studies that used commercially available test kits for tuberculosis , hiv , and malaria , and found that all of the 238 articles fulfilling the study inclusion criteria had design issues . in only 10 % of the studies the reference standard was adequately described . nine of the 25 indicators of the standards for reporting of diagnostic accuracy ( stard ) ( bossuyt et al . 2004 ) were reported in fewer than 25 % of the studies , even though the studies reviewed were published immediately following publication of the stard statement . ( 2006 ) have effectively demonstrated for therapeutic studies , if reporting guidelines are adopted by journals , and in consequence by authors , the quality of reporting is measurably improved . in addition , it is substantially simpler to read articles if authors follow reporting statements . therefore , authors of biomarker studies are strongly encouraged to report their work using appropriate reporting guidelines , such as stard , strengthening the reporting of observational studies in epidemiology molecular epidemiology ( strobe - me ; gallo et al . 2011 ) , reporting recommendations for tumor marker prognostic studies ( remark ; mcshane et al . 2010 ) or the preferred reporting items for systematic reviews and meta - analyses ( prisma , formerly quorum ; moher et al . 2009 ) ; for a complete list , the reader may refer to the equator network at http://equator-network.org . while validation of diagnostic and prognostic biomarkers does not need to be linked to a specific therapy but rather to standard of care , validation of predictive biomarkers must be closely linked to the specific therapy under consideration . in order to gain marketing approval , pivotal drug trials , i.e. , randomized controlled phase iii clinical trials generally operate from the paradigm recruiting a broad spectrum of patients , not only to achieve higher enrollment numbers , but also to investigate the generalizability of findings . if , however , a drug has different levels of efficacy and safety in different ethnicities , as in for example gemcitabine , tamoxifen ( sai and saito 2011 ) or warfarin ( el rouby et al . 2004 ) , it might be possible to leverage the power to detect efficacy and demonstrate safety of a drug as a rationale to conduct trials in ethnically homogenous populations . although the ich e5 guideline ( european medicines agency 1998a ) clearly outlines a framework to evaluate the impact of ethnic factors , the use of an ethnically homogeneous population to evaluate the effect of a biomarker might be more promising if the aim is detection of different effects between biomarkers ( see the study of link et al . if recruitment is done on a global level in a phase iii clinical trial , one has to expect substantial population stratification , i.e. , heterogeneity in the patient population , and as sample sizes per ethnic group are eventually small , identification of biomarkers might be hindered . even if a biomarker or a set of biomarkers has been identified to be a good diagnostic , prognostic , or predictive marker , application to clinical routine although predictive biomarkers may be readily applied , as specific therapies rely on biomarker results , clinical applications for diagnostic or prognostic biomarkers are more complex . here , use in clinical routine will depend on the ease of applicability , including the media for biomarker measurement , and whether clinicians can measure the biomarker in their own laboratory , a local laboratory , or a specialized laboratory at some distance from their practices . also important if clinicians are able to perform calculations by hand and the rules are easy to interpret , acceptance of the biomarker(s ) is more likely than if some kind of black box is required . therefore , classifications and probabilities estimated by a logistic regression model are more likely to be accepted by clinicians than results obtained by machine learning methods , such as artificial neural networks or support vector machines we conclude that a priori planning of research strategies is vital for the identification and validation of biomarkers .

biomarkers are of increasing importance for personalized medicine , with applications including diagnosis , prognosis , and selection of targeted therapies . their use is extremely diverse , ranging from pharmacodynamics to treatment monitoring . following a concise review of terminology , we provide examples and current applications of three broad categories of biomarkers dna biomarkers , dna tumor biomarkers , and other general biomarkers . we outline clinical trial phases for identifying and validating diagnostic and prognostic biomarkers . predictive biomarkers , more generally termed companion diagnostic tests predict treatment response in terms of efficacy and/or safety . we consider suitability of clinical trial designs for predictive biomarkers , including a detailed discussion of validation study designs , with emphasis on interpretation of study results . we specifically discuss the interpretability of treatment effects if a large set of dna biomarker profiles is available and the number of therapies is identical to the number of different profiles .

pubmed

myosin va ( myova ) is a processive molecular motor involved in intracellular cargo transport along the actin cytoskeleton [ 2 , 3 ] . each dimerized molecule consists of a pair of n - terminal motor domains ( i.e. , heads ) connected to lever arms , followed by a coiled - coil rod domain , ending in a globular c - terminal cargo - binding domain . myova 's directed movement is accomplished by a conformational change in the motor domain following atp hydrolysis [ 5 , 6 ] . this motion is amplified by the lever arms , resulting in the motor 's processive motion as each head takes 72 nm steps in a hand - over - hand fashion [ 79 ] . each lever arm consists of six tandem -helix motifs , stabilized by calmodulin or calmodulin - like light chains . calcium is required to activate the atpase activity of myova ; however in the presence of high calcium ( > 1 m ) , motor processivity is reduced and one or more calmodulins may dissociate from each lever arm . calmodulin dissociation may render the lever arm highly compliant , which could reduce the efficiency of both the lever arm displacement amplification and the strain - dependent gating between the two heads , which are necessary for the motor 's processivity . single myova are adept at maneuvering through actin intersections and traveling along actin bundles in vitro [ 1417 ] . myova must have sufficient structural flexibility to permit the unbound leading head to undergo a broad diffusive search [ 7 , 1820 ] , allowing myova to switch actin tracks even when the angle between intersecting actin filaments is ~150 . the structural domains that are critical to myova 's maneuverability have yet to be determined but may reside within the lever arms or at the lever arm / coiled - coil junction . here we used tethered particle microscopy ( tpm ) to directly measure the flexural stiffness of individual myova motors and their subdomains . tpm has been used extensively to characterize the contour length of dna tethers [ 2225 ] . tpm has additionally been coupled with monte carlo simulations of nanoparticles attached to wormlike chain tethers to estimate , with high precision , the persistence length ( a measure of flexural stiffness ) of double - stranded dna . our utilization of tpm involved anchoring a myova - actin complex to a microscope slide and then attaching a visible particle ( i.e. , fluorescent quantum dot ( qdot ) ) to a desired location along the motor . thermal energy caused the qdot to be randomly displaced within bounds defined by the length and stiffness of the effective tether ( i.e. , the myova ) . the resulting motion was measured by comparing the difference in apparent image size of the tethered qdot to a stationary , reference qdot , both of which are viewed over a long ( ~2.5 s ) exposure time [ 24 , 25 ] . while a qdot held in a fixed location has an apparent image size ( sr ) which is dependent on its diffraction limited point spread function , tethered qdots will appear larger ( st ) depending on the length and flexibility of the tether ( figure 1 ) . by attaching qdots to expressed myova constructs of different lengths ( figure 2 ) , we isolated the contributions of individual structural domains , for example , the lever arm and rod domain , to the overall flexibility and flexural stiffness of the myova molecule . in addition , we hypothesized that calmodulin disassociation from the myova lever arm decreases the lever arm 's flexural stiffness , thus contributing to the motor 's compromised processivity in the presence of calcium . various myova constructs , listed as follows , were expressed in the baculovirus / sf9 cell system and purified as described previously . these constructs allowed the flexural stiffness of myova subdomains to be defined by tpm ( figure 2 ) . full - length murine myova ( myova - fl ) was expressed as was a double - headed short construct ( myova - hmm ) , engineered by truncating the myova - fl heavy chain at residue 1098 , followed by a biotin tag and a flag epitope tag at the c - terminus to facilitate purification [ 14 , 28 ] . double - headed myova constructs were coexpressed with calcium - insensitive calmodulin ( camall ) as described previously . single - headed myova ( myova - s1 ) was truncated at amino acid 908 with a biotin tag at the c - terminal end and coexpressed with wild - type calmodulin and purified as described . the myova - s1 and its wild - type calmodulins are sensitive to calcium in that calmodulin(s ) will dissociate from the lever arm in the presence of calcium in the solution . the biotin tag is an 88-amino - acid segment from the escherichia coli biotin carboxyl carrier protein , which was biotinylated at a single lysine residue ( located 35 amino acids from the c - terminus ) during expression in sf9 cells . n - ethylmaleimide- ( nem- ) modified skeletal muscle myosin was prepared as previously described and was used to strongly adhere actin filaments to the glass surface . actin filaments were isolated from chicken pectoralis ; labeled with tetramethyl rhodamine isothiocyanate ( tritc ) phalloidin overnight in buffer a ( 25 mm imidazole , ph 7.4 , 4 mm mgcl2 , 1 mm egta , 25 mm kcl , and 10 mm dtt ) prior to experiments . carboxylated qdots , emitting at 655 nm ( invitrogen - molecular probe , eugene , or ) , were attached to the myova - fl cargo - binding domain by incubating a 4 : 1 mixture of qdots to myova - fl for 15 min on ice in buffer b ( 25 mm imidazole , ph 7.4 , 4 mm mgcl2 , 1 mm egta , 25 mm kcl , 10 mm dtt , oxygen scavenger composed of 0.1 mg / ml glucose oxidase , 0.020.18 mg / ml catalase , and 3.0 mg / ml glucose ) and 0.1 mg / ml bsa . streptavidin - conjugated qdots , also emitting at 655 nm ( invitrogen - molecular probe , eugene , or ) , were attached to the c - termini of the myova - hmm and myova - s1 constructs by mixing qdots and construct ( 4 : 1 ratio ) and incubating for 15 minutes on ice in buffer b plus 0.5 mg / ml bsa . the mixture was further diluted to a final myova construct concentration of ~0.1 nm before imaging . to attach streptavidin - conjugated qdots to actin filaments , biotinylated actin filaments were prepared by mixing 1 m actin filament , 500 nm tritc - phalloidin , and 500 nm biotin - phalloidin ( invitrogen ) in buffer a and then incubating overnight at 4c . biotinylated actin filaments ( 50 nm ) were attached to the glass surface through nem - modified myosin ( see section 2.3 for details ) . after washing the flow cell with buffer then 24 nm streptavidin - conjugated qdots ( emitting at 655 nm ) were introduced into the flow cell . to remove the unbound qdots , the flow cell was washed by buffer a. a 20 l flow cell was constructed as described . first , 20 l of nem - modified myosin at 0.5 mg / ml in buffer c ( 25 mm imidazole , ph 7.4 , 4 mm mgcl2 , 1 mm egta , 300 mm kcl , and 10 mm dtt ) was introduced and incubated for 2 minutes at room temperature . nem - modified myosin was used to fix the actin filaments to the glass surface , as it has strong binding affinity to actin . after washing the flow cell using buffer b , 20 l of 0.5 mg / ml bsa in buffer b was infused and incubated for 2 min , followed by a buffer b wash . tritc - phalloidin labeled actin in buffer b was then introduced into the flow - cell chamber and incubated for 2 min , followed by a buffer b wash containing a myova : qdot complex . all myova constructs were strongly bound to actin in the absence of mgatp , that is , in rigor . it is assumed that myova constructs bound to the top surface of the actin filaments due to the high entropy required to approach the filaments from the sides . to measure the calcium - dependent flexibility of the myova - s1 construct , calcium was added to the myova - s1:qdot complex in buffer b , to a final concentration of 1 m , and then introduced into the flow cell . single molecule imaging was performed at room temperature ( 23 1c ) using a nikon eclipse ti - u inverted microscope equipped with a planapo objective lens ( 100x , 1.49 n.a . ) through the objective tirf microscopy . images were acquired using piper software ( piper control v2.3.14 software , stanford photonics , stanford , ca ) and a high resolution ( 95 nm per pixel ) digital camera ( standard photonics , turbo - z , stanford , ca ) . qdots were excited with a 473 nm argon laser line and images obtained using a 655 20 nm emission filter ( chroma technologies , rockingham , vt ) at 8.3 ms integration time for 300 frames . following imaging of the qdots , a single image of the actin filaments in the same visual field was obtained by exciting the tritc - phalloidin labeled actin with a 532 nm laser line and the emission acquired through a 605 35 nm filter ( chroma technologies , rockingham , vt ) and a 67 ms exposure time . first , each series of frames was converted to double precision and summed for an effective integration time of 2.49 s , which was determined to be suitably long to sample the full extent of particle motion ( see results and discussion ; figure 3 ) . summation of short exposures was chosen for this study rather than a single long exposure in order to reduce noise and confirm that the microscope stage did not drift during the experimental protocol . qdots were automatically identified ( figures 4(a ) and 4(b ) ) by thresholding the summed images to binary and recording the centroids of regions which fit three different selection criteria . regions were included based on size ( < 144 pixels ) and elliptical eccentricity ( < 0.86 , which is an aspect ratio of approximately 2 : 1 ) to reject regions likely representing multiple qdots in close proximity to each other . additionally , distance from edges of the image qdot apparent image sizes were then measured by extracting a twelve - pixel square subwindow around each qdot ( figures 4(c ) and 4(d ) ) . this window size was suitably large to fully encompass the qdot image that on average was approximately seven pixels in diameter . each window was then fit , using the least squares method , by a two - dimensional gaussian function ( figure 4(e ) ) , in local coordinates x - y of the form(1)g = a12x2ex+x2/2x212y2ey+y2/2y2+b , where a is the intensity of the qdot , b is the mean intensity of the background , x and y are the x - axis and y - axis standard deviations of the gaussian function , and x and y are offsets between the center of the two - dimensional gaussian function and the center of the window . an image size parameter , s , for each qdot was then defined as the average of x and y multiplied by a microscope image calibration factor ( 95 nm per pixel ) . surface bound qdots ( i.e. , stationary and fixed ) were used as an internal reference and control for the tethered qdot image size parameters in the same field of view , whether they be on an actin filament or a myova construct . the reference and tethered qdots were differentiated by merging images of qdots and actin filaments and manually selecting the qdots not colocalized with actin as reference qdots ( figure 4(a ) ) . when two orthogonal ( i.e. , not correlated ) signals oscillating around a central point are summed , the resulting variance is the sum of the variances of the two signals . given two signals with standard deviations 1 and 2 , the sum of the two signals will thus have a deviation , 3 , given by the square root of the sum of squares:(2)3=12+22 . both the reference image size parameter , sr( ) , and the tethered particle motion , t( ) ( where may refer to images with qdots bound to actin filaments , a , or myova constructs , m ) , are defined as the deviations of normally distributed random signals , which , added together , result in an apparent image size parameter for the tethered qdot ( i.e. , st( ) ) . therefore , st( ) results from letting 1 = sr( ) and 2 = t( ) in ( 2 ) . this then allows the motion of the tethered particle to be determined when median values of reference ( sr( ) ) and tethered qdot ( st( ) ) image size parameters are known as in ( 3 ) . due to nonnormality of image size parameter frequency distributions ( figure 5 ) , equation ( 2 ) may also be used to relate the deviations of any two points on a randomly oscillating structure that is fixed at one end to a rigid structure . this is particularly useful for canceling out the compliance of the nem - modified myosin , which results in motion of the actin filaments , t(a ) . the motion of a qdot tethered by a myova construct , corrected for actin filament motion , ma , may then be determined knowing the myosin tethered qdot motion , t(m ) , and the baseline actin filament motion , t(a):(4)ma=tm2ta2 . by substituting ( 3 ) into ( 4 ) , the motion of a particle tethered by a myova construct relative to actin , ma , was related to the various median measured image size parameters:(5)ma = stm2srm2sta2+sra2 . for median values of st( ) and sr( ) measured for each construct , the standard error of the median ( sem ) was calculated from the standard deviation ( sd ) and sample size ( n ) assuming an approximately gaussian distribution:(6)sem=1.25sdn . error propagation through ( 5 ) ( ema ) was calculated from the median image size parameters of tethered and actin - bound qdots ( st(m ) and st(a ) ) along with the sizes of their associated reference qdots ( sr(m ) and sr(a ) ) and their respective standard errors ( et(m ) , et(a ) , er(m ) , and er(a)):(7)ematastm2etm2+tasrm2erm2+tasta2eta2+tasra2era2=stm2etm2+srm2erm2sta2eta2+sra2era2stm2srm2sta2+sra2 . the flexural stiffness for the various myova constructs , k , was determined by equipartition due to thermal energy , resulting in brownian motion of the tethered qdot relative to the actin filament , ma , where kb is boltzmann 's constant and t is temperature in degrees kelvin:(8)k = kbtma2 . the persistence length of the tether , lp , is defined as ei / kbt , where ei is the flexural rigidity of a prismatic rod . assuming that the tether acts as a cantilevered beam , with load imparted primarily through the qdot at the end , the effective spring constant , k , can be related to flexural rigidity by k = 3ei / l . combining these two expressions and substituting into ( 8) allow lp to be calculated directly from the motion of the tethered qdot relative to actin ( ma ) and the length of the molecule , l ( see figure 2):(9)lp = l33ma2 . in addition to being used to measure the flexural stiffness of myova constructs , the effective motions of the distal rod domain were isolated by using the procedure above with the motion of qdots bound to myova - hmm constructs rather than actin filaments as the baseline of comparison for myova - fl . the validity of the tethered particle motion approach was confirmed by defining the relationship between myosin tethered qdot motion relative to the actin filament ( ma ) ( see ( 5 ) ) and the assumed tether length , l , for the various myova constructs , which was estimated to span from the actin filament surface to the center of the attached 20 nm qdot , with the length of the myova construct based on its structure ( see figure 2 ) . to test the significance or the trend in ma with l , a linear regression was performed using prism 6 ( graphpad inc . ) . in general , ma was expected to increase with increasing l , which in fact was the case ( see figure 6 ) . a pilot study showed no significant effect of up to 100 nm of displacement out of the focal plane on qdot image size parameters ( see figure s1 in supplementary material available online at http://dx.doi.org/10.1155/2015/465693 ) , confirming that the trend in increasing ma with l was , in fact , the result of tether compliance and not focal separation between tethered and reference qdots . to estimate the flexural stiffness of myova - fl and its subdomains , we utilized long exposure time tpm imaging of qdots attached to various myova constructs that served as flexible tethers ( figure 2 ) . the long exposure times were necessary since the brownian excursion time for a qdot tethered to a myova construct should have been on the order of microseconds . based on our experience , imaging qdots with acceptable signal : thus , accurately assigning the tethered qdot 's location at a single point in time was effectively impossible . however , at the other extreme , millisecond exposures do not fully sample the qdot 's excursion overall times . therefore , to accurately estimate myova 's flexural stiffness , the full range of tethered qdot motion is needed ; otherwise the ambiguity in tracking the qdot 's position results in a significant underestimate of motion . imaging for relatively long exposure times ( 100 ms ) by summing sequentially acquired images ( see section 2 ) ( figure 3 ) circumvents this issue by sampling the full distribution of particle locations and becomes insensitive to exact exposure times . a further advantage of tpm is the ability to efficiently estimate the flexural stiffness of a large number of myova constructs simultaneously within each visual field . we measured image size parameters for qdots bound to actin and tethered by various myova constructs . before relating these parameters to estimates of tether motion and thus flexural stiffness , it was important to control for the effective motion ( i.e. , noise ) in the imaging system , the mode of qdot attachment , and the actin filament movement to which the various myova constructs were bound ( figure 2 ) . to control for the imaging system noise , image size parameters were obtained for qdots bound to the microscope slide and the nem - modified myosin layer under identical imaging conditions ( figure 2 ) . these image size parameters were no different ( 185.8 2.8 nm and 184.3 2.3 nm mean sem , resp . ) , indicating that image size parameters for qdots in the visual field that were not bound to actin and are suitable for use as stationary , surface - bound qdot references , sr . however , qdots bound directly to actin filaments showed 17 nm of motion , t(a ) , relative to reference qdots , r(a ) ( table 1 ) . this amount of motion indicates substantial compliance in the nem - modified myosin attachment to the actin filaments , requiring the use of actin - bound qdots and their motion as a corrective baseline for myova stiffness estimates ( table 1 ) . using a range of different length myova constructs ( figure 2 ) , we observed a proportional increase in tethered qdot motion relative to actin , ma , with increasing tether length , that is , the length of the myova construct , l ( table 1 ; figure 6 ) . the linear regression of ma with respect to l was statistically significant ( slope = 0.341 , p < 0.01 ) , with all points falling between the theoretical bounds of 1 ( which assumes a tether with zero stiffness ) and 0 ( perfectly rigid tether ) . in addition , to the flexural stiffness of the various myova constructs , we could estimate the flexural stiffness of myova subdomains , that is , the lever arms , lever arm / coiled - coil junction , and distal rod ( table 1 , figure 7 ) as presented below . myova 's lever arms and their elasticity are critical to the motor 's processive movement . differential strain in the motor 's lever arms while undergoing processive movement is apparent in electron and atomic force microscopic images in which bending of the leading head 's lever arm is observed as it attempts its power stroke against the resistive load of the strongly bound trailing head . with the kinetics of the chemomechanical cycle of the individual heads being load - dependent , the difference in strain experienced by the leading and trailing heads is believed to underlie the gating mechanism that assures forward displacement of the motor along actin [ 21 , 36 ] . therefore , the lever arm flexural stiffness may be tuned via calcium signaling to effectively allow or inhibit this necessary intramolecular communication between heads . using tpm , the motion of qdots attached to the c - termini of single - headed myova - s1 constructs relative to actin - bound baseline qdots was used to estimate the lever arm stiffness ( see ( 5 ) ) . based on electron microscopic images , the position of a strongly bound myova head domain appears fixed on the actin filament relative to the observed swinging motion of the lever arm during the power stroke . therefore , we assumed that the tethered qdot motion predominantly reflects the 0.0346 pn / nm flexural stiffness of the lever arm ( figure 7 ; table 1 ) with a corresponding persistence length of 242 nm . a significantly higher stiffness value of 0.21 pn / nm for the myova - s1 was estimated using oscillatory length perturbations in the laser trap assay . however , this approach most likely imposed a combination of bending and axial tension , which would be expected to lead to higher stiffness values . the tpm method more closely approximates pure bending , so that the tpm estimate for persistence length agrees with our previous estimate of 310 nm , based on myova - s1 step size measurements in a laser trap assay . in these earlier studies , the motor itself imposed an internal bending strain on the lever arm due to the power stroke working against the stiffness of the laser trap and thus would have been dominated by bending rather than tensile forces . following the addition of calcium , the average motion of qdots tethered to the myova - s1 c - terminus increased by 10% , indicating a 40% reduction in lever arm flexural stiffness ( figure 7 ; table 1 ) . a substantial decrease in stiffness is expected , given that the addition of calcium can disassociate as many as three calmodulins from each lever arm , exposing the underlying alpha - helix , which by itself is assumed to be unstable [ 12 , 13 , 37 ] . it is important to note that although calmodulins could dissociate from the lever arm in the absence of calcium without excess calmodulin in the assay buffer , as in our experimental conditions , we do not believe this was the case . it has been shown that myova - hmm processivity , as defined by the motor 's run length , is a sensitive measure of interhead communication that relies on rigid lever arms [ 12 , 37 ] . thus , the addition of calcium leads to an abrupt decline in processivity as calmodulins dissociate and the lever arms become compliant [ 12 , 37 ] . however , in control experiments ( figure s4 in supplemental materials ) , run lengths for myova - hmm with and without excess calmodulin in the assay buffer were unchanged , confirming that the lever arms ' calmodulin occupancy under our zero calcium conditions was fully intact . interestingly , in addition to an increase in the average tethered qdot motion with calcium , there was a large increase in the variance of the qdot motion , with the sem increasing from 3 nm to 21 nm ( figure 6 ) . the number of calmodulins dissociated from each individual lever arm is variable , with an average of 2.2 per lever [ 13 , 37 ] , which may account for the broad distribution of observed tethered qdot motions for the myova - s1 construct in the presence of calcium ( figure 6 ; table 1 ) . the observed decrease in lever arm flexural stiffness is likely large enough to negatively impact the interhead communication and gating necessary for force production and processivity . the myova rod domain lies between the lever arms and the globular tail domain ( figure 7 ) . this rod is predominantly -helical coiled - coil and contains two pest sites , with the -helical coiled - coil in the proximal rod prior to the first pest site necessary for myova dimerization ( figure 7 ) . to estimate the flexural stiffness of the distal rod domain , we compared the tethered qdot motion of the myova - fl construct to that of the myova - hmm construct , which was truncated just before the first pest site ( figures 2 and 7 ) . our measurements suggest that the distal rod segment , itself , has a flexural stiffness of 0.0326 pn / nm . unlike the lever arm , however , it is difficult to apply ( 9 ) and calculate a persistence length for the rod segment , as the exact length and structure of the distal rod are unknown , with estimates of the distal rod ranging up to 38 nm not including the 7 nm globular tail domain [ 4 , 27 ] . the observed increase in tethered particle excursion requires the distal rod to have some effective length , though the measured stiffness requires a structure composed of either a long ( ~35 nm ) segment of coiled - coil ( lp = 100 nm ) or a series of coiled - coil segments interspersed with highly compliant disordered segments . both the regulatory interaction between the globular tail and motor domains [ 27 , 40 , 41 ] and the variation in apparent rod lengths in electron micrographs strongly suggest the latter . interestingly , the flexural stiffness for the myova - hmm and myova - fl constructs is an order of magnitude lower than both the distal coiled - coil rod domain and lever arms ( table 1 , figure 7 ) . with the tpm measurements for both of the myova - hmm and myova - fl constructs obtained in rigor , we assume that for either construct both heads are strongly bound to the actin filament . the two - headed bound state with the two lever arms in a triangular arrangement should give a combined stiffness for the two levers , which is greater than a single lever arm and greater than the flexural stiffness of the rod . thus , the extremely low myova - hmm and myova - fl flexural stiffness require that the lever arm / rod junction must be highly compliant . this compliance must contribute to the free diffusive search of the unbound leading head [ 18 , 19 ] , allowing myova to maneuver through actin - actin intersections and to switch tracks on actin bundles . we have developed a unique system , utilizing long exposure tpm , to measure the flexural stiffness of single molecules with lengths less than 100 nm . the estimates of flexural stiffness for various myova constructs and subdomains by tpm offer direct insight into the structural components of the myova motor that are critical to its functional capacities . specifically , the lever arms must be rigid enough to allow intramolecular strains to be communicated between heads for proper gating and control of the motor 's processivity . the compliance of the lever arm / rod junction must have sufficient flexibility to allow myova to avoid intracellular obstacles and deliver cargo efficiently to its destination in vivo through the dense actin meshwork . in addition , the potential for alterations in the lever arm stiffness with changes in intracellular calcium may provide a measure of tunability in regulating cargo transport and delivery linked to changes in the cell 's ionic environment .

myosin va ( myova ) is a processive molecular motor involved in intracellular cargo transport on the actin cytoskeleton . the motor 's processivity and ability to navigate actin intersections are believed to be governed by the stiffness of various parts of the motor 's structure . specifically , changes in calcium may regulate motor processivity by altering the motor 's lever arm stiffness and thus its interhead communication . in order to measure the flexural stiffness of myova subdomains , we use tethered particle microscopy , which relates the brownian motion of fluorescent quantum dots , which are attached to various single- and double - headed myova constructs bound to actin in rigor , to the motor 's flexural stiffness . based on these measurements , the myova lever arm and coiled - coil rod domain have comparable flexural stiffness ( 0.034 pn / nm ) . upon addition of calcium , the lever arm stiffness is reduced 40% as a result of calmodulins potentially dissociating from the lever arm . in addition , the flexural stiffness of the full - length myova construct is an order of magnitude less stiff than both a single lever arm and the coiled - coil rod . this suggests that the myova lever arm - rod junction provides a flexible hinge that would allow the motor to maneuver cargo through the complex intracellular actin network .

pubmed

the aim of this study was to determine whether clinical outcome after surgical resection of esophageal adenocarcinoma ( eac ) or esophageal squamous cell carcinoma ( escc ) could be predicted by functional polymorphisms in different proto - oncogenes and tumor suppressor genes . six single nucleotide polymorphisms ( snps ) in the aurka ( rs2273535 ) , erbb2 ( rs1136201 ) , mdm2 ( rs2279744 ) , cdh1 ( rs5030625 ) , cdkn2a ( rs11515 ) , and tp73 ( rs2273953 ) genes were genotyped in a consecutive cohort of 346 esophageal cancer patients , who had underwent surgical resection with curative intent . associations with disease - free survival ( dfs ) were analyzed with kaplan meier curves and cox regression , adjusting for potential confounders . univariate analysis showed no significant associations between the tested polymorphisms and dfs in patients with eac or escc . however , in a multivariate analysis , patients with eac carrying the heterozygous mdm2 ( rs2279744 ) t / g genotype had significantly improved dfs compared with patients carrying the wild - type genotype ( adjusted hazard ratio ( ahr ) , 0.63 ; 95% confidence interval ( ci ) [ 0.450.88 ] ) . patients with eac harboring the homozygous cdh1 ( rs5030625 ) ga / ga genotype had a significantly reduced survival as compared with patients carrying the wild - type genotype ahr 4.0 , 95% ci [ 1.411 ] . in a large cohort of esophageal cancer patients , the mdm2 t / g and cdh1 ga / ga genotype confer risk of death in patients with eac . these data suggest that inter - individual differences in germ - line dna have an impact on dfs in patients with eac . many therapeutic options are used to treat esophageal cancer , but traditionally , surgery is used most frequently to obtain loco - regional control and long - term survival.1,2 comprehensive preoperative staging has improved selection of patients for potentially curative surgery ; however , many patients present with recurrent disease within 2 years after operation . the majority of these patients develop not only loco - regional recurrences , but also distant metastases ( such as liver , lung , pleural , and/or peritoneal disease recurrences ) are common.35 despite attempts to improve the outcome of patients with esophageal cancer , prognosis remains poor with a 5-year overall survival of 2030%.5,6 well - known prognostic factors for esophageal cancer are summarized in tumor node metastasis ( tnm ) staging.7,8 although tnm parameters have the advantage of simplicity , they do not seem to completely reflect the biologic diversity of esophageal cancer.9,10 the true drivers of this clinical biologic diversity include the molecular aberrations of the cancer and the genetic make - up of the patient . in this respect , the study of host genetic variability offers worthwhile potential to identify individuals that may have the best chance of survival . single nucleotide polymorphisms ( snps ) in the germ - line are the most common type of host genetic variations . gene - related functional snps can potentially lead to differences in protein expression and/or function . in this way , snps in proto - oncogenes and tumor suppressor genes can potentially alter the risk for metastatic or aggressive tumor , resulting in differences in clinical outcome . altered expression of the aurka , erbb2 , mdm2 , cdh1 , cdkn2a , and tp73 proteins has been correlated to disease progression and clinical outcome in patients with esophageal cancer.1117 in addition , polymorphisms with effects on protein function have been identified in these proto - oncogenes and tumor suppressor genes.1823 based on these results , we postulated that functional snps in the aurka ( aurka_nm_003600.2 ; rs2273535 c.449 t > a ) , erbb2 ( erbb2_nm004448.2 ; rs1136201 c.655 a > g ) , mdm2 ( mdm2_nm002392.2 ; rs2279744 309 t > g ) , cdh1 ( cdh1_nm004360.3 ; rs5030625 -347 g > ga ) , cdkn2a ( cdkn2a_nm000077.3 ; rs11515 c.712 c > g ) , and tp73 ( tp73_nm005427.1 ; rs2273953 81 g > a ) genes may serve as molecular markers for clinical outcome in patients with esophageal adenocarcinoma ( eac ) or esophageal squamous cell carcinoma ( escc ) who underwent surgical resection . patients between 1996 and 2001 , a total of 632 consecutive patients with esophageal cancer were evaluated for surgery with curative intent at the erasmus university medical center ( fig . 1 ) . outcome for all patients with esophageal cancer referred to our hospital are collected prospectively and stored in a database by a data manager . all patients were staged using esophago - gastroscopy with biopsies , ultrasonography of the cervical and upper abdominal region , and computed tomography of the thorax and abdomen . endoscopic ultrasonography for evaluation of t - stage and nodal status was routinely performed . 1flowchart of patients with esophageal cancer referred to the erasmus mc for treatment between january 1996 and december 2001 . patients excluded from the present study are shown flowchart of patients with esophageal cancer referred to the erasmus mc for treatment between january 1996 and december 2001 . patients excluded from the present study are shown surgery for carcinomas of the upper half of the intra - thoracic esophagus , a right - sided thoracotomy was performed . for carcinomas of the lower half of the intra - thoracic esophagus , the tumor and its adjacent lymph nodes were dissected en bloc ; however , no extended lymph node dissection was performed . the continuity of the digestive tract was restored by means of a gastric tube reconstruction or colonic interposition with a cervical anastomosis . resections were considered radical ( r0 ) if microscopic examination revealed no tumor tissue at or less than 1 mm from the circumferential , proximal , or distal margins . pathological staging was done according to the union for international cancer control ( uicc ) sixth edition . the tumor stage after resection was classified according to the tnm classification of the international union against cancer . snp genotyping to determine the individual genotype for each snp , genomic dna was extracted from frozen or formalin - fixed and paraffin - embedded ( ffpe ) tissues . normal tissue was obtained from the resection specimens ( i.e. , tumor - negative lymph nodes or tumor - negative resection margins ) . all the archival tissue samples were used according to the code for adequate secondary use of tissue , code of conduct : proper secondary use of human tissue established by the dutch federation of medical scientific societies ( http://www.federa.org).polymerase chain reactions ( pcr ) were carried out in a volume of 15 l containing genomic dna , 8.3 l h2o , 5 l mgfree buffer , 25 mm mgcl2 , 0.3 l of 10 mm deoxynucleotide triphosphates , 20 pmol of each primer , and 1 u taq polymerase ( promega , madison wi , usa ) . pcr conditions were standardized at 35 cycles of 95c for 45 s , 61c for 45 s , 72c for 30 s , with a 10-min extension at 72c for 10 min following the last cycle . for the polymorphism in cdh1 ( rs5030625 ) , amplified pcr products were visualized on a denaturing polyacrylamide gel . for detection of the restriction length polymorphisms in erbb2 ( rs1136201 ) and aurka ( rs2273535 ) , pcr products were digested for 16 h at the appropriate temperature with10 u of restriction endonuclease bsmai , mspi , or apoi ( promega , madison , wi , usa ) , respectively . the polymorphisms in cdkn2a ( rs11515 ) , mdm2 ( rs2279744 ) , and tp73 ( rs2273953 ) were genotyped by bi - directional sequencing . table 1description of the polymorphisms located in different oncogenes and tumor suppressor genesgenerefsnp idchangeminor alleleminor allele frequencypotential effect on protein functionforward primerreverse primeraurkars2273535c.449 t > a ( phe > ile)a - allele0.25preferentially amplified ; faster growth of cultured cells175-tccattctaggctacagctc-35-aagaatttgaaggacacaagac-3erbb2rs1136201c.655 a > g ( ile > val)g - allele0.16increased dimerization , autophosphorylation of her-2 and tyrosine kinase activity185-agccctctgacgtccatc-35-ctgcagcagtctccgcatc-3mdm2rs2279744c.309 t > gg - allelendassociated with higher levels of mdm2 expression195-gcggaggttttgttggactg-35-ctgagtcaacctgcccact-3cdh1rs5030625c.-347 g > gaga - allele0.14associated with decreased transcriptional activity205-ggccagaggaccgcttgag-35-gtttgttcgttttggaga-3cdkn2ars11515c.712 c > gg - allele0.13potential detrimental effect on rna stability215-ccccgattgaaagaaccagaga-35-aggaccttcggtgactgatgat-3tp73rs2273953c.-30 g > aa - allele0.19influences the p73 translation225-gagcacgagttcccagggtg-35-ccaagcgcactcacagagag-3nd not determinedrefsnp i d ( http://www.ncbi.nlm.nih.gov/snp)minor allele frequency according 102 controls of the snp500cancer cohort ( http://snp500cancer.nci.nih.gov/snp.cfm ) description of the polymorphisms located in different oncogenes and tumor suppressor genes refsnp i d ( http://www.ncbi.nlm.nih.gov/snp ) minor allele frequency according 102 controls of the snp500cancer cohort ( http://snp500cancer.nci.nih.gov/snp.cfm ) statistical analysis data on follow - up were collected from the prospective database and the medical charts . all patients were followed at an interval of 3 to 4 months during the first year , every 6 months for the second year , and then at the end of each year until 5 years after treatment . recurrence or disease progression was diagnosed on clinical grounds . whenever a relapse was suspected , radiologic , endoscopic , or histologic confirmation was sought . recurrent disease was classified as local regional ( occurring in the upper abdomen or mediastinum ) or distant ( including cervical recurrences).study end - point was disease - free survival ( dfs ) that was defined as the time from surgery until recurrent disease or death from any cause . the kaplan meier survival function and log - rank tests were used to assess clinical outcome in relation to patient s characteristics and individual polymorphisms . cox proportional hazard ratios for patients with eac were adjusted for weight loss prior to operation , tumor length , presence of barrett s epithelium , radicality of resection , and pathological tumor stage . for patients with escc , cox proportional hazard ratios were adjusted for location of tumor , resection type , post - operative tnm stage , and radicality of resection . we did not adjust for multiple testing since each gene outcome was prespecified and of interest in itself . patients a total of 346 esophageal cancer patients underwent surgical resection with curative intent . of these , 25 patients were excluded from the current follow - up study because no tissue samples were available ( n = 9 ) , genotyping failure ( n = 3 ) , or incomplete follow - up ( n = 13 ) ( fig . 1 ) . of the 214 eac and 97 escc patients remaining for analysis , the majority were male , 85% in eac and 60% in escc . of all patients with eac , 84% underwent primary surgery and 16% received preoperative chemotherapy with or without radiotherapy . in contrast , 71% of patients with escc received preoperative chemotherapy with or without radiotherapy and 29% underwent primary surgical resection ( table 2 ) . table 2survival according to patients and tumor characteristicsvariablepatients with eac ( n = 214)patients with escc ( n = 97)no . ( % ) median dfsp valuesage in years0.230.33<65 years111 ( 52)1964 ( 66)2165 years103 ( 48)1233 ( 34)12gender0.410.44male182 ( 85)1658 ( 60)15female32 ( 15)1139 ( 40)20weight loss before operation0.0130.47no loss or < 5%127 ( 59)1946 ( 47)15510%35 ( 16)1131 ( 32)27>10%32 ( 15)817 ( 18)10not recorded20 ( 10)103 ( 3)5smoking status0.980.70current smoker54 ( 25)1443 ( 44)16no current smoker146 ( 68)1547 ( 49)20not recorded14 ( 7)87 ( 7)4location of tumor0.1500.008upper one third thoracic esophagus3 ( 3)4middle one third thoracic esophagus3 ( 1)1139 ( 40)12lower one third thoracic esophagus68 ( 32)2445 ( 47)20gej86 ( 40)1310 ( 10)60gastric cardia57 ( 27)12tumor length ( cm)0.0280.270234 ( 16)418 ( 8)373452 ( 24)1524 ( 25)104567 ( 31)1431 ( 32)24646 ( 22)925 ( 26)9not recorded15 ( 7)169 ( 9)11barrett s epithelium0.086no127 ( 59)12yes87 ( 41)24treatment0.830.18surgery alone180 ( 84)1428 ( 29)8chemotherapy + surgery23 ( 11)1565 ( 67)15chemoradiotherapy + surgery11 ( 5)214 ( 4)not reachedresection type0.830.042transhiatal187 ( 87)1553 ( 55)37transthoracic27 ( 13)944 ( 45)11post - operative uicc stage<0.001<0.001complete response8 ( 4)4013 ( 13)not reachedi24 ( 11)9813 ( 13)86iia43 ( 20)3734 ( 35)12iib8 ( 4)154 ( 4)26iii74 ( 34)1117 ( 18)8iv57 ( 27)716 ( 17)4radicality of resection<0.001<0.001r0141 ( 66)3465 ( 67)41r170 ( 33)729 ( 30)6 r23 ( 1)93 ( 3)5 survival according to patients and tumor characteristics snp genotyping genotyping was complete in 95% to 100% of eac and escc patients . the genotype distributions did not deviate from hwe ( p > 0.05 ) . the genotype distribution of each snp is listed in table 3 . tumor stage distributions were similar across all snp genotypes , and there was no association between genotypes and age at diagnosis , sex , weight loss , smoking status , or preoperative treatment . table 3polymorphisms and clinical outcome in patients with resected eacgenotypedisease - free survival in eac patientsdisease - free survival in escc patientsnmps ( months)log - rank pahr[95% ci]nmps ( months)log - rank pahr[95% ci]aurka_rs2273535t / t129150.83reference62120.72referencea / t75141.1[0.761.4]29200.60[0.172.1]a / a9210.92[0.422.0]5550.63[0.182.1]erbb2_rs1136201a / a113140.25reference66120.66referencea / g86150.92[0.671.3]23260.73[0.411.3]g / g14120.68[0.331.4]681.3[0.493.2]mdm2_rs2279744t / t100110.076reference40100.63referencet / g84190.63[0.450.88]45210.98[0.591.6]g / g24120.95[0.581.6]7160.81[0.282.4]cdh1_rs5030625g / g166170.1468110.13referenceg / ga41111.2[0.781.7]18270.63[0.321.3]ga / ga474.0[1.411]1not reachedcdkn2a_rs11515c / c162130.79reference74120.67referencec / g47190.94[0.651.3]20200.67[0.361.3]g / g4191.7[0.525.6]1not reachedtp73_rs2273953g / g138160.44reference62240.48referenceg / a69130.98[0.711.4]32101.1[0.661.8]a / a5111.1[0.413.1]241.7[0.407.3]adjusted hazard ratio for weight loss prior to operation , tumor length , presence of barrett s epithelium , post - operative tnm stage , and radicality of resectionadjusted hazard ratio for location of the tumor , type of resection , post - operative tnm stage , and radicality of resection polymorphisms and clinical outcome in patients with resected eac adjusted hazard ratio for weight loss prior to operation , tumor length , presence of barrett s epithelium , post - operative tnm stage , and radicality of resection adjusted hazard ratio for location of the tumor , type of resection , post - operative tnm stage , and radicality of resection dfs and pattern of disease recurrence the median dfs of eac patients was 14 months ( range , 0.07138 months ) and for escc patients 16 months ( range , 0.5148 months ) . at the time of analysis , 37 ( 17% ) eac and 27 ( 28% ) escc patients were alive with a median dfs time of 93 months ( range , 62138 months ) and 104 months ( range 79148 months ) , respectively ( fig 2a ) . 2a kaplan meier analysis of dfs in patients with esophageal cancer , by histological subgroup . b kaplan meier analysis of dfs in patients with eac , by mdm2 ( rs2279744 ) . c kaplan meier analysis of dfs in patients with eac , by cdh1 genotype ( rs5030625)the pattern of disease recurrence is depicted in table 3 . loco - regional recurrences were mediastinal or abdominal lymph node metastases and recurrences in the gastric tube . distant metastases were found in liver , lung , brain , bone , adrenal gland , pleura , peritoneum , and skin.recurrent disease after surgery was found in 138 ( 78% ) eac patients ; 40 patients had loco - regional recurrence , 51 had distant metastasis , and 46 had both loco - regional recurrence and distant metastasis . diseases recurrences were found in 51 escc patients ; 28 patients had loco - regional recurrence , 10 had distant metastasis , and 10 had both loco - regional recurrence and distant metastasis . three patients had disease recurrence , but site of failure was not recorded . a kaplan meier analysis of dfs in patients with esophageal cancer , by histological subgroup . b kaplan meier analysis of dfs in patients with eac , by mdm2 ( rs2279744 ) . c kaplan meier analysis of dfs in patients with eac , by cdh1 genotype ( rs5030625 ) snp genotype and dfs univariate analysis showed no significant associations between dfs in patients with eac or escc and the genotype distributions of the aurka , erbb2 , mdm2 , cdh1 , cdkn2a , and the tp73 gene polymorphisms ( table 4 ; fig 2b and c ) . however , in a multivariate analysis , patients with eac carrying the heterozygous mdm2 ( rs2279744 ) t / g genotype had significantly improved dfs compared with patients carrying the wild - type t / t genotype ( adjusted hazard ratio ( ahr ) 0.63 , 95% confidence interval ( ci ) [ 0.450.88 ] , p = 0.007 ) . the post - operative tnm stage of the tumor and the radicality of resection were also found important factors for dfs ( hr 1.4 , 95% ci [ 1.21.6 ] , p < 0.0001 and hr 2.3 , 95%ci [ 1.73.1 ] , p < 0.0001 respectively ) . table 4pattern of failureeac ( n = 214)escc ( n = 97)alive37 ( 17)27 ( 28)nature of first failure local recurrence40 ( 29)28 ( 55 ) distant metastases51 ( 37)10 ( 20 ) local recurrence and distant metastases46 ( 33)10 ( 20 ) disease recurrence but site of failure not reported1 ( 1)3 ( 5)total deaths177 ( 83)70 ( 72)cause of death cancer - related138 ( 78)51 ( 71 ) surgery - related14 ( 8)7 ( 11 ) 2nd primary5 ( 3)6 ( 8) death from other cause ( not cancer - related)20 ( 11)6 ( 8)also , patients with eac harboring the homozygous cdh1 ( rs5030625 ) ga / ga genotype had a significantly reduced survival as compared with patients carrying the wild - type g / g genotype ahr 4.0 , 95% ci [ 1.411 ] , p = 0.008 . in multivariate analysis , the post - operative tnm stage of the tumor and the radicality of resection were found as important factors for dfs ( hr 1.4 , 95% ci [ 1.21.5 ] , p < 0.0001 and hr 2.4 , 95%ci [ 1.73.2 ] , p < 0.0001 respectively ) . in the present study , we determined the relationship between inter - individual dna variations in six bona fide proto - oncogenes and tumor suppressor genes and dfs in a large cohort of caucasian patients with esophageal cancer . after adjustment for potential confounders , the variant genotypes of snps located in the promoter region of the mdm2 and cdh1 gene were significantly associated with dfs in patients with eac . the results of the present study showed a significant survival benefit for patients harboring the mdm2 t / g as compared with patients carrying the wild - type t / t genotype . the mdm2 protein is a nuclear phosphoprotein that binds and inhibits the tumor suppressor tp53 as part of an autoregulatory negative feedback loop . the most intensively characterized mdm2 polymorphism is the t309 g promoter snp located in the first intron.20 the g variant of this snp is known to increase promoter - binding affinity , leading to up - regulation of mdm2 and consequent inhibition and down - regulation of the p53 pathway . therefore , it could be expected that the variant mdm2 genotypes ( t / g and g / g ) are associated with adverse outcome in esophageal cancer patients ( as shown in other cancer types).24 however , the present study showed improved survival in patients with the mdm2 t / g genotype compared with the wild - type t / t genotype . a possible explanation for our findings is provided by a large study in breast cancer patients that reported strong interaction between the mdm2 snp status and tumor tp53 status , which appeared to modify the association between tp53 status and breast cancer survival.25 among breast cancer patients with the wild - type mdm2 genotype ( t / t ) , a mutant tp53 status and aberrant tp53 expression in breast tumors were associated with poor survival . the tumor tp53 status was not associated with breast cancer survival among carriers of the variant mdm2 allele ( t / g or g / g ) . since tp53 is the most frequently mutated gene in eac , it could be hypothesized that the tumors of most patients with the t / t genotype harbor a tp53 mutation , which could lead to a reduced survival as observed in the present study . in a previously well - conducted study , the known tp53 codon 72 arg / pro and mdm2 polymorphisms were genotyped in 300 patients with eac and 63 patients with escc.26 as in concordance with the results of the present study , patients with eac harboring the mdm2 t / g genotype had a borderline improved overall survival as compared with patients carrying the wild - type genotype ( ahr for death 0.70 , 95% ci [ 0.500.99 ] , p = 0.04 ) . but unlike the present study , the mdm2 variant genotype did correlate with marked reduced survival in patients with escc . this could be due to differences in study samples size , population selection , tissue handling , and genotyping methods . in this study , patients carrying the cdh1 ga / ga genotype had a significantly reduced survival as compared with patients with the wild - type g / g genotype . however , it should be noted that only four eac patients carried the ga / ga genotype , which may represents a chance finding . nevertheless , this -347 g / ga insertion polymorphism located in the promoter of the cell cell adhesion gene cdh1 has been reported to suppress cdh1 gene expression and was found to be associated with familial gastric cancer and sporadic colorectal cancer.27 the ga - allele has been associated with significant suppression of cdh1 promoter activity in colorectal and gastric cancer cell lines.27 it can be hypothesized that the ga - allele might enhance the progression of esophageal cancer by reducing cdh1 transcription resulting in a decrease in cdh1 protein expression and impairment of cell cell adhesion . all four patients harboring the ga / ga genotype died of recurrent disease ; three had loco - regional and distant metastasis , and one had only loco - regional disease recurrence . to our knowledge , this is one of the first studies that investigated the relationship between polymorphisms and esophageal cancer outcome . here , we studied ( only ) six polymorphisms , whereas snp arrays can determine more than a million of snps in one sample . although this technique is widely used on blood or fresh frozen samples , it is not very suitable for ffpe tissue samples ( our series consisted primarily of ffpe samples ) . therefore , collection of blood samples or fresh frozen tissue samples of esophageal cancer patients is necessary and should become standard procedure in order to perform genome - wide association studies . in this study , the majority of polymorphisms were not associated with dfs after esophagectomy . it could be well that our study , among a relatively large population ( n = 346 ) of esophageal cancer patients , failed to observe a difference due to under powering . since esophageal cancer has a relatively low incidence , consortia ( of multiple hospitals ) recurrent cancer is the leading cause of death in patients undergoing surgical resection.35 although treatment options for esophageal cancer recurrences are limited , it could be proposed that early detection of recurrent disease is desirable because aggressive treatment may result in prolonged tumor - free survival or occasional cure . in this light , our findings could contribute to the identification of patients who are at high or low risk for the development of disease recurrences . it can also be proposed that patients with a certain genetic constitution that is associated with a high chance of ( distant ) disease recurrence should be given systemic adjuvant treatment after surgical resection . furthermore , identification of polymorphisms associated with dfs could serve well as hypothesis generating for prospective studies that evaluate the prognostic significance of germ - line variants . in summary , our results indicate that two of six investigated functional polymorphisms are associated with dfs in patients who underwent esophagectomy for eac . patients with eac carrying the heterozygous mdm2 t / g genotype had twofold reduced risk of disease recurrence , and patients with the homozygous cdh1 ga / ga had a fourfold increased risk of disease recurrence . additional prospective studies are necessary to validate both associations and to study the prognostic significance of both germ - line variants .

purposethe aim of this study was to determine whether clinical outcome after surgical resection of esophageal adenocarcinoma ( eac ) or esophageal squamous cell carcinoma ( escc ) could be predicted by functional polymorphisms in different proto - oncogenes and tumor suppressor genes.experimental designsix single nucleotide polymorphisms ( snps ) in the aurka ( rs2273535 ) , erbb2 ( rs1136201 ) , mdm2 ( rs2279744 ) , cdh1 ( rs5030625 ) , cdkn2a ( rs11515 ) , and tp73 ( rs2273953 ) genes were genotyped in a consecutive cohort of 346 esophageal cancer patients , who had underwent surgical resection with curative intent . associations with disease - free survival ( dfs ) were analyzed with kaplan meier curves and cox regression , adjusting for potential confounders.resultsunivariate analysis showed no significant associations between the tested polymorphisms and dfs in patients with eac or escc . however , in a multivariate analysis , patients with eac carrying the heterozygous mdm2 ( rs2279744 ) t / g genotype had significantly improved dfs compared with patients carrying the wild - type genotype ( adjusted hazard ratio ( ahr ) , 0.63 ; 95% confidence interval ( ci ) [ 0.450.88 ] ) . patients with eac harboring the homozygous cdh1 ( rs5030625 ) ga / ga genotype had a significantly reduced survival as compared with patients carrying the wild - type genotype ahr 4.0 , 95% ci [ 1.411].conclusionsin a large cohort of esophageal cancer patients , the mdm2 t / g and cdh1 ga / ga genotype confer risk of death in patients with eac . these data suggest that inter - individual differences in germ - line dna have an impact on dfs in patients with eac .

pubmed

oral cavity is colonized by candida albicans or other candida species in 40 - 60% of healthy persons . in immune - compromised patients , candida species can trigger a variety of disease manifestations ranging from localized mild oral lesion to a disseminated candidiasis.1 many factors contribute to the development of oropharyngeal candidiasis ( opc ) including malnutrition , poor oral hygiene , dental malocclusion , and immunosuppression.2 diagnosis and treatment of oral lesions caused by candida species are of utmost importance in human immunodeficiency virus ( hiv)-positive patients who , despite the initiation of triple antiretroviral therapy ( art ) , continue to suffer from significant candida associated morbidity.3 - 5 according to the joint united nations program on hiv / acquired immune deficiency syndrome ( aids ) as of 2013 , approximately 35.3 millions people have hiv worldwide with the number of new infections that year being about 2.1 millions.6 opc has been described as the most frequent opportunistic fungal infection among hiv - positive patients , and it has been estimated that more than 90% of hiv - positive patients develop this infection at some time during the progression of their disease.7 - 9 opc is an opportunistic infection of soft buccal mucosa . opc can appear as erythematous patches or white , scrapable lesions and is often one of the first clinical signs of hiv infection.9,10 opc is observed with a higher prevalence in patients with cd4 + counts below 200/mm or a high viral load ( > 10,000 copies / ml).4,5,7 - 9 opc caused by c. albicans is generally managed by judicious use of fluconazole.2,4,7 - 9 a rise in resistant organisms may be due to prolonged or frequent treatment with azoles.10 an epidemiologic shift of candida species could significantly impact the utility of fluconazole as empiric treatment for candidiasis in patients with hiv / aids.11 the present study was directed to evaluate the forms of opc and their correlation with cd4 + cell counts in hiv patients . this was a descriptive and analytical cross - sectional study carried out for a 2-year period , in which quantitative data collection methods were used . patient records , available at the odontology unit of saint - antoine hospital , were initially studied and then the patients were asked to visit the clinic for a further evaluation . a complete medical history was taken and a physical examination of the oral cavity , head , and neck area was performed on each patient . the variables studied , including medical history , physical examination , socio - demographic characteristics , socio - behavioral factors , experience with oral lesions , and laboratory tests , were reviewed . based on the findings of a physical examination and laboratory tests , patients were prescribed essential medication and repeated examination , and follow - up visits were considered . one dental surgeon , who was blinded to the clinical staging , carried out all oral examinations . patients were examined while seated in the dental chair and a well - illuminated room . extra oral and perioral areas were examined first , followed by intraoral tissues for any abnormalities . diagnosis of oral lesions was implemented using european community clearinghouse guidelines for presumptive diagnosis of opc.12 blood samples were obtained on the same day as the oral examinations , and their results were recorded onto each participant s questionnaire . a single oral swab was collected from each study participant by passing a sterile swab firmly across buccal mucosa , floor of mouth , dorsal tongue in cases of asymptomatic patients , and from the base of the oral lesion in cases of symptomatic patients . candida was identified by conventional tests and species identification was performed using the germ tube test , growth on chrom agar candida medium ( drg international inc . dehydrated media , springfield , u.s.a ) , and sugar assimilation tests.12,13 all patients who had oral lesions and from whom candida species were isolated were considered to have opc . recent cd4 + levels were analyzed by the flow cytometer method , and a history of intake of art was noted . based on the who classification14 the cd4 + cell counts 500 cells / mm was classified as type 1 , cd4 + cell count of > 201 to < 499 cells / mm as type 2 and cd4 + cell count of 200 cells / mm as type 3 . patient data , microbiological results , and cd4 + counts were collected and protected with a password . data included age , gender , socio - behavioral factors , opc , antifungal use , art , cd4 + count , were entered in excel data sheet ( microsoft corporation seattle , usa ) . all statistical calculations were performed using spss software version 20 ( ibm spss , chicago , usa ) . one dental surgeon , who was blinded to the clinical staging , carried out all oral examinations . patients were examined while seated in the dental chair and a well - illuminated room . extra oral and perioral areas were examined first , followed by intraoral tissues for any abnormalities . diagnosis of oral lesions was implemented using european community clearinghouse guidelines for presumptive diagnosis of opc.12 blood samples were obtained on the same day as the oral examinations , and their results were recorded onto each participant s questionnaire . a single oral swab was collected from each study participant by passing a sterile swab firmly across buccal mucosa , floor of mouth , dorsal tongue in cases of asymptomatic patients , and from the base of the oral lesion in cases of symptomatic patients . candida was identified by conventional tests and species identification was performed using the germ tube test , growth on chrom agar candida medium ( drg international inc . dehydrated media , springfield , u.s.a ) , and sugar assimilation tests.12,13 all patients who had oral lesions and from whom candida species were isolated were considered to have opc . recent cd4 + levels were analyzed by the flow cytometer method , and a history of intake of art was noted . based on the who classification14 the cd4 + cell counts 500 cells / mm was classified as cd4 + cell count of > 201 to < 499 cells / mm as type 2 and cd4 + cell count of 200 cells / mm as type 3 . patient data , microbiological results , and cd4 + counts were collected and protected with a password . data included age , gender , socio - behavioral factors , opc , antifungal use , art , cd4 + count , were entered in excel data sheet ( microsoft corporation seattle , usa ) . all statistical calculations were performed using spss software version 20 ( ibm spss , chicago , usa ) . of the 50 consenting participants , 5 ( 10% ) were females and 45 ( 90% ) males . the median age in this study group was 39 years ( 28 - 57 ) . of the participants , 38 ( 76% ) had a history of sexual contamination , 11 ( 22% ) had a history of sharing intravenous needles , and one ( 2% ) had a history of homosexuality and sharing intravenous needles . most participants had cd4 + count ( cells / mm ) was < 200 , 201 - 499 , and > 500 in 32 cases ( 64% ) , 16 cases ( 32% ) and 2 cases ( 4% ) , respectively , and the mean cd4 + count ( cells / mm ) was 167.12 . median duration of art was 2 years ( range , 1.5 months to 6 years ) . overall , candida species was isolated from the oral cavity in 38/50 ( 76% ) . hiv - associated oral lesions were observed in all patients ( periodontal disease , herpetic lesions , hairy leukoplakia , gingivitis , oral ulceration , kaposi s sarcoma , and non - hodgkin lymphoma ) . pseudomembranous candidiasis 20/38 ( p ) was the most common clinical presentation of opc ( 52.6% ) followed by erythematous 5/38 ( 13.15% ) , angular cheilitis 5/38 ( 13.15% ) ( ac ) and a combination of ac and e 4/38 ( 10.52% ) or ac , e and p 4/38 ( 10.52% ) ( figure 1 ) . clinical forms of oropharyngeal candidiasis : ( a ) pseudomembranous , ( b ) erythematous ( c ) angular cheilitis . c. albicans was the most frequent species isolated in 35 cases of opc ( 92% ) . candida tropicalis was isolated in 2 cases ( 5.26% ) and candida glabrata in 1 case ( 2.64% ) . the distribution of oral lesions based on the cd4 + count of hiv - infected patients showed that 62% of the oral lesions occurred at cd4 + count < 200 cells / mm ( mean cd4 + ) , about 26% oral lesions were seen at cd4 + count of 201 - 499 cells / mm ( mean cd4 + ) whereas 12% cases of oral lesions were seen at cd4 + count > 500 cells / mm ( mean cd4 ) . the majority of patients with opc had cell counts 28/38 ( 73% ) < 200 cells / mm , followed by 9/38 ( 23% ) at cd4 + cell counts of 201 - 499 cells / mm ( table 1 ) . profile of hiv patients with opc form distribution , cd4 + count and candida species opc is the most common fungal infection in hiv patients and fluconazole efficiency in treating this infection has been proven . however , certain non - albican species ( c. glabrata , candida krusei ) proved to be less susceptible to flunconazole than c. albicans . since they were isolated in hiv patients.7,13 impact of art on opportunistic infections in hiv patients is continuously evolving : patel and co - workers ( 2012 ) studied the changing aspect of opc epidemiology in 215 hiv / aids patients by assessing yeast colonization from oral rinse samples . c. albicans was the most prevalent and c. glabrata and candida dubliniensis were identified in 29% of cultures . decrease susceptibility to flunonazole was studied and detected in 10% of isolates.9 in a prospective cross - sectional study of predisposing factors for oropharyngeal colonization of yeasts in hiv - infected patients , lin et al . ( 2013 ) reviewed 105 patients , among whom , 54 ( 51.4% ) were colonized with yeasts and among the 68 isolates , c. albicans accounted for 73.5 % , c. tropicalis for 5.95% , c. glabrata for 5.9% , and c. dubliniensi for 4.4% , and there 7.5% candida isolates resistant to flunonazole and a higher prevalence of yeast colonization was observed in patients with cd4 + count < 200 cells / mm.15 thompson et al . ( 2013 ) studied opc in 122 hiv - infected patients in whom infection was observed in one - third : c. albicans was the most implicated pathogen and oral yeast colonization in 81.1% , despite the availability of art , and resistant yeasts occurred in 25.3% of patients.4 in an observational cohort , arribas et al . ( 2000 ) determined the relationship between art and changes in prevalence and amount of opc . they found that the majority of 99 observed patient presented opc with a cd4 + count < 200 cells / mm.16 in our study , the mean age was 39 years , and male gender was 90% , and female was 10% . about 76% of our patients were contaminated with hiv by sexual transmission and 22% by sharing intravenous needles and 2% by a combination of sexual and intravenous sharing needles . similar figures were recorded by thompson et al.,4 castro et al.13 and lin et al.15 earlier studies2,5,15,16 showed that pseudomembranous candidiasis was the most common in clinical appearance of opc . these findings shared our clinical observation . c. albicans was isolated in 92% of our cases followed by c. tropicalis and c. glabrata . some of the recent report1,4,8 have also reported the same species while others12,13 described other species as c. dubliniensis beside the mentioned species . these variations could be related to the differences in the socio - demographic characteristics and socio - behavioral factors or may be due to increased identification of the species , which can be mistaken phenotypically as c. albicans.17 distribution of opc lesions and the cd4 + cell counts , 73% associated with < 200 cells / mm is a common observation with other studies.4,5,18 the only given explication may be the decrease of the immunodeficiency of the immune system . current guidelines note that for aids patients , 200 mg / day of fluconazole is acceptable and does not lead to resistance.19 oral candida colonization and invasive infection occur more frequently in hiv - positive patient and are significantly more common in patients with cd4 + cell counts < 200 cell / mm . art reduces opc . c. albicans continues to be the most frequently isolated species in opc . our results emphasize the need of continued awareness of opc as a major clinical sign in hiv population .

background : the present study was directed to evaluate the forms of oropharyngeal candidiasis ( opc ) and their correlation with cd4 + cell counts in human immunodeficiency virus ( hiv ) patients.materials and methods : this was a descriptive and analytical cross - sectional study carried out for a 2-year period , in which quantitative data collection methods were used . 50 patients with hiv infection were evaluated . relationship between opc and cd4 + was investigated.results:five different clinical forms were noticed on examination : pseudomembranous candidiasis 20/38 ( p ) was the most common one ( 52.6% ) followed by erythematous 5/38 ( 13.15% ) , angular cheilitis 5/38 ( 13.15% ) ( ac ) , a combination of ac and e 4/38 ( 10.52% ) or ac , e and p 4/38 ( 10.52% ) . candida albicans was the most frequent specie isolated in 35 cases of opc ( 92% ) . candida tropicalis was isolated in 2 cases ( 5.26% ) and candida glabrata in 1 case ( 2.64% ) . the majority of patients with opc had cell counts 28/38 ( 73% ) < 200 cells / mm3 , followed by 9/38 ( 23% ) at cd4 + cell counts of 201 - 499 cells / mm3.conclusion : oral candida colonization and invasive infection occur more frequently in hiv - positive patient and is significantly more common in patients with cd4 + cell counts < 200 cell / mm3 .

pubmed

breast cancer is the most common cancer among women , with approximately 1.67 million new patients diagnosed annually worldwide and accounting for 25% of all cancer cases.1 breast cancer predominantly spreads via the lymphatic system to locoregional lymph nodes , meaning that the status of these lymph nodes is important for staging of the disease and determining prognosis.24 the sentinel lymph nodes ( slns ) are defined as the first draining nodes from a primary tumor and , therefore , are most likely to be the first site of lymphatic metastasis . sentinel lymph node biopsy ( slnb ) with the combined technique of radioisotope and blue dye is the standard method for determining axillary staging in early - stage breast cancer patients with a clinically and radiologically negative axilla.3,59 the combined technique is performed by injecting a radioactive tracer ( tc nanocolloid ) and blue dye interstitially , either peritumorally or periareolarly . the tracers are then detected within the slns by the surgeon using a handheld scintillation counter ( gamma probe ) and/or visually . the gamma probe is first used to transcutaneously localize the slns and determine the optimal incision site . postincision both the blue dye and gamma probe guide the surgeon to the slns . after identification , the slns are removed and sent for histopathological examination . with an identification rate of 96% and a false - negative rate of 5%10% , it is an effective procedure with low morbidity.3 although the currently used technique performs well , it has drawbacks . the use of radioisotopes is subject to stringent regulations on training of staff and handling and disposal of radioactive material . most importantly , many hospitals in the world do not have access to radioisotopes . consequently , only approximately 60% of the patients in the western world and negligible numbers in the rest of the world have access to slnb.10 this has led to the search for other techniques , not reliant on the use of radioisotopes.11 recently the use of a magnetic tracer and a handheld magnetometer was evaluated against the combined technique as a radiation - free alternative in two separate clinical trials.12,13 a superparamagnetic iron oxide ( spio ) tracer was administered interstitially in the breast , followed by identification of the slns with a handheld magnetometer . the magnetic technique was found to be noninferior to the combined technique with identification rates of 94.4%98.0%.12,13 however , there was a discordance in slns identified between the magnetic and combined techniques that ranged between 2.0%13 and 6.9%,12 potentially leading to false - negative staging with the magnetic technique . a smaller study by shiozawa et al14 used a similar spio tracer and different magnetometer , comparing spio and blue dye only no radioisotope was used . they found an sln identification rate of 90% for the spio in combination with blue dye , with a discordance rate of 16.7% and demonstrated the viability of the technique for performing slnb in the absence of radioisotopes . physical and chemical properties including shape , coating material , and particle size influence the distribution of nanoparticles in vivo.15,16 tc nanocolloid has a mean particle size of approximately 8 nm,17,18 while the particle size of the magnetic tracer used for the magnetic technique ( sienna+ , endomagnetics ltd , uk ) is much larger with a mean size of approximately 60 nm . because particle size is the most important property influencing lymphatic uptake and lymph node retention,19 the identification of different slns by the two techniques could possibly arise due to differences in particle size of the radioisotope and magnetic tracer . in order to optimize the clinical application of the magnetic technique for slnb , we evaluated the performance of three different - sized spio - based formulations licensed for human use within an in vivo porcine model . three spio - based licensed formulations with a broad range in particle size were used as magnetic tracers in this study . feraheme / rienso ( takeda , japan ) consists of ferumoxytol , 30 mg fe / ml and is an food and drug administration approved drug used for the intravenous treatment of iron deficiency anemia in adult patients with chronic kidney disease . the particles have a magnetic core of maghemite ( -fe2o3 ) surrounded by a polyglucose sorbitol carboxymethylether coating . sienna+ contains 27 mg fe / ml and is a ce - marked magnetic tracer intended to mark and locate slns in cancer patients . the magnetic core consists of magnetite and maghemite ( fe3o4/-fe2o3 ) , with a carboxydextran coating . finally , endorem / feridex ( guerbet , france ) consisting of ferumoxide , 11.2 mg fe / ml is licensed as a magnetic resonance imaging ( mri ) contrast agent for liver imaging , with a magnetite core coated with dextran . all magnetic tracers were diluted to the same iron concentration of 11.2 mg fe / ml to facilitate comparison of performance . the hydrodynamic particle size of all tracers was determined by dynamic light scattering using a zetasizer nano zs ( malvern instruments , uk ) . ethical permission for animal experimentation was granted by the research institute against digestive cancer ( ircad ) ethics review board ( strasbourg , france ) under reference number 38.2012.01.047 . a previously developed and validated porcine model that closely resembles human lymphatic drainage was used to evaluate the performance of the different magnetic tracers for slnb.20 at the ircad institute ( strasbourg , france ) , after induction of anesthesia , mini - pigs were injected with one of the three different magnetic tracers subcutaneously bilaterally into the areolas of the third inguinal mammary glands . a total of 0.5 ml ( 5.6 mg fe ) of each diluted tracer was injected on each side , in six mini - pigs , for each tracer ( 18 mini - pigs in total ; 36 injections ) . transcutaneous magnetometer measurements using a handheld magnetometer ( sentimag , endomagnetics ltd , uk ) were undertaken in the inguinal region , where the draining lymph nodes are located . measurements were performed at 5 minutes , 10 minutes , 15 minutes , 30 minutes , 45 minutes , 60 minutes , and 240 minutes after administration of the tracer . the sites at which uptake of spio was successfully detected ( hotspots ) were marked using a permanent marker . after identification , lymph nodes demonstrating spio uptake were removed , and the ex vivo magnetometer count was recorded . lymph nodes with a magnetometer count higher than 10% of the hottest node were defined as slns , as was used in the sentimag multicentre trial,12 and in accordance with standard clinical practice.21 after completion of the slnb procedure , a groin node clearance was performed . ex vivo magnetometer measurements were performed on the groin node clearances to determine if there were any further slns that were missed initially . all resected lymph nodes were fixed in formalin and sent to the university of twente ( enschede , the netherlands ) for iron quantification and subsequently to king s college london ( london , uk ) for histopathological evaluation . the quantification of magnetic tracer in the excised and formalin - fixed sentinel lymph nodes was performed using vibrating sample magnetometry ( vsm ) on a physical properties measuring system ( quantum design inc . , san diego , ca , usa ) . an applied magnetic field of 4.0 t was used to bring the magnetic iron oxide nanoparticles to saturation . the amount of magnetic tracer in the lymph nodes was determined by comparing the obtained amplitude of the saturation magnetization to known calibration samples of each of the tracers . the iron content was reported as the mass of iron ( fe ) in the node , present in the form of maghemite or magnetite . vsm allows quantification with 0.5 g fe accuracy and is nondestructive , which allows subsequent histopathological analysis of the same samples.22 the nodes were then transferred intact to king s college london ( london , uk ) where they were embedded in wax , thinly sliced , and stained with hematoxylin and eosin and with perl s prussian blue for iron . iron staining within each node was graded using a previously validated 5-point grading scale ( 0 = none , 1 = minimal , 2 = mild , 3 = moderate , and 4 = marked),20 by an experienced pathologist and a second observer ( sp , ba , or ma ) . we conducted a two - sided test ( alpha = 0.05 ) expecting a difference of 50 g ( standard deviation [ sd ] = 30 ) in iron content readings between each magnetic tracer . when performing a total of 12 procedures ( six mini - pigs ) for each magnetic tracer , these 36 procedures provided us with a power of 82% to detect this difference . the relationship between continuous variables was calculated using pearson s correlation coefficient , associations between categorical data using the test , and associations between categorical and continuous variables using analyses of variance . all statistical analyses were performed using statistical analysis systems ( sas ) release 9.3 ( sas institute , cary , nc ) . three spio - based licensed formulations with a broad range in particle size were used as magnetic tracers in this study . feraheme / rienso ( takeda , japan ) consists of ferumoxytol , 30 mg fe / ml and is an food and drug administration approved drug used for the intravenous treatment of iron deficiency anemia in adult patients with chronic kidney disease . the particles have a magnetic core of maghemite ( -fe2o3 ) surrounded by a polyglucose sorbitol carboxymethylether coating . sienna+ contains 27 mg fe / ml and is a ce - marked magnetic tracer intended to mark and locate slns in cancer patients . the magnetic core consists of magnetite and maghemite ( fe3o4/-fe2o3 ) , with a carboxydextran coating . finally , endorem / feridex ( guerbet , france ) consisting of ferumoxide , 11.2 mg fe / ml is licensed as a magnetic resonance imaging ( mri ) contrast agent for liver imaging , with a magnetite core coated with dextran . all magnetic tracers were diluted to the same iron concentration of 11.2 mg fe / ml to facilitate comparison of performance . the hydrodynamic particle size of all tracers was determined by dynamic light scattering using a zetasizer nano zs ( malvern instruments , uk ) . ethical permission for animal experimentation was granted by the research institute against digestive cancer ( ircad ) ethics review board ( strasbourg , france ) under reference number 38.2012.01.047 . a previously developed and validated porcine model that closely resembles human lymphatic drainage was used to evaluate the performance of the different magnetic tracers for slnb.20 at the ircad institute ( strasbourg , france ) , after induction of anesthesia , mini - pigs were injected with one of the three different magnetic tracers subcutaneously bilaterally into the areolas of the third inguinal mammary glands . a total of 0.5 ml ( 5.6 mg fe ) of each diluted tracer was injected on each side , in six mini - pigs , for each tracer ( 18 mini - pigs in total ; 36 injections ) . transcutaneous magnetometer measurements using a handheld magnetometer ( sentimag , endomagnetics ltd , uk ) were undertaken in the inguinal region , where the draining lymph nodes are located . measurements were performed at 5 minutes , 10 minutes , 15 minutes , 30 minutes , 45 minutes , 60 minutes , and 240 minutes after administration of the tracer . the sites at which uptake of spio was successfully detected ( hotspots ) were marked using a permanent marker . after identification , lymph nodes demonstrating spio uptake were removed , and the ex vivo magnetometer count was recorded . lymph nodes with a magnetometer count higher than 10% of the hottest node were defined as slns , as was used in the sentimag multicentre trial,12 and in accordance with standard clinical practice.21 after completion of the slnb procedure , a groin node clearance was performed . ex vivo magnetometer measurements were performed on the groin node clearances to determine if there were any further slns that were missed initially . all resected lymph nodes were fixed in formalin and sent to the university of twente ( enschede , the netherlands ) for iron quantification and subsequently to king s college london ( london , uk ) for histopathological evaluation . the quantification of magnetic tracer in the excised and formalin - fixed sentinel lymph nodes was performed using vibrating sample magnetometry ( vsm ) on a physical properties measuring system ( quantum design inc . , san diego , ca , usa ) . an applied magnetic field of 4.0 t was used to bring the magnetic iron oxide nanoparticles to saturation . the amount of magnetic tracer in the lymph nodes was determined by comparing the obtained amplitude of the saturation magnetization to known calibration samples of each of the tracers . the iron content was reported as the mass of iron ( fe ) in the node , present in the form of maghemite or magnetite . vsm allows quantification with 0.5 g fe accuracy and is nondestructive , which allows subsequent histopathological analysis of the same samples.22 the nodes were then transferred intact to king s college london ( london , uk ) where they were embedded in wax , thinly sliced , and stained with hematoxylin and eosin and with perl s prussian blue for iron . iron staining within each node was graded using a previously validated 5-point grading scale ( 0 = none , 1 = minimal , 2 = mild , 3 = moderate , and 4 = marked),20 by an experienced pathologist and a second observer ( sp , ba , or ma ) . we conducted a two - sided test ( alpha = 0.05 ) expecting a difference of 50 g ( standard deviation [ sd ] = 30 ) in iron content readings between each magnetic tracer . when performing a total of 12 procedures ( six mini - pigs ) for each magnetic tracer , these 36 procedures provided us with a power of 82% to detect this difference . the relationship between continuous variables was calculated using pearson s correlation coefficient , associations between categorical data using the test , and associations between categorical and continuous variables using analyses of variance . all statistical analyses were performed using statistical analysis systems ( sas ) release 9.3 ( sas institute , cary , nc ) . the z - averaged hydrodynamic diameter of the magnetic tracers ferumoxytol , sienna+ , and ferumoxide was determined to be 32 nm , 59 nm , and 111 nm , respectively . when the size distributions were evaluated graphically ( figure 1 ) , significant intratracer heterogeneity in particle size was observed . this is reflected by a polydispersity index of 0.179 , 0.181 , and 0.266 for the 32 nm tracer , 59 nm tracer , and 111 nm tracer , respectively . a total of 36 slnb procedures were performed in 18 mini - pigs , and sln identification was successful in all cases . in vivo magnetic hotspots from the draining inguinal lymph nodes were successfully identified transcutaneously prior to surgical excision using the handheld magnetometer in all cases ( 12/12 ) for the 32 nm tracer and the 59 nm tracer and in all but one case ( 11/12 ) for the 111 nm tracer ( 240 minutes after injection ) . the number of procedures in which a transcutaneous hotspot was successfully identified increased with time after injection for all three tracers ( figure 2 ) . there were 7/12 and 8/12 successfully identified magnetic hotspots after 30 minutes using the 32 nm tracer and 111 nm tracer , respectively , compared to the 59 nm tracer with magnetic hotspots in all cases ( 12/12 ) . a total of 77 slns were identified with the handheld magnetometer during surgery and subsequently excised . thirty - five slns were identified using the 32 nm tracer ( mean 2.9 , sd 1.2 ) , 20 slns with the 111 nm tracer ( mean 1.7 , sd 0.9 ) , and 22 slns ( mean 1.8 , sd 0.8 ) with the 59 nm tracer ( figure 3a ) . a statistically significant difference between the tracer used , and the number of slns identified more nodes per basin were excised with the 32 nm tracer compared to the 111 nm tracer ( p<0.001 ) and the 59 nm tracer ( p=0.03 ) . the distribution of magnetometer counts demonstrated a statistically significant difference among the three tracers overall ( p<0.0001 ) ( figure 3b ) . multicomparisons demonstrated that ex vivo magnetometer counts were significantly higher with the 59 nm tracer compared to the other two tracers ( p<0.05 ) . the iron content of the ex vivo slns was recorded using vsm ( figure 3c ) . multiple comparisons between the tracers demonstrated a significant higher iron content for the 59 nm tracer compared to the 32 nm tracer ( p<0.05 ) only . the mean iron content of the slns was determined to be 106 ( sd 70 ) g , 179 ( sd 159 ) g , and 265 ( sd 206 ) g for the 32 nm tracer , 111 nm tracer , and 59 nm tracer , respectively . a linear relationship between the iron content of the ex vivo slns and the handheld magnetometer counts was observed for all tracers ( figure 4 ) 32 nm tracer ( r=0.93 ; p<0.001 ) , 111 nm tracer ( r=0.95 ; p<0.001 ) , and 59 nm tracer ( r=0.93 ; p<0.001 ) . figure 4 demonstrates a difference in the sensitivity of the magnetometer for the different tracers . the magnetometer count is 2.8 times higher for the 59 nm tracer compared to the same amount of iron for the 111 nm tracer and 1.3 times higher for the 32 nm tracer compared to the 111 nm tracer . the iron was distributed predominantly in the subcapsular space peripherally within the cortex , subcapsular space , and sinuses ( figure 5 ) . the iron deposition from the 111 nm tracer was mostly confined to macrophages within the nodes ( figure 5a ) , whereas the 59 nm tracer demonstrated more free iron granules ( particularly peripherally ) ( figure 5b ) . only very sparse , small islands of iron within macrophages were visible for the 32 nm tracer ( figure 5c ) . there was a significant difference in the grade of the iron content of excised slns between the three different tracers overall ( p<0.0001 ) ( figure 6 ) . the amount of iron deposition was significantly greater with the 111 nm tracer and 59 nm tracer ( p<0.05 ) compared to the 32 nm tracer . the z - averaged hydrodynamic diameter of the magnetic tracers ferumoxytol , sienna+ , and ferumoxide was determined to be 32 nm , 59 nm , and 111 nm , respectively . when the size distributions were evaluated graphically ( figure 1 ) , significant intratracer heterogeneity in particle size was observed . this is reflected by a polydispersity index of 0.179 , 0.181 , and 0.266 for the 32 nm tracer , 59 nm tracer , and 111 nm tracer , respectively . a total of 36 slnb procedures were performed in 18 mini - pigs , and sln identification was successful in all cases . in vivo magnetic hotspots from the draining inguinal lymph nodes were successfully identified transcutaneously prior to surgical excision using the handheld magnetometer in all cases ( 12/12 ) for the 32 nm tracer and the 59 nm tracer and in all but one case ( 11/12 ) for the 111 nm tracer ( 240 minutes after injection ) . the number of procedures in which a transcutaneous hotspot was successfully identified increased with time after injection for all three tracers ( figure 2 ) . there were 7/12 and 8/12 successfully identified magnetic hotspots after 30 minutes using the 32 nm tracer and 111 nm tracer , respectively , compared to the 59 nm tracer with magnetic hotspots in all cases ( 12/12 ) . a total of 77 slns were identified with the handheld magnetometer during surgery and subsequently excised . thirty - five slns were identified using the 32 nm tracer ( mean 2.9 , sd 1.2 ) , 20 slns with the 111 nm tracer ( mean 1.7 , sd 0.9 ) , and 22 slns ( mean 1.8 , sd 0.8 ) with the 59 nm tracer ( figure 3a ) . a statistically significant difference between the tracer used , and the number of slns identified more nodes per basin were excised with the 32 nm tracer compared to the 111 nm tracer ( p<0.001 ) and the 59 nm tracer ( p=0.03 ) . the distribution of magnetometer counts demonstrated a statistically significant difference among the three tracers overall ( p<0.0001 ) ( figure 3b ) . multicomparisons demonstrated that ex vivo magnetometer counts were significantly higher with the 59 nm tracer compared to the other two tracers ( p<0.05 ) . the iron content of the ex vivo slns was recorded using vsm ( figure 3c ) . multiple comparisons between the tracers demonstrated a significant higher iron content for the 59 nm tracer compared to the 32 nm tracer ( p<0.05 ) only . the mean iron content of the slns was determined to be 106 ( sd 70 ) g , 179 ( sd 159 ) g , and 265 ( sd 206 ) g for the 32 nm tracer , 111 nm tracer , and 59 nm tracer , respectively . a linear relationship between the iron content of the ex vivo slns and the handheld magnetometer counts was observed for all tracers ( figure 4 ) 32 nm tracer ( r=0.93 ; p<0.001 ) , 111 nm tracer ( r=0.95 ; p<0.001 ) , and 59 nm tracer ( r=0.93 ; p<0.001 ) . figure 4 demonstrates a difference in the sensitivity of the magnetometer for the different tracers . the magnetometer count is 2.8 times higher for the 59 nm tracer compared to the same amount of iron for the 111 nm tracer and 1.3 times higher for the 32 nm tracer compared to the 111 nm tracer . the iron was distributed predominantly in the subcapsular space peripherally within the cortex , subcapsular space , and sinuses ( figure 5 ) . the iron deposition from the 111 nm tracer was mostly confined to macrophages within the nodes ( figure 5a ) , whereas the 59 nm tracer demonstrated more free iron granules ( particularly peripherally ) ( figure 5b ) . only very sparse , small islands of iron within macrophages were visible for the 32 nm tracer ( figure 5c ) . there was a significant difference in the grade of the iron content of excised slns between the three different tracers overall ( p<0.0001 ) ( figure 6 ) . the amount of iron deposition was significantly greater with the 111 nm tracer and 59 nm tracer ( p<0.05 ) compared to the 32 nm tracer . rapid uptake of a magnetic tracer after interstitial injection is important to maximize transcutaneous hotspot detection and enable injection in the theater suite , facilitating theater scheduling . transcutaneous identification of hotspots was possible in most procedures with the 59 nm tracer within 10 minutes of the injection and in all except one procedure ( with the 111 nm tracer ) within 240 minutes . within 30 minutes of injection , all hotspots were already identifiable using the 59 nm tracer . the greater sensitivity of the magnetometer for the 59 nm tracer for a given quantity of iron compared to the other two tracers ( figure 4 ) facilitates hotspot detection . discoloration of the skin was present at the injection site using all tracers , but this was not quantified during this study . our animal model did not allow to evaluate whether the skin discoloration would fade away over time or persist as a tattoo . although superficial administration of an spio tracer can result in skin tattooing , the current clinical studies using spio have also not reported adequate follow - up data to fully quantify this.1214 however , any tattooing is anticipated to be temporary rather than permanent but may persist for several months similar to that expected for blue dye.23 any tracer used for slnb must be retained by the slns and not pass on to higher echelon nodes in order to avoid excessive excision of normal nodes . within this study , a statistically significantly greater number of nodes were excised using the 32 nm tracer compared to the other two tracers . it suggests that rather than being retained within the sln after entering through the afferent duct , it passes through the efferent system and onto higher echelon nodes . this is reflected by the small islands of sparsely distributed iron within the nodes after the administration of the 32 nm tracer on histopathology ( figure 5c ) . by applying the 10% rule and excising those nodes with only 10% of the ex vivo count of the hottest excised node using radioisotope , martin et al24 demonstrated that a mean sln harvest of 1.96 nodes was associated with a false - negative rate of 5.8% . in the sentimag multicentre trial using the 59 nm tracer , excising more than two slns during sentinel node biopsy is not proven to be beneficial except after primary systemic therapy.25 our data suggest that the small - sized 32 nm tracer would result in removal of an excessive number of normal nodes . in addition to retention , the accumulation of magnetic tracer within the slns is essential for intraoperative identification . the 59 nm tracer demonstrated statistically significant higher ex vivo magnetometer counts compared to the other two tracers , but only a statistically significant greater iron content over the 32 nm tracer . this is explained by the higher sensitivity of the used magnetometer for the 59 nm tracer compared to the other two tracers . the higher sensitivity for the 59 nm tracer is due to differences in magnetic properties of the tracers , arising from differences in size and material of the magnetic cores.26 by injecting the magnetic tracer 24 hours before surgery , one may be able to improve the iron uptake by the slns and thereby improve the performance of a tracer . however , extending the period between injection and surgery could allow adverse spio migration to higher echelon nodes beyond the sln . therefore , this is likely only suitable for the larger 111 nm tracer within this context . however , to optimize the time between injection and surgery for each tracer , formal assessment of individual tracers for slnb when injected at different times before surgery is needed . the variation in the accumulation of iron within nodes , between different tracers was demonstrated on histopathology . there was significantly less iron deposition in nodes from the 32 nm tracer compared to the other tracers , with only sparse , small islands of iron derived from the 32 nm tracer being observed in the nodes . there was no difference in the grading of nodes between the 59 nm tracer and the 111 nm tracer on histopathology . in both cases , the iron was distributed in the subcapsular space and sinuses of nodes , consistent with previous histopathological studies.20,27 however , the 111 nm tracer was found to be deposited mainly within macrophages demonstrating its ability to activate the mononuclear phagocytic system compared to the 59 nm tracer , which although demonstrating phagocytosis within the node also displayed extensive free iron granules peripherally . these two processes of activating the mononuclear phagocytic system and free iron granule deposition facilitate iron accumulation and hence sln identification using a handheld magnetometer . different studies have recently evaluated the influence of magnetic nanoparticle size on lymphatic uptake in rodent models.2830 tracers with particle sizes ranging from 4 to 1,000 nm were used in these studies . the purpose of these studies was to optimize tracer uptake in the slns to facilitate preoperative localization with mri , rather than intraoperative detection . mori et al30 used 50 nm , 100 nm , 200 nm , and 1,000 nm sized particles and concluded that particles of 200 nm and larger are not suitable in view of lack of uptake within 24 hours . iida et al28 compared the performance of 4 nm , 8 nm , and 20 nm citrate - coated nanoparticles and concluded that the 20 nm particles were best retained in the slns and accumulated in the highest concentration . however , the 20 nm tracer was the largest used , and therefore , it is possible that uptake could be further optimized by increasing the particle size . kjellman et al29 used monodisperse magnetic nanoparticles with a polyethylene glycol coating with sizes of 15 nm , 27 nm , and 57 nm . the 15 nm particles were observed to accumulate in the slns the fastest and in the highest concentrations . a 15 nm polyethylene glycol coated tracer approved for use in humans is currently not available and was not used in our study . therefore , the efficiency of the 15 nm tracer can not be compared to our results . during the course of the animal experimentation , there were no signs of adverse events developing within the animals as a consequence of the magnetic tracer administration . when magnetic tracers are used as mri contrast agents , doses of 25100 mg are typically administered . animal studies have demonstrated no acute or subacute toxicity when 150 times this dose has been administered.31 consequently , iron overload would not be possible within this model due to the small amounts of iron injected ( 5.6 mg ) . in this study however , only the 59 nm tracer is currently ce - marked for the purpose of slnb in europe . the 111 nm and 32 nm tracers are licensed as an mri contrast agent and an intravenous treatment of iron deficiency anemia , respectively . apart from the dilution of the tracers to standardize iron concentrations , no changes were made to the constituents to facilitate the translation of the results to clinical practice . the formulation of the tracers is very similar ; all are aqueous suspensions of magnetic nanoparticles in water , with a ph level ranging from 5 to 9 . the most remarkable difference between the tracers is the particle size ; however , differences in coating and other constituents are also evident . the coating of the 111 nm tracer and 59 nm tracer are both dextran based ( dextran and carboxydextran , respectively ) ; however , a polyglucose sorbitol carboxymethylether coating is used in the 32 nm tracer . although the coating is known to be a factor influencing in vivo nanoparticle distribution , particle size was demonstrated to be the most significant factor for interstitially administered tracers.19,29,3234 the poor performance of the polyethylene glycol coated 32 nm tracer compared to the other tracers is therefore likely explained by a combination of the particle size distribution and the coating . the observed differences between the dextran - coated tracers are most likely explained by the different particle size distributions of these tracers . currently , the 59 nm tracer is the best performing magnetic nanoparticle tracer , approved for human use , in slnb with a handheld magnetometer . the particles of the 59 nm tracer distribute rapidly from the injection site to the slns , allowing transcutaneous localization within 30 minutes in contrast to the 240 minutes for the 32 nm tracer and 111 nm tracer . the particles of the 59 nm tracer are retained in the first nodes reached in contrast to the particles of the 32 nm tracer . finally , the particles accumulate in high concentrations , facilitating intraoperative localization . the rapid distribution , retention in the first nodes reached , and accumulation in high concentration make the 59 nm tracer the most suitable magnetic tracer for slnb .

introductionbreast cancer staging with sentinel lymph node biopsy relies on the use of radioisotopes , which limits the availability of the procedure worldwide . the use of a magnetic nanoparticle tracer and a handheld magnetometer provides a radiation - free alternative , which was recently evaluated in two clinical trials . the hydrodynamic particle size of the used magnetic tracer differs substantially from the radioisotope tracer and could therefore benefit from optimization . the aim of this study was to assess the performance of three different - sized magnetic nanoparticle tracers for sentinel lymph node biopsy within an in vivo porcine model.materials and methodssentinel lymph node biopsy was performed within a validated porcine model using three magnetic nanoparticle tracers , approved for use in humans ( ferumoxytol , with hydrodynamic diameter dh = 32 nm ; sienna+ , dh = 59 nm ; and ferumoxide , dh = 111 nm ) , and a handheld magnetometer . magnetometer counts ( transcutaneous and ex vivo ) , iron quantification ( vibrating sample magnetometry ) , and histopathological assessments were performed on all ex vivo nodes.resultstranscutaneous hotspots were present in 12/12 cases within 30 minutes of injection for the 59 nm tracer , compared to 7/12 for the 32 nm tracer and 8/12 for the 111 nm tracer , at the same time point . ex vivo magnetometer counts were significantly greater for the 59 nm tracer than for the other tracers . significantly more nodes per basin were excised for the 32 nm tracer compared to other tracers , indicating poor retention of the 32 nm tracer . using the 59 nm tracer resulted in a significantly higher iron accumulation compared to the 32 nm tracer.conclusionthe 59 nm tracer demonstrated rapid lymphatic uptake , retention in the first nodes reached , and accumulation in high concentration , making it the most suitable tracer for intraoperative sentinel lymph node localization .

pubmed

visceral adipose tissue - derived serine proteinase inhibitor ( vaspin ) was identified in the visceral adipose tissue of oletf ( otsuka long - evans tokushim a fatty ) rats , an animal model of obesity and type 2 diabetes mellitus ( t2 dm ) . vaspin is also expressed in the skin , hypothalamus , pancreatic islets , and stomach . circulating levels of adiponectin , a hormone produced predominantly by adipocytes , are highly heritable and are inversely associated with t2 dm and other metabolic traits . some studies have shown that plasma vaspin concentrations are significantly higher in men with the metabolic syndrome compared with those without the metabolic syndrome . the serum vaspin levels in the patients with type 2 diabetes have been demonstrated to be higher than [ 5 , 6 ] or similar to those observed in subjects with normal glucose tolerance . in recent , inoue et al . reported that the serum vaspin levels are negatively correlated with the creatinine levels and were significantly reduced in the japanese chronic hemodialysis ( hd ) patients . thus , it was interesting to ask whether serum vaspin levels are elevated or reduced in diabetic patients with renal insufficiency . we measured serum vaspin and adiponectin levels in subjects with normal renal function and renal insufficiency with or without t2 dm and compared various clinical parameters . all subjects were local residents of han ethnicity in shanghai and consecutively recruited in department of endocrinology and cardiology , xinhua hospital and shanghai tenth people 's hospital from june 2011 to december 2011 . the study was approved by the ethical committee of shanghai tenth people 's hospital and xinhua hospital . clinical characteristics were recorded with respect to age , sex , body mass index ( bmi ) , risk factors for renal function , and diabetic mellitus ( dm ) . dm was defined as fasting serum glucose level 7.0 mmol / l or 2-hour postprandial glucose 11.1 mmol / l , or taking hypoglycemic drugs . those with type 1 diabetes , acute myocardial infarction , systemic inflammatory disorders , severe heart failure ( left ventricular ejection fraction < 30% ) , advanced renal insufficiency , and malignant tumor were excluded . human total adiponectin ( r&d systems , minneapolis , american ) and vaspin ( adipogen , seoul , south korea ) plasma levels were measured with commercially available elisas as indicated before . while high - sensitive c - reactive protein ( hscrp ) , fibrinogen ( fib ) , creatinine ( cr ) , blood urea nitrogen ( bun ) , uric acid ( ua ) , fasting blood glucose ( fbg ) , 2-hour postprandial glucose ( pg2h ) , hemoglobin a1c ( hba1c ) , and lipid profiles , including total cholesterol ( tc ) , triglyceride ( tg ) , high - density lipoprotein cholesterol ( hdl - c ) , low - density lipoprotein cholesterol ( ldl - c ) , apolipoprotein a ( apoa ) , and apolipoprotein b ( apob ) were measured by colorimetric enzymatic assay systems ( roche modular p-800 , swiss confederation ) . 416 subjects were divided into 4 groups according to the presence or absence of ri and t2 dm : 247 individuals without t2 dm who had ri ( n = 27 ; group i ) or normal renal function ( n = 220 ; group ii ) and 169 patients with t2 dm who had ri ( n = 23 ; group iii ) or normal renal function ( n = 146 ; group iv ) . renal function was assessed by the estimated creatinine clearance ( crcl ) derived from cockroft - gault formula , where crcl ( ml / min ) = ( [ 140 age ( years ) ] weight ( kg))/(0.818 serum creatinine ( mol / l ) ) , corrected in women by a factor of 0.85 . a calculated crcl less than 60 ml / min was defined as ri , according to guidelines . statistical analyses were performed using the pasw statistics 20 software ( ibm corporation , armonk , ny , usa ) . comparisons of variables with a normal distribution were made using student 's t - test , and values were provided as mean s.d . for parameters with an abnormal distribution , the mann - whitney u test was used for comparisons , and values were given as median ( interquartile range ) . categorical variables were presented as frequencies . to evaluate the correlation between serum vaspin and adiponectin levels with other variables such as age , bmi , hscrp , crcl , cr , fbg , and tc single linear univariate correlations and multivariate regression analyses were performed . in the population either with t2 dm or without t2 dm , patients with ri ( group i or group iii ) were older and had lower bmi , higher systolic blood pressure ( sbp ) than those without ri ( group ii or group iv ) ( all p < 0.05 ) ( table 1 ) . the serum vaspin showed no significant differences either between non - t2 dm patients with ri ( group i ) and without ri ( group ii ) ( 0.548 ( 0.4281.136 ) ng / ml versus 0.517 ( 0.2321.324 ) ng / ml , p = 0.402 ) or between diabetic patients with ri ( group iii ) and without ( group iv ) ( 0.497 ( 0.2880.907 ) ng / ml versus 0.381 ( 0.2320.740 ) ng / ml , p = 0.245 ) . interestingly , the serum vaspin was lower in 169 patients with t2 dm than in 247 individuals without t2 dm ( 0.399 ( 0.2240.737 ng / ml versus 0.523 ( 0.1821.172 ) ng / ml , p < 0.05 ) ( table 2 ) . however , no significant difference for the serum vaspin level was observed between 50 patients with ri ( combined by group i and group iii ) and 366 individuals without ri ( combined by group ii and group iv ) ( 0.540 ( 0.3991.020 ) ng / ml versus 0.432 ( 0.2340.925 ) ng / ml , p = 0.141 ) ( table 2 ) . compared with the nondiabetic individuals without ri ( group ii ) , the serum adiponectin was higher in the non - diabetic individuals with ri ( group i ) ( 16082.437 9926.203 ng / ml , p < 0.01 ) but did not differ between diabetic patients with ri ( group iii ) and without ri ( group iv ) ( 12987.336 14623.762 ng / ml versus 8744.920 5628.952 ng / ml , p = 0.349 ) . regardless of the individuals with or without t2 dm , the serum adiponectin level was significantly higher in 50 individuals with ri ( combined by group i and iii ) than 366 individuals without ri ( combined by group ii and iv ) ( 10549.706 ( 5589.71822429.025 ) ng / ml versus 6955.130 ( 2590.05213533.955 ) ng / ml , p < 0.01 ) ( tables 1 and 2 ) . when compared with the individuals without t2 dm ( combined by group i and ii ) in our tall 416 samples , the serum adiponectin was significantly higher in 169 patients with t2 dm ( combined by group iii and iv ) ( 7599.068 ( 4100.63313827.261 ) ng / ml versus 5553.476 ( 12.09614225.162 ) ng / ml , p < 0.05 ) . spearmen 's analysis showed that serum vaspin level was only related to cr in patients without t2 dm . in patients with t2 dm , serum vaspin levels were related to age , sbp , and ua . in all subjects , serum vaspin levels were related to crcl , tc , and hba1c ( tables 3 and 4 ) . the serum adiponectin levels were related to age , sbp , hscrp , crcl , and cr in subjects without t2 dm . in patients with t2 dm , serum adiponectin levels were related to fbg , ldl - c . in all subjects , serum adiponectin levels were related to age , sbp , crcl , cr , bun , and hba1c ( tables 3 and 4 ) . multiple regression analyses were performed to test whether any of the following factors influence the serum vaspin and adiponectin concentrations : age , sbp , fbg , ua , crcl , cr , tg , tc , hba1c levels , and so on . when correcting for these variables using stepwise regression analyses after adjustment for age and bmi , the level of hba1c was found to be independently and significantly associated with the vaspin concentration in 427 total subjects . however , no significant correlations between the serum vaspin level and any parameter were detected in both groups of the subjects with t2 dm and without t2 dm . the level of crcl was found to be independently and significantly associated with the adiponectin concentration in all subjects . however , in the groups of the subjects with or without t2 dm , no significant correlations were detected between the serum adiponectin level and any parameter ( tables 5 and 6 ) . mann - whitney u test showed that the serum vaspin and adiponectin levels were both higher in women than in men whether in total subjects or in patients with t2 dm ( tables 7 and 8) . adipose tissue is a highly active endocrine organ secreting a number of bioactive molecules called adipokines [ 5 , 6 , 12 ] , such as vaspin and adiponectin . vaspin is a member serpin a12 of the serine protease inhibitor family [ 1315 ] , and a large number of clinical data have showed that vaspin played an important role in occurrence and development in the metabolic syndrome [ 1 , 16 , 17 ] . upregulation of vaspin can improve the insulin resistance , which also inhibit inflammation and progression of arthrosclerosis . some studies show that vaspin increases nitric oxide bioavailability through the reduction of asymmetric dimethylarginine in vascular endothelial cells or inhibits platelet - derived growth factor - bb - induced migration of vascular smooth muscle cells . the decrease of vaspin in the ri patients will increase the risk of coronary heart disease ( chd ) . therefore , measuring the levels of adipokines in patients with ri may be beneficial for predicting cardiovascular events and patient survival . in patients with ckd , the levels of adipokines appear to increase in association with declines in the gfr . this is most likely due to reduced renal metabolism of adipokines , which may not increase vascular risks in patients with a reduced renal function . since vaspin is a small protein size with the molecular mass 50 kda , which would be freely filtered by the kidneys ; thus it is reasonable to presume that vaspin levels should be increased in patients with renal insufficiency . but our data suggested that the serum vaspin levels have no significant difference between patients with ri and subjects without ri due to the fact that many factors might affect the serum levels of vaspin concentration . previous studies have shown that , in the diabetic patients , serum vaspin level is higher than that in normal glucose tolerance subjects in women , and rosiglitazone therapy decreased plasma vaspin levels through glucose and insulin sensitivity regulation . our study also shows that the serum vaspin level was higher in women than in men . however , we have found that the serum vaspin level was lower in 169 diabetic patients than 247 individuals without t2 dm . the paradox results may be caused by two reasons : one is the sex difference in the subjects between their study and our study ; another reason is that there are differences in the diabetic patients with vascular complications between the two population , since some reports have demonstrated that low vaspin serum concentrations have been found to be correlated with recently experienced ischemic events in patients with carotid stenosis and coronary artery disease ( cad ) . adiponectin is a fat - derived hormone whose reduction plays central roles in obesity - linked diseases including insulin resistance , type 2 diabetes , and atherosclerosis . adiponectin multimers are secreted by white adipose tissue [ 2527 ] , which is associated with body weight and markers of cardiovascular risk in adolescence . adiponectin has recently attracted much attention because of its antidiabetic and antiatherogenic effects as well as its antiproliferative effects in cancer cells ; thus , adiponectin is expected to be a therapeutic tool for diabetes , metabolic syndrome , cardiovascular diseases , and cancers [ 29 , 30 ] . plasma adiponectin levels have also been reported to be reduced in obese humans , particularly those with visceral obesity [ 31 , 32 ] . so , in the ri patients , lower bmi may lead to the high level of adiponectin but in the diabetic patients , there are no differences between ri and non - ri . some studies have shown that rosiglitazone can increase serum adiponectin levels in patients with type 2 diabetes mellitus [ 33 , 34 ] . in all patients with diabetes in our current study , only 19 patients have been treated with thiazolidinediones . after removing the patients , both of t2 dm and renal insufficiency were correlated with the serum level of adiponectin . although the serum vaspin levels showed no significant difference between the patients with renal insufficiency which did not required treatment by chronic hemodialysis and subjects without renal insufficiency , we have found that the serum vaspin level was lower in 169 diabetic patients than in 247 individuals without t2 dm .

vaspin and adiponectin are two adipocytokines with antidiabetic effects . some studies reported that levels of adiponectin and vaspin were correlated with decreased glomerular filtration rate ( fgr ) and increased albuminuria . we therefore evaluated the vaspin and adiponectin levels in renal insufficiency ( ri ) patients with or without t2 dm . serum vaspin , adiponectin levels were measured in 416 subjects with or without t2 dm . analysis was made between groups divided by these subjects presence or absence of ri . we found that serum adiponectin level was significantly higher in nondiabetic patients with ri than in nondiabetic subjects without ri ; however , there were no statistical differences between the diabetic patients with ri and without ri . in all the subjects , the serum adiponectin level was also higher in 50 individuals with ri than that in 366 subjects without ri . the serum vaspin levels showed no significant differences between the diabetic patients or nondiabetics subjects with ri and without ri . contrary to adiponectin , the serum vaspin level was lower in 169 patients with t2 dm than in 247 individuals without t2 dm . our data suggested that both of t2 dm and renal insufficiency were correlated with the serum level of adiponectin . however , the serum vaspin levels showed no significant difference between the individuals with renal insufficiency and without renal insufficiency .

pubmed

data from two multicenter , double - blind , randomized clinical trials were used to test the surrogate variables proposed for neuropathic dfus and to explore the correlation between granulation tissue development ( the main efficacy variable of the studies ) and complete healing during follow - up for 1 year after the eot ( median time to complete closure , 15 weeks ; 25th percentile , 9.6 weeks ; 75th percentile , 21.4 weeks ) . in the first study , a dose - exploratory trial in five hospitals ( 13 ) , patients were randomized to receive intralesional injections of rhegf at 75 ( 23 subjects ) or 25 g ( 18 subjects ) , three times per week . in the second study , a confirmatory trial in 20 hospitals ( 16 ) , 53 subjects were randomized to rhegf 75 g , 48 to rhegf 25 g , and 48 to a placebo group . each patient had only one lesion , so the total number of ulcers included in the analyses was 190 . for confirmation in a larger sample from common clinical practice , the results of an active postmarketing surveillance of rhegf treatment in 1,440 dfus in 60 healthcare units ( 41 hospitals and 19 primary care polyclinics ) were taken into account ( unpublished data ) . in this series , the 25- and 75-g dose levels were used , according to the product label ( 17 ) . in all series , rhegf treatment was administered up to complete granulation or a maximum of 8 weeks . diabetic patients ( type 1 or 2 ) , both sexes , > 18 years of age were included in the trials , with wagner grade 3 or 4 dfus > 1 cm . intralesional rhegf was administered adjuvant to standard care , which included thorough debridement ( sometimes minor amputation ) , moist dressing , pressure offloading , and antibiotics , if necessary . patients with chronic , uncontrolled illness , psychiatric or neurologic diseases that could impair proper reasoning for consent , or presence or suspicion of neoplasia or who were pregnant or breastfeeding were excluded . ( 2 ) as surrogate markers of complete healing were generated : percent change in area , log healing rate , and ratio of log areas at the 2nd week and eot ( median , 5 weeks).additionally the variablewas taken into account . considering that the median treatment duration was 5 weeks , and that there was no area evaluation at 4 weeks for most of the patients ( > 50% missing values ) , the eot data were taken as the comparison points with the 4-week area change proposed by other authors ( 24 ) . ulcer areas and percent granulation were measured by planimetry from manual tracing on a transparent grid sheet , using a validated portable wound measurement device ( visitrack ; smith & nephew , london , u.k . ) ( 18 ) . for comparability with the results in margolis et al . ( 2 ) , the variables defined in eqs . 2 and 3 were calculated using the area in mm and t in days . continuous variables are expressed as mean sd or median ( 25th and 75th percentiles ) . comparison between healed and nonhealed was assessed by the test for categorical variables and mann - whitney u test for continuous variables . since missing data can constitute deviations from the intention - to - treat principle , clinical trial analyses require the use of imputation ( 19 ) . for the purpose of this work , 18 missing data at the 2nd week ( 9.5% ) were imputed using the last observation carried forward ( locf ) or the average between the two contiguous evaluations that included the missing one . the area under the receiver operating characteristic ( roc ) curve was calculated ( 20 ) for each candidate surrogate variable to evaluate its capacity and quality of discrimination among individuals . cutoff points were generated in order to maximize both sensitivity ( probability to detect a positive result if the outcome is positive ) and specificity ( probability to detect a negative result if the outcome is negative ) . the candidate surrogates were then codified , and their association with the final outcome ( complete ulcer closure ) was assessed using the sensitivity , specificity , positive predictive values ( pv+ , conditional probability to have a positive result if the test was positive ) , negative pvs ( pv , conditional probability to have a negative result if the test was negative ) , and the correct classification index ( percent of individuals correctly classified ) , also named validity index . test validation and sensitivity were performed only for the placebo - controlled trial ( 16 ) for the sake of homogeneity and balance of the samples compared . to quantify the contribution of the candidate surrogates to the final outcome , the product coefficient ( pc ) this is one of the methods to assess mediation in the case of binary - dependent outcome that treats it as the product of two regression coefficients : the regression of the independent treatment variable on the mediator ( t in eq . 6 , below ) and the partial regression coefficient of the independent treatment variable on the final outcome adjusted for the mediator ( s in eq . 7 , below ) . in order to estimate the pc , closure ( 1 , yes ; 0 , no ) , x the baseline covariates x1 ( etiology : neuropathic and ischemic ) and x2 ( initial area ) , t the intervention status ( 1 for rhegf treatment and 0 for placebo ) , and s the granulation tissue covering the wound area ( at 2nd week or at the eot ) . the following models were adjusted : which measures the effect of treatment on healing without the influence of the surrogate , which measures the effect of treatment on the surrogate , taking into account the same covariates , andwhich measures the effect of treatment on wound closure , mediated by the surrogate , taking into account the covariates . the pc estimator and the proportion mediated and its 95% ci ( 21 ) were used to quantify the contribution of the surrogate ( s : granulation and area change variables ) on ulcer closure . the association with some baseline influencing factors between healing and candidate surrogate variables was estimated using the odds ratio and the 95% cis in binary logistic regression . confirmation was performed only for the granulation variable percent of granulation tissue covering the ulcer area ( > 75% or not ) , since during medical practice it was not possible to standardize a precise measurement of the wound area and partial percent of granulation . variables proposed by margolis et al . ( 2 ) as surrogate markers of complete healing were generated : percent change in area , log healing rate , and ratio of log areas at the 2nd week and eot ( median , 5 weeks).additionally the variablewas taken into account . considering that the median treatment duration was 5 weeks , and that there was no area evaluation at 4 weeks for most of the patients ( > 50% missing values ) , the eot data were taken as the comparison points with the 4-week area change proposed by other authors ( 24 ) . ulcer areas and percent granulation were measured by planimetry from manual tracing on a transparent grid sheet , using a validated portable wound measurement device ( visitrack ; smith & nephew , london , u.k . ) ( 18 ) . for comparability with the results in margolis et al . ( 2 ) , the variables defined in eqs . 2 and 3 were calculated using the area in mm and t in days . continuous variables are expressed as mean sd or median ( 25th and 75th percentiles ) . comparison between healed and nonhealed was assessed by the test for categorical variables and mann - whitney u test for continuous variables . since missing data can constitute deviations from the intention - to - treat principle , clinical trial analyses require the use of imputation ( 19 ) . for the purpose of this work , 18 missing data at the 2nd week ( 9.5% ) were imputed using the last observation carried forward ( locf ) or the average between the two contiguous evaluations that included the missing one . the area under the receiver operating characteristic ( roc ) curve was calculated ( 20 ) for each candidate surrogate variable to evaluate its capacity and quality of discrimination among individuals . cutoff points were generated in order to maximize both sensitivity ( probability to detect a positive result if the outcome is positive ) and specificity ( probability to detect a negative result if the outcome is negative ) . the candidate surrogates were then codified , and their association with the final outcome ( complete ulcer closure ) was assessed using the sensitivity , specificity , positive predictive values ( pv+ , conditional probability to have a positive result if the test was positive ) , negative pvs ( pv , conditional probability to have a negative result if the test was negative ) , and the correct classification index ( percent of individuals correctly classified ) , also named validity index . test validation and sensitivity were performed only for the placebo - controlled trial ( 16 ) for the sake of homogeneity and balance of the samples compared . to quantify the contribution of the candidate surrogates to the final outcome , the product coefficient ( pc ) this is one of the methods to assess mediation in the case of binary - dependent outcome that treats it as the product of two regression coefficients : the regression of the independent treatment variable on the mediator ( t in eq . 6 , below ) and the partial regression coefficient of the independent treatment variable on the final outcome adjusted for the mediator ( s in eq . ( 1 , yes ; 0 , no ) , x the baseline covariates x1 ( etiology : neuropathic and ischemic ) and x2 ( initial area ) , t the intervention status ( 1 for rhegf treatment and 0 for placebo ) , and s the granulation tissue covering the wound area ( at 2nd week or at the eot ) . the following models were adjusted : which measures the effect of treatment on healing without the influence of the surrogate , which measures the effect of treatment on the surrogate , taking into account the same covariates , andwhich measures the effect of treatment on wound closure , mediated by the surrogate , taking into account the covariates . the pc estimator and the proportion mediated and its 95% ci ( 21 ) were used to quantify the contribution of the surrogate ( s : granulation and area change variables ) on ulcer closure . the association with some baseline influencing factors between healing and candidate surrogate variables was estimated using the odds ratio and the 95% cis in binary logistic regression . confirmation was performed only for the granulation variable percent of granulation tissue covering the ulcer area ( > 75% or not ) , since during medical practice it was not possible to standardize a precise measurement of the wound area and partial percent of granulation . continuous variables are expressed as mean sd or median ( 25th and 75th percentiles ) . comparison between healed and nonhealed was assessed by the test for categorical variables and mann - whitney u test for continuous variables . since missing data can constitute deviations from the intention - to - treat principle , clinical trial analyses require the use of imputation ( 19 ) . for the purpose of this work , 18 missing data at the 2nd week ( 9.5% ) were imputed using the last observation carried forward ( locf ) or the average between the two contiguous evaluations that included the missing one . the area under the receiver operating characteristic ( roc ) curve was calculated ( 20 ) for each candidate surrogate variable to evaluate its capacity and quality of discrimination among individuals . cutoff points were generated in order to maximize both sensitivity ( probability to detect a positive result if the outcome is positive ) and specificity ( probability to detect a negative result if the outcome is negative ) . the candidate surrogates were then codified , and their association with the final outcome ( complete ulcer closure ) was assessed using the sensitivity , specificity , positive predictive values ( pv+ , conditional probability to have a positive result if the test was positive ) , negative pvs ( pv , conditional probability to have a negative result if the test was negative ) , and the correct classification index ( percent of individuals correctly classified ) , also named validity index . test validation and sensitivity were performed only for the placebo - controlled trial ( 16 ) for the sake of homogeneity and balance of the samples compared . to quantify the contribution of the candidate surrogates to the final outcome , the product coefficient ( pc ) this is one of the methods to assess mediation in the case of binary - dependent outcome that treats it as the product of two regression coefficients : the regression of the independent treatment variable on the mediator ( t in eq . 6 , below ) and the partial regression coefficient of the independent treatment variable on the final outcome adjusted for the mediator ( s in eq . 7 , below ) . in order to estimate the pc , ( 1 , yes ; 0 , no ) , x the baseline covariates x1 ( etiology : neuropathic and ischemic ) and x2 ( initial area ) , t the intervention status ( 1 for rhegf treatment and 0 for placebo ) , and s the granulation tissue covering the wound area ( at 2nd week or at the eot ) . the following models were adjusted : which measures the effect of treatment on healing without the influence of the surrogate , which measures the effect of treatment on the surrogate , taking into account the same covariates , andwhich measures the effect of treatment on wound closure , mediated by the surrogate , taking into account the covariates . the pc estimator and the proportion mediated and its 95% ci ( 21 ) were used to quantify the contribution of the surrogate ( s : granulation and area change variables ) on ulcer closure . the association with some baseline influencing factors between healing and candidate surrogate variables was estimated using the odds ratio and the 95% cis in binary logistic regression . confirmation was performed only for the granulation variable percent of granulation tissue covering the ulcer area ( > 75% or not ) , since during medical practice it was not possible to standardize a precise measurement of the wound area and partial percent of granulation . the median age was > 60 years , median ulcer size was 22 cm ( 75% > 10 cm ) , lesions were 57% ischemic , and wagner classification grades 3 and 4 were 73.7 and 26.3% , respectively . the inferential summary of baseline characteristics and candidate surrogates by healing is shown in table 1 . globally , 58.4% of the wounds achieved closure . complete healing showed dependence on all variables except for sex , ethnicity , treatment duration , and wagner classification . patient characteristics in terms of healing ( all treatment groups combined ) all variables tested had a good discrimination capacity , with roc curve areas > 0.7 at the 2nd week and > 0.8 at the eot ( see supplementary table 2 for details ) . those with the lower limit of the 95% ci > 0.7 were selected as the best ones : the three variables based on the area change at the eot and granulation tissue formation at the 2nd week and at the eot . table 2 shows these , as well as the sensitivity , specificity , positive and negative pvs , and percent correct classification calculated from them . the 95% cis were not shown for simplicity of the table , but they were all < 2% around the estimators , showing high precision . diagnostic test for candidate surrogate variables and complete healing the most efficient predictor was the granulation tissue covering > 75% of the ulcer area at the end of the treatment , with 79.5% correct classification and a high negative pv ( 93.5% ) . an earlier evaluation of this surrogate marker ( granulation tissue covering > 50% of the ulcer area at 2 weeks ) was also adequate , with 73.7% correct classification , pvs ( positive and negative ) > 70% , and acceptable sensitivity and specificity values . the best cutoff points for variables based on the area change at the eot were selected too . the correct classification was also high ( > 70% ) , with suitable diagnostic indicators . at this point , all candidates can be used to discriminate healing . the proportion - mediated ( using pc estimators ) for 2 weeks > 50% granulation and eot > 75% granulation were 1.008 ( 95% ci 0.9331.083 ) and 1.043 ( 0.9461.142 ) , respectively , both very close to 1 , suggesting that almost 100% of the effect of the treatment on complete healing may have been mediated by the formation of granulation tissue . with the area change the mediation explained by this variable was very low : 0.003 ( 95% ci 0.002 to 0.008 ) . the effect of the treatment on healing of this type of complicated ulcers is not necessarily mediated by an area reduction in the earlier weeks . the models given by eqs . 6 and 7 for percent area change and ratio of log areas table 3 shows the association between healing and candidate surrogate variables with some baseline influencing factors . all the models with the granulation - based candidates were adequate ( hosmer - lemeshow goodness - of - fit statistics ) . treatment had a significant influence on healing and the two granulation - dependent variables considered . for simplicity , only the 75-g rhegf is shown , since the 25-g dose had no significant effect on healing in the clinical trial ( 16 ) . the effect of the intervention on healing was lost when either 2 weeks > 50% granulation or eot > 75% granulation is incorporated in the model . the odds ratios for the influence of the covariates ( initial area and etiology ) were similar for the three dependent outcomes ( 2-week and eot granulation and healing ) . association between covariates and outcomes ( granulation and healing ) the results of the model with the eot percent area change are also shown in table 3 as an example . models with the log healing rate variable are not shown , but their interpretation was the same . no effect of treatment on the surrogate was found ( 95% ci 0.714.03 ) , and the effect of treatment on complete healing was not lost when the percent area change was incorporated in the model . the results of the granulation - healing correlation analysis in the postmarketing study in current medical practice were obtained from the data of 1,440 ulcers . the result confirms that granulation tissue development at the eot was an early predictor of final healing , with a 72.9% certainty and positive / negative pvs of 71.7/75.8% , respectively ( details in supplementary table 4 ) . figure 1 illustrates the discrimination capacity of the granulation variables in clinical trials and in the postmarketing series . discrimination capacity of the granulation variables in clinical trials ( cts ) and in the postmarketing ( pm ) series . the results of this study show that several surrogate variables can be taken into consideration for the final outcome in advanced dfus , including ischemic , wagner grade 3 or 4 , and median area > 20 cm . granulation tissue development has been taken previously as a secondary outcome in dfu trials ( 14,15 ) . it was also proposed as a potential intermediate variable ( 9 ) , but its use as a surrogate of complete wound closure has not been proposed before , despite being part of the healing process and necessarily preceding final re - epithelization . the granulation process showed good capacity to discriminate healers from nonhealers with roc curve areas > 70% at the 2nd week and eot . the validation tests using multivariate analyses were developed with the data of the randomized placebo - controlled clinical trial of intralesional rhegf in advanced dfus ( 16 ) . the proportion mediated for granulation variables ( at 2 weeks and at the eot ) was very high , which agrees with the idea that granulation constitutes an essential part of the healing process , particularly in larger and advanced ulcers . the egf - induced mitogenic , motogenic , and cytoprotective actions are instrumental for healing events that may be summarized as follows : 1 ) stimulation of productive cell migration toward the injured area ; 2 ) stimulation of granulation tissue outgrowth , including extracellular matrix accumulation , maturation , and de novo angiogenesis ; 3 ) stimulation of wound contraction by myofibroblast activation and proliferation ; and 4 ) stimulation of the damaged area resurfacing by epithelial cell migration and proliferation ( 23 ) . since treatment with rhegf was given only up to complete granulation , mechanisms 1 and 2 took place completely but not 3 and 4 , which require a previous granulating wound bed . for area change dependent candidates , the proportion mediated was small , which could be explained by a more exigent condition for these lesions . had the treatment continued after complete granulation , maybe a better proportion mediated would be obtained , given that mechanisms 3 and 4 could have worked longer . the models for final cicatrization and the granulation surrogates were adequate and fulfilled the original prentice ( 24 ) criteria for the definition of a surrogate end point : 1 ) treatment had a significant effect upon the surrogate end point ; 2 ) treatment had a significant effect upon the true end point ; 3 ) the surrogate end point had a significant effect upon the true end point ; and 4 ) the full effect of treatment upon the true end point was mediated by the surrogate . additionally , the effect of the covariates ( wound area and ulcer etiology ) was similar in all models . criterion 4 is highly dependent on the power of the model and the sample size used . patients with such advanced dfus are usually not included in clinical trials , which limits the possibility of meta - analysis at present . the larger sample of the postmarketing series could not be included since it was not a controlled study . nevertheless , it was useful to confirm the predictive character of the eot granulation on final wound closure . on the other hand , as a measure of statistical power , 10 events per predictor is considered suitable , taking all the candidates and the other covariates ( 25,26 ) . in this work , only in the placebo - controlled confirmatory trial ( 16 ) , there were 93 healing events and 9 variables , so the criterion is fulfilled . eot > 75% granulation had a higher pv with respect to final re - epithelization and thus should be a better surrogate for clinical trial design . this can be useful since it would not be necessary to wait for complete re - epithelization due to treatment , and alternative wound closure procedures could be used after complete granulation , such as grafts or engineered skin . the 2 weeks > 50% granulation , being an earlier surrogate , may also have practical value for treatment algorithm decisions and/or pharmacoeconomic considerations . several variables based on wound area change have been proposed as surrogates for complete healing in neuropathic noncomplicated lesions . ( 2 ) described the predictive capacity of 61% area reduction at 4 weeks as well as several transformations of the area change . sheehan ( 27 ) , based on previous work ( 3 ) , proposes 50% wound closure at 4 weeks as a good surrogate for final healing and thus a decision point in a foot ulcer management algorithm . the results of the present work suggest that the predictive property of percent area change , log healing rate , and ratio of log area can be extrapolated to more complicated , ischemic , larger , wagner grade 3 or 4 ulcers . however , the cutoff points for classification were not the same . with the cutoff proposed by the above - mentioned authors , the correct classification percent in the present series was < 70% , with negative pvs < 60 . this is reasonable since larger and more complex ulcers take longer to heal , and thus the value to expect as predictive around 4 weeks should be smaller , 16% area change , and much lower for the other transformed variables . it was not possible to validate the surrogate property for these variables in this series since the percent area change did not fulfill the prentice criteria ( 24 ) 1 and 4 . additionally , the proportion mediated by these variables was very low ( discussed above ) . the extrapolation of the cutoff points proposed in the present work has the limitation of the small sample size analyzed , so it requires further research . it could not be performed in the confirmatory , postmarketing series since the partial areas were not measured in routine clinical practice . this work provides the first evidence for the use of granulation tissue development as a predictor of wound healing in advanced dfus . these results can be useful for clinical trial design , particularly during the exploratory phase of development of new products . the surrogate markers that we report can easily be applied by clinicians because the measurement of the proportion of wound area covered by granulation tissue is part of routine clinical care . correctly classifying patients with advanced diabetic foot ulcers into those who will heal or not heal by the 2nd week of care or at the end of granulation may have relevant implications in new wound healing products and for healthcare programs managing diabetic foot ulcers .

objectiveto determine if partial wound closure surrogate markers proposed for neuropathic , small diabetic foot ulcers ( dfus ) can be extended to advanced lesions and if the development of granulation tissue can be used to predict complete healing.research design and methodsdata from two multicenter , double - blind , randomized clinical trials ( one of them placebo controlled ) that used intralesional recombinant human epidermal growth factor ( rhegf ) to promote granulation and healing were used . for confirmation in a larger sample from common clinical practice , the results of an active postmarketing surveillance of rhegf treatment of dfus in 60 healthcare units was included . the surrogates evaluated were percent area change , log healing rate , ratio of log areas , and percent of granulation tissue covering the wound area . the tests used were surrogate final end point correlation , receiver operating characteristic curves to discriminate healers from nonhealers , validation tests using logistic regression models , and the proportion - mediated estimation.resultstwo weeks > 50% granulation , end of treatment > 75% granulation , and 16.1% area change showed significant predictive value ( > 70% correct classification ) for final wound closure . the granulation - based variables fulfilled the criterion that the effect of rhegf treatment on wound closure was mediated by the surrogate.conclusionsthis work provides the first evidence for the use of granulation tissue development as a predictor of wound healing in advanced dfus . these results can be useful for clinical trial design , particularly during the exploratory phase of new products .

pubmed

to date , laparoscopic transabdominal preperitoneal ( tapp ) mesh repair has been regarded as one of the standard treatments for inguinal hernia in adults . however , patients who undergo the procedure experience a significantly higher incidence of early postoperative pain , which is associated with subsequent chronic pain . surgical trauma to the abdominal wall , mesh implantation , and mesh fixation in the groin region have been indicated as possible causes of the pain . the ideal mesh material is still being debated , but mesh fixation with fibrin glue or the alternate choice of self - fixating mesh with microhooks has obvious advantages in reducing chronic groin pain . in the context of surgical tissue trauma at trocar sites , guidelines suggest that the use of a 10-mm trocar in the umbilical region or in the oblique abdominal wall predisposes patients to local nerve or vessel injury and port - site hernia . thus , an improvement in current laparoscopic hernia surgery has been the further reduction of the size of the abdominal wall incision for trocar insertion . modified tapp repairs by needlescopic surgery ( ntapp ) , with the use of smaller trocars than the standard 10-mm optical trocars and 5-mm working trocars , have been shown to be superior in patient outcomes over conventional tapp repairs . also tapp repairs in single - incision laparoscopic surgery ( stapp ) , with a reduction in the number of ports to only 1 umbilical port , have been put forward with good initial results . however , a direct comparison between these 2 advanced minimally invasive approaches is still missing . the purpose of this study was to compare the perioperative extent of surgical trauma and its postoperative impact on the abdominal wall after ntapp and stapp repair . for this retrospective cohort study with institutional review board approval , 85 patients with a single - sided primary inguinal hernia were recruited at a community hospital between november 1 , 2009 , and july 31 , 2012 . in this period of 32 months demographic and surgical data , including gender , age , body mass index , location and size of the inguinal hernia , length of the umbilical skin incision for the trocar port , operative time , and length of hospital stay were retrospectively retrieved from surgical records . on postoperative day 3 , all patients were scheduled for a wound check and removal of skin staples . in october 2013 , the patients were asked to complete a questionnaire and undergo a physical examination in the outpatient clinic . the following information was then recorded : hernia recurrence ; postoperative pain , according to the visual analog scale ( vas ) ( 0 = no pain ; 10 = worst pain ) ; postoperative mobility of the abdominal wall , determined by janda 's test , a sit - up exercise ( 0 = no recovery of normal muscle power ; 5 = full recovery of normal muscle power ) ; postoperative cosmetic satisfaction , according to the cosmetic vas ( 0 = completely dissatisfied ; 10 = completely satisfied ) ; and period of sick leave after hospital discharge for employed workers . the operative procedure in laparoscopic tapp mesh repair was described in depth in our previous study . in brief , it involved establishing pneumoperitoneum , dissecting the peritoneal flap and the preperitoneal space with an electric hook , placing a 15 9-cm parietene progrip self - fixating monofilament polypropylene mesh ( covidien , dublin , ireland ) , and closing the peritoneal flap with a v - loc barbed suture ( covidien ) . in the ntapp group , the length of the transverse skin incision at the umbilicus was first measured , and an 8-mm trocar was inserted for the use of a 5-mm optical instrument . under optical view , a 5-mm trocar for the electric hook on the right side of the abdomen and a 2-mm trocar for the grasping forceps on the left side of the abdomen were placed at the level of the umbilicus on the midclavicular line . in the stapp group , the umbilical skin incision length was also first measured , and a gelpoint advanced - access platform ( applied medical , rancho santa margarita , california ) was placed through the umbilicus . a 5-mm optical trocar and two 5-mm working trocars were then positioned in this single - port - access device . the self - fixating parietene progrip mesh , with microhooks on its underside , was folded in a special manner , as demonstrated in our previous publication . it was then inserted into the abdominal cavity through the 8-mm umbilical trocar in the ntapp group or through the 510-mm compatible cannula of the gelpoint advanced - access platform in the stapp group . once hernia repair was completed , the umbilical wound was closed with an absorbable fascia suture and skin staples in all ntapp and stapp procedures . the lateral 5- and 2-mm trocar wounds in the ntapp group were closed with steri - strips adhesive skin closures ( 3 m health center , st . all operations were performed on an elective basis in patients under general anesthesia by the same experienced surgeon . spss 22 for windows ( ibm corp . , somers , new york ) was used for statistical analysis . data are expressed as percentages for categorical variables and as the mean sd for continuous variables . comparisons between the 2 operative approaches were performed with the test and student 's t test , as applicable . for this retrospective cohort study with institutional review board approval , 85 patients with a single - sided primary inguinal hernia were recruited at a community hospital between november 1 , 2009 , and july 31 , 2012 . in this period of 32 months demographic and surgical data , including gender , age , body mass index , location and size of the inguinal hernia , length of the umbilical skin incision for the trocar port , operative time , and length of hospital stay were retrospectively retrieved from surgical records . on postoperative day 3 , all patients were scheduled for a wound check and removal of skin staples . in october 2013 , the patients were asked to complete a questionnaire and undergo a physical examination in the outpatient clinic . the following information was then recorded : hernia recurrence ; postoperative pain , according to the visual analog scale ( vas ) ( 0 = no pain ; 10 = worst pain ) ; postoperative mobility of the abdominal wall , determined by janda 's test , a sit - up exercise ( 0 = no recovery of normal muscle power ; 5 = full recovery of normal muscle power ) ; postoperative cosmetic satisfaction , according to the cosmetic vas ( 0 = completely dissatisfied ; 10 = completely satisfied ) ; and period of sick leave after hospital discharge for employed workers . the operative procedure in laparoscopic tapp mesh repair was described in depth in our previous study . in brief , it involved establishing pneumoperitoneum , dissecting the peritoneal flap and the preperitoneal space with an electric hook , placing a 15 9-cm parietene progrip self - fixating monofilament polypropylene mesh ( covidien , dublin , ireland ) , and closing the peritoneal flap with a v - loc barbed suture ( covidien ) . in the ntapp group , the length of the transverse skin incision at the umbilicus was first measured , and an 8-mm trocar was inserted for the use of a 5-mm optical instrument . under optical view , a 5-mm trocar for the electric hook on the right side of the abdomen and a 2-mm trocar for the grasping forceps on the left side of the abdomen were placed at the level of the umbilicus on the midclavicular line . in the stapp group , the umbilical skin incision length was also first measured , and a gelpoint advanced - access platform ( applied medical , rancho santa margarita , california ) was placed through the umbilicus . a 5-mm optical trocar and two 5-mm working trocars were then positioned in this single - port - access device . the self - fixating parietene progrip mesh , with microhooks on its underside , was folded in a special manner , as demonstrated in our previous publication . it was then inserted into the abdominal cavity through the 8-mm umbilical trocar in the ntapp group or through the 510-mm compatible cannula of the gelpoint advanced - access platform in the stapp group . once hernia repair was completed , the umbilical wound was closed with an absorbable fascia suture and skin staples in all ntapp and stapp procedures . the lateral 5- and 2-mm trocar wounds in the ntapp group were closed with steri - strips adhesive skin closures ( 3 m health center , st . all operations were performed on an elective basis in patients under general anesthesia by the same experienced surgeon . spss 22 for windows ( ibm corp . , somers , new york ) was used for statistical analysis . data are expressed as percentages for categorical variables and as the mean sd for continuous variables . comparisons between the 2 operative approaches were performed with the test and student 's t test , as applicable . patient characteristics in the ntapp and stapp groups were comparable at baseline ( male : female 5.4:1 vs 5.3:1 ; age 52.5 y vs 48.3 y ; bmi 25.3 kg / m vs 24.4 kg / m ; direct : indirect : pantaloon hernia , 32:58:10% vs 31.4:54.3:14.3% ; and hernia diameter , 2.4 cm vs 2.3 cm ) . only 2 patients in the stapp group required an additional 5-mm trocar port in the lateral abdomen , to create a better view of the operative field . the mean measured umbilical skin incision length was 13 4 mm in the ntapp group , compared with 27 3 mm in the stapp group ( p < .001 ) . the ntapp procedure required significantly less operating time than the stapp procedure ( ntapp 54.8 16.9 minutes vs stapp 85.9 19.7 minutes ; p < .001 ) . the reduction in operative time was particularly seen in cases of direct hernia ( figure 1 ) . the mean length of postoperative hospital stay was 1.8 0.7 days for the ntapp group and 2.0 0.9 d for the stapp group ( p = .21 ) . by the time of the wound check on postoperative day 3 , one patient in the stapp group had a superficial hematoma in the umbilical region that required only conservative treatment . operative time of ntapp and stapp hernia repair . the mean operative time for a direct or an indirect hernia was significantly shorter in ntapp repair ( direct : ntapp .001 ; indirect : ntapp 59.4 minutes vs stapp 88.7 minutes ; p < .001 ) . after a mean follow - up time of 33.5 months for the ntapp group and 30.1 months for the stapp group , 40 ntapp patients ( 80% ) and 26 stapp patients ( 74% ) were re - examined in the outpatient clinic . neither inguinal nor port - site hernia occurred in either group . regarding immediate postoperative pain , there was a tendency for longer pain duration in the wound region after the stapp procedure ( ntapp , 5.6 5.5 days vs stapp 9.8 8.4 days ; p = .072 ) . also stapp patients experienced greater wound pain intensity when they began to resume their regular daily activities after hospital discharge ( ntapp 2.7 2.1 vas vs stapp 4.4 1.9 vas ; p = .016 ) . one patient in the ntapp group had chronic discomfort , with paresthesia in the groin region . one patient in the stapp group developed postherniorrhaphy pain syndrome and remained on paid sick leave at home for approximately 2 months . at the time of the follow - up examination , both groups achieved similar results in janda 's clinical test for abdominal wall mobility ( ntapp 4.7 0.5 vs stapp 4.1 1.1 ; p = .101 ) . however , 7 ( 20% ) patients who underwent stapp repair noted a subjective deterioration in abdominal wall mobility in the questionnaire , whereas no patients who underwent ntapp repair had such an experience . cosmetic satisfaction was equally high , with a vas score of 9.7 0.6 in the ntapp group and 9.6 0.7 in the stapp group ( p = .836 ) . sick leave records showed that the ntapp group had a significantly shorter mean period of sick leave , compared with the stapp group ( ntapp 11.2 8.4 days vs stapp 24.1 20.1 days ; p = .02 ) . notably , even with cessation of postoperative wound pain , 27 ntapp patients ( 54% ) and 32 ( stapp patients 91.4% ) remained on sick leave at the follow - up assessment ( p = .045 ) . patient characteristics in the ntapp and stapp groups were comparable at baseline ( male : female 5.4:1 vs 5.3:1 ; age 52.5 y vs 48.3 y ; bmi 25.3 kg / m vs 24.4 kg / m ; direct : indirect : pantaloon hernia , 32:58:10% vs 31.4:54.3:14.3% ; and hernia diameter , 2.4 cm vs 2.3 cm ) . only 2 patients in the stapp group required an additional 5-mm trocar port in the lateral abdomen , to create a better view of the operative field . the mean measured umbilical skin incision length was 13 4 mm in the ntapp group , compared with 27 3 mm in the stapp group ( p < .001 ) . the ntapp procedure required significantly less operating time than the stapp procedure ( ntapp 54.8 16.9 minutes vs stapp 85.9 19.7 minutes ; p < .001 ) . the reduction in operative time was particularly seen in cases of direct hernia ( figure 1 ) . the mean length of postoperative hospital stay was 1.8 0.7 days for the ntapp group and 2.0 0.9 d for the stapp group ( p = .21 ) . by the time of the wound check on postoperative day 3 , one patient in the stapp group had a superficial hematoma in the umbilical region that required only conservative treatment . operative time of ntapp and stapp hernia repair . the mean operative time for a direct or an indirect hernia was significantly shorter in ntapp repair ( direct : ntapp 47.1 minutes vs stapp 82.5 minutes ; p < .001 ; indirect : ntapp 59.4 minutes vs stapp 88.7 minutes ; p < .001 ) . after a mean follow - up time of 33.5 months for the ntapp group and 30.1 months for the stapp group , 40 ntapp patients ( 80% ) and 26 stapp patients ( 74% ) were re - examined in the outpatient clinic . neither inguinal nor port - site hernia occurred in either group . regarding immediate postoperative pain , there was a tendency for longer pain duration in the wound region after the stapp procedure ( ntapp , 5.6 5.5 days vs stapp 9.8 8.4 days ; p = .072 ) . also stapp patients experienced greater wound pain intensity when they began to resume their regular daily activities after hospital discharge ( ntapp 2.7 2.1 vas vs stapp 4.4 1.9 vas ; p = .016 ) . one patient in the ntapp group had chronic discomfort , with paresthesia in the groin region . one patient in the stapp group developed postherniorrhaphy pain syndrome and remained on paid sick leave at home for approximately 2 months . at the time of the follow - up examination , both groups achieved similar results in janda 's clinical test for abdominal wall mobility ( ntapp 4.7 0.5 vs stapp 4.1 1.1 ; p = .101 ) . however , 7 ( 20% ) patients who underwent stapp repair noted a subjective deterioration in abdominal wall mobility in the questionnaire , whereas no patients who underwent ntapp repair had such an experience . cosmetic satisfaction was equally high , with a vas score of 9.7 0.6 in the ntapp group and 9.6 0.7 in the stapp group ( p = .836 ) . sick leave records showed that the ntapp group had a significantly shorter mean period of sick leave , compared with the stapp group ( ntapp 11.2 8.4 days vs stapp 24.1 20.1 days ; p = .02 ) . notably , even with cessation of postoperative wound pain , 27 ntapp patients ( 54% ) and 32 ( stapp patients 91.4% ) remained on sick leave at the follow - up assessment ( p = .045 ) . in the present study , only patients with uncomplicated inguinal hernias without strangulation or acute pain were included in the investigation , to avoid the possibility that memory of preoperative pain would influence the postoperative pain assessment . also needlescopic trocars and instruments , per se , are not suitable for bowel resections and removal of any extensive tissue from the abdominal cavity . to avoid technical errors and the effects of the learning curve , the operating surgeon in this study was experienced , having performed approximately 20 ntapp repairs with an 8-mm optical trocar and two 5-mm working trocars . before the study , the same surgeon had also performed at least 20 single - incision laparoscopic surgeries for inguinal hernia or cholecystectomy . the results presented in this study should therefore apply to cases involving common hernia and treatment by an experienced surgeon . for the assessment of perioperative surgical trauma to the abdominal wall , we focused on the umbilical skin incision length and total operative time for each procedure . in the ntapp group , a larger ( 8-mm ) umbilical trocar the purpose was to allow for gentle mesh insertion without inadvertent adherence of the folded parietene progrip mesh to itself within the trocar cannula . still , the incisional trauma at the umbilicus in stapp patients was twice that in ntapp patients . although we encountered only 1 stapp patient with postoperative hematoma , there is evidence that a longer surgical skin cut in single - incision laparoscopic surgery is associated with wound complications . furthermore , the operative time for stapp repair of a unilateral hernia ( 85 minutes ) was also significantly longer on average than that required for ntapp ( 54 minutes ) or conventional tapp ( 60 minutes in our previous publication ) repair . the increased operative time for stapp repair indicates an increase in technical difficulty without obvious benefits in surgical capability , as 2 stapp cases ( 5.7% ) in our study still needed an additional working trocar for the creation of a sufficient preperitoneal space . as mentioned in the literature , operative difficulties for single - incision laparoscopic surgery lie in the lack of triangulation and the restricted movement of surgical instruments . in this study , we did not use curved or articulating instruments to improve triangulation in the stapp procedure , as such instruments would add another factor of variation of surgical equipment between the 2 procedures . the smaller jaws and lesser contact area would theoretically exert more force and tear tissue , particularly under the control of the nondominant left hand in our study . some surgeons in our department prefer to perform ntapp repairs with one 5-mm optical trocar and two 5-mm working trocars , with mesh fixation via absorbable tacks and closure of the peritoneum via extracorporeally knotted running sutures . particularly in such procedures , no fascial closure ( a potential source of postoperative wound pain ) was needed for the 5-mm trocar wounds . such ntapp repair variation also seems to be safe and feasible , according to unpublished data . concerning technique variations in single - incision laparoscopic surgery , one study group reported using a single skin incision with 3 separate adjacent fascia punctures in laparoscopic ventral hernia operations . this single - incision multiport technique reduces incisional trauma on the abdominal wall , but preformed proximate fascia defects still have a greater potential to cause a swiss cheese hernia . apart from investigating the surgical trauma in ntapp and stapp repair , we investigated postoperative pain , mobility , and cosmesis of the abdominal wall , as well as length of sick leave . we found that the ntapp procedure was able to further reduce immediate postoperative pain in wound regions . these results of wound pain reduction in ntapp repair are in concurrence with available studies comparing pain experienced after needlescopic and conventional laparoscopic inguinal hernia repair . in contrast , a current review referenced 4 available stapp trials and could not verify superiority in pain reduction over conventional tapp repair . however , reduction of postoperative wound pain in patients who underwent ntapp did not significantly improve incidence of chronic groin pain . such pain still occurred in 2% of our ntapp group , compared with 2.8% in the stapp group . moreover , functional mobility of the abdominal wall is the result of intact motor activity and simultaneous load transfer . scar tissues with less loading capacity at the umbilicus in the midline therefore suggests a mechanical mismatch in the latter component and predisposes the patient to a functional impairment of the abdominal wall . despite the greater extent of such scar tissue in the stapp group , the groups regained equal abdominal wall mobility and satisfactory cosmetic results after an approximate observation time of 30 months . taking into account the length of hospital stay added to the subsequent sick leave days , the total time until return to work in the ntapp group was half that in the stapp group . it is also worth noting that more stapp than ntapp patients remained on sick leave , even with an absence of postoperative wound pain . there are nonetheless multiple factors that influence the period of sick leave , including psychological factors related to the larger scar caused by stapp repair . finally , such patient - related outcomes in pain , cosmetic satisfaction , and length of disability are motivation dependent and therefore are subjective measurements . this effect is especially seen in our data on pain duration and sick leave time , with high standard deviations indicating more polarized responses . in a direct comparison of 2 advanced techniques for laparoscopic tapp hernia repair , the ntapp procedure with smaller caliber trocars can further minimize surgical trauma to the abdominal wall tissue . it also requires significantly less operating time than the stapp procedure , as it still adheres to the basic principles of triangulation in laparoscopic technique . in uncomplicated hernia repair , the ntapp procedure has distinct advantages in the immediate postoperative phase in reduced postoperative wound pain and shorter sick leave . however , it offers comparable long - term results in hernia recurrence , abdominal wall mobility , and cosmetic satisfaction . randomized controlled trials comparing ntapp , stapp , and conventional tapp repairs are still needed to validate the benefits of needlescopic surgery .

background and objectives : in recent years , 2 modifications of laparoscopic transabdominal preperitoneal ( tapp ) inguinal hernia repair needlescopic ( ntapp ) surgery and single - port ( stapp ) surgery have greatly improved patient outcomes over traditional approaches . for a comparison of these 2 modifications , we sought to investigate and compare the extent of surgical trauma and postoperative consequences for the abdominal wall in these two procedures.methods:in a retrospective study , 50 ntapp and 35 stapp procedures occurring at a community hospital from november 1 , 2009 , through july 31 , 2012 were reviewed . intraoperative data , including length of the umbilical skin incision and operative time , were recorded . a follow - up evaluation included investigation of hernia recurrence , postoperative pain , abdominal wall mobility , cosmetic satisfaction , and period of sick leave.results:the mean umbilical skin incision was 13 4 mm in ntapp vs 27 3 mm in stapp ( p < .001 ) . the ntapp procedure required less operating time than the stapp procedure ( 54.8 16.9 minutes vs 85.9 19.7 minutes ; p < .001 ) . the mean immediate postoperative pain score on the visual analog scale was 2.7 2.1 in the ntapp group and 4.4 1.9 in the stapp group ( p = .016 ) . in addition , patients who underwent ntapp had a shorter period of sick leave ( 11.2 8.4 days vs 24.1 20.1 days ; p = .02 ) . at the follow - up evaluation after approximately 30 months , abdominal wall mobility and cosmetic satisfaction were equally positive , with no hernia recurrence.conclusion:in patients with uncomplicated inguinal hernia , the ntapp procedure , with less surgical trauma and operating time , has distinct advantages in reduction of immediate postoperative pain and sick leave time .

pubmed

the breathing process , which begins at the upper airway and culminates in gas exchange inside the alveoli , is essential for the human organism to survive . when breathing begins through the nose , the air is prepared in order to reach the lungs with the ideal conditions , that is , warmed , moistened , and filtered , thus activating immunological processes such as mucociliary transport and microbicidal activity that protect the lower airway . when breathing begins through the mouth , despite air warming and moistening , no filtering or immunological activity occurs1 . when mouth breathing occurs in childhood , a period during which intense facial muscular and skeletal growth occurs , it promotes a pathological adaptation of the structures of the stomatognathic system , to the detriment of the usually harmonious morphological and functional growth of these structures2 . the main causes of mouth breathing in childhood are the hypertrophy of the pharyngeal and/or palatine tonsil , nasal mucosa and turbinate edema , nasal septal deviation , extended suction habits , and others3 . morphological adaptations in mouth - breathing children occur with the aim of facilitating the necessary arrival of the air to the alveoli . thus , it is possible to observe maxillary hypoplasia and posterior mandibular demotion / rotation , which lead to dental occlusion alterations , higher mandibular inclination , and a vertical facial growth pattern , with alterations in normal facial proportions and hard palate elevation , head anteriorization , and muscular deharmonization , occurring mainly in the nasofacial region . these adaptations generate functional changes in the stomatognathic system , which are observed through alterations in speech , chewing , and deglutition4 6 . the complexity of the consequences of mouth breathing that are associated with various etiologies justifies the participation of several professionals , including otorhinolaryngologists , odontologists , phonoaudiologists , physiotherapists , pediatricians , among others , along the different phases of the care of mouth - breathers , including the diagnosis , treatment , rehabilitation , and the prevention of mouth breathing in childhood . multidisciplinary staff integration is essential , as it is desirable to use a uniform categorization of mouth breathers , consisting of the same terminology and the same complementary exams . accurate diagnosis of the cause of mouth breathing is essential to the effectiveness of treatment . in phonoaudiology , the type of care that is used for mouth - breathing patients is typically determined by the etiology of the altered respiratory mode . therefore , the accurate use of the available diagnostic options contributes to early diagnosis , and will aid in the establishment of a multidisciplinary therapeutics that are more appropriate to each case and minimize relapse during mouth breathing rehabilitation7 8 . most causes of mouth breathing are diagnosed through a disarmed otorhinolaryngologic exam , except for the diagnosis of hyperplasia of the pharyngeal tonsils , which demands that complementary exams be used3 . in the diagnosis of pharyngeal hyperplasia , nasopharyngoscopy is a procedure that is used to visualize the pharyngeal tonsil and its relation to other nasopharyngeal structures , and is considered by many authors as the most reliable exam in the diagnosis of nasopharyngeal obstruction9 10 11 12 13 . cephalometry is an exam that is similar to cavum radiography ; however , it is carried out through the use of a cephalostat , which enables the most appropriate patient positioning , and provides data related to craniofacial growth and the myofunctional status of these structures14 . since it is a noninvasive exam , it is more comfortable for the patient , and is often more accepted by children . moreover , is associated with a lower cost and greater availability relative to nasopharyngoscopy . with the aim of contributing to the accurate diagnosis of mouth breathing , this study was carried out with the purpose of verifying the correlation between nasopharyngoscopy and cephalometry in the diagnosis of hyperplasia of the pharyngeal tonsils . the preset study was approved by the research ethics committee of the institution of origin under the protocol number 220.0.243.000 - 8 . the children agreed to take part in the study and the informed consent forms were signed by their representatives . the study sample consisted of children from 3 public schools who were evaluated from september 2008 through december 2009 . the inclusion criteria were included complaints related to mouth breathing , such as night drooling , snoring , and restless sleep ; the group age ranged from 7.0 to 11.11 years . children that presented evident neurological disorders , craniofacial malformation , syndromes , or history of pharyngeal surgery were excluded from the study . after employing the inclusion and exclusion criteria , 55 children , including 25 males and 30 females , with an average age 9.8 years were selected . these children underwent otorhinolaryngologic assessment , as well as nasopharyngoscopy and cephalometry to check for the presence or absence of nasopharyngeal obstruction . any other causes of obstruction of the upper airway , such as nasal septal deviation , primary hypertrophy of the nasal turbinates , polyps , and malformations were not diagnosed in the study sample . for the patients with bacterial rhinosinusitis and/or symptomatic allergic rhinitis , appropriate treatments were prescribed and subsequent revaluation was carried out during a period ranging from 30 to 60 days . after this period was completed , the nasopharyn - goscopy and cephalometry exams were performed . otorhinolaryngologic evaluation was carried out in the presence of and with the help of the parents or sponsors . after a general and specific otorhinolaryngologic anamnesis was conducted , giving emphasis to the aspects related to mouth / nasal breathing , a physical exam consisting of otoscopy , anterior rhinoscopy , cervical palpation , and nasoscopy was carried out . this evaluation allowed for the determination of whether the patient had either an oral or mouth - breathing mode . immediately after the completion of the anamnesis and the otorhinolaryngologic clinical exam , nasopharyngoscopy was carried out , always by the same assessor and in the same environment , employing topical anesthesia ( lidocaine 5% ) and a nasal vasoconstrictor ( oxymetazoline 0.05% ) , with a flexible nasofibroscope ( 3.2 mm mashida brand , microcamera asap ) , and recorded on dvd . through this exam , apart from studying the size and the relation of the pharyngeal tonsil to the nasopharynx , the nasal septum positioning , turbinate size , upper pole of the palatine tonsils , presence of secretions , and other kinds of lesions in the nasal cavity and hypopharynx were assessed . when using nasopharyngoscopy to determine the level of hyperplasia of the pharyngeal tonsils , we used a graded scale in which the relation of the pharyngeal tonsils to the other structures that are present in the nasopharynx was assessed15 , as follows : grade 1tonsil without contact with the nasopharyngeal structures ( figure 1 ) ; grade 2tonsil in contact with the torus tubarius ( figure 2 ) ; grade 3tonsil in contact with the torus tubarius and vomer ( figure 3 ) ; grade 4tonsil in contact with the torus tubarius , vomer , and the soft palate at rest ( figure 4 ) . grade 1 hyperplasia of the pharyngeal tonsils by nasofibropharyngoscopy and cephalometry ( cephalometric analysis ) . grade 2 hyperplasia of the pharyngeal tonsils by nasofibropharyngoscopy and cephalometry ( cephalometric analysis ) . grade 3 hyperplasia of the pharyngeal tonsils by nasofibropharyngoscopy and cephalometry ( cephalometric analysis ) . grade 4 hyperplasia of the pharyngeal tonsils by nasofibropharyngoscopy and cephalometry ( cephalometric analysis ) subsequently , children were referred to the odontological radiology service center to undergo a cephalometric assessment , through which it was possible to obtain a left profile cranium teleradiograph by using the cephalostat for positioning the patient at a distance of 1.5 meters . for the standard lateral radiography assessment , computerized cephalometric tracing was performed with cef x software ( a computerized cephalometry system version 2.4.0.0 from cdt software for informatics consulting , development , and training ) . the exam was carried out with the same equipment in each patient , and was analyzed by the same professional expert in odontological radiology . when using cephalometric analysis , in order to determine the level of hyperplasia of the pharyngeal tonsils , we used a graded scale in which the airway between the palate pharynx border and the point that was most proximal to the pharynx was measured in millimeters16 , as follows : grade 1nasopharyngeal space larger than 6 mm ( figure 1 ) ; grade 2nasopharyngeal space between 4.1 and 6 mm ( figure 2 ) ; grade 3nasopharyngeal space between 2.1 and 4 mm ( figure 3 ) ; grade 4nasopharyngeal space between 0 and 2 mm ( figure 4 ) . in order to verify the correlation between the nasopharyngoscopy and cephalometry examinations , the test results in a correlation coefficient ranging from -1 to + 1 , with zero indicating that there is no correlation , -1 indicating a perfect negative correlation , and + 1 denoting a perfect positive correlation ; the closer to 1 or + 1 , the greater the correlation between the variables that are being tested . the possible results for a positive correlation are17 : 0 when there is no correlation ; between 0 and 0.3 - poor correlation ; between 0.3 and 0.6 - regular correlation ; between 0.6 and 0.9 - a very strong correlation ; equal to 1a perfect correlation between the data . for calculating the sensitivity , specificity , the positive predictive value , and the negative predictive value of radiologic exams , nasopharyngoscopy was considered to be the reference exam ( the gold standard ) . it was also necessary to establish limit values in order to determine whether the exams that were studied were either positive or negative . therefore , the grade 1 and 2 nasopharyngoscopic and cephalometric exams were considered to be negative , and the grade 3 and 4 exams were considered to be positive . table 1 shows the results of the correlation analysis between the nasopharyngoscopy and cephalometry examinations in diagnosing the grade of hyperplasia in the pharyngeal tonsils . n = number of children ; p = significance level at 5% ; r = spearman 's rank correlation coefficient . from the analysis of the results presented in table 1 , it was possible to verify that the spearman 's rank correlation coefficient , rho ( r ) , was equal to 0.52 . this result indicates a significant regular and positive association between the nasopharyngoscopy and cephalometry assessments ( p = 0.000 ) at a significance level of 5% . table 2 describes the nasopharyngoscopy and cephalometry distribution results that were used for assessing the diagnostic tests . in table 2 , using nasopharyngoscopy as the gold standard for the diagnosis of hyperplasia of the pharyngeal tonsils , it was possible to verify that the sensitivity of cephalometry ( determined by the proportion between the number of correct positive radiographic cephalometry diagnoses and the total number of positive cases ) was 35% . the negative predictive value of cephalometry ( determined by the proportion between the number of correct negative diagnoses and the total number of negative cases ) was 97% . the positive predictive value of the cephalometric exam ( determined by the ratio of the number of correct positive cephalometric diagnoses and the total number of positive cephalometric diagnoses ) was found to be 87% . the negative predictive value ( determined by the ratio of the number of correct negative cephalometric diagnoses and the total number of negative cephalometric diagnoses ) was 72% . hyperplasia of the pharyngeal tonsils is one of the main causes of upper airway obstruction . for this reason , studies have been carried out with the objective of assessing the reliability of the available diagnostic procedures that are used for the detection of nasopharyngeal obstruction resulting from the increased size of the pharyngeal tonsils . the most commonly used exams are cavum radiographs , flexible and rigid nasopharyngoscopy , and cephalometry11 13 18 . reports in the literature indicate that there is concern regarding the best method for diagnosing and treating children in which hyperplasia of pharyngeal tonsils is suspected , which a frequently occurring situation in the otorhinolaryngological practice13 19 . therefore , this study aimed to establish a correlation between the nasopharyngoscopy and cephalometry examinations in the diagnosis of hyperplasia of the pharyngeal tonsils , while highlighting their main contributions . the findings of this study demonstrated that it was possible to verify a regular and positive correlation , r = 0.52 ; however , a perfect correlation between the exams was not found . the results revealed that most of the children showed grade 2 and 3 hyperplasia of the pharyngeal tonsils , followed by grade 1 in the nasopharyngoscopic exam , and most of the children showed grade 1 hyperplasia of the pharyngeal tonsils , followed by grade 2 in the cephalometric assessment ( table 1 ) . after analyzing these results , it was possible to infer that cephalometry , as compared to nasopharyngoscopy , presented a tendency to underestimate the pharyngeal tonsil size in the nasopharynx . the sensitivity of cephalometric exam , which indicates the probability of a positive result when the patient presents with the condition in question , was 35% . the negative predictive value , which expresses the probability that the individual does not present with the condition in question when the exam result is negative , was 72% . these results suggested that a negative cephalometric exam might not provide a proper assessment of the nasopharynx when compared to the nasopharyngoscopic exam . similar results were obtained when cavum radiography and flexible nasopharyngoscopy for the assessment of the grade of nasopharyngeal obstruction were compared in other similar study13 . the authors concluded that children with classical symptoms of respiratory obstruction , in the absence of hypertrophy of the pharyngeal tonsils by a radiological exam , should undergo flexible nasopharyngoscopy in order to achieve a more accurate diagnosis . several published studies consider nasopharyngoscopy to be the gold standard exam for assessing the nasopharynx12 13 20 21 22 . this is justified , among other reasons , by the dynamic and tridimensional nasopharyngeal view provided by this assessment , which makes it possible to analyze not only the pharyngeal tonsil size , but also its relation with other structures in the region . this may explain the reason why , in the comparison between nasopharyngoscopy and cephalometry , the latter tended to underestimate the size and position of the pharyngeal tonsils in relation to the nasopharynx . frequently in clinical practice , it is possible to verify that patients with mouth breathing symptoms who show normal or next to normal pharyngeal tonsil size with radiological exams , may present hyperplasia of the pharyngeal tonsils and other alterations , such as hypertrophy of the lower turbinate and posterior nasal septal deviation , when assessed by nasopharyngoscopy . this statement is reinforced by another study11 in which 45 children between 4 and 12 years of age were assessed with nasopharyngoscopy ; all of them presented chronic nasal obstruction due to hyperplasia of the pharyngeal tonsils , and cavum radiography revealed no alterations . the results demonstrated that 27% ( 17 cases ) of the pharyngeal tonsils were estimated to be large , 42% ( 24 cases ) of the pharyngeal tonsils were of moderate size , and 31% ( 19 cases ) of the pharyngeal tonsils were small . furthermore , the exams detected 8 cases of hypertrophy of the lower turbinate ( 13.3% ) and 4 cases of posterior nasal septal deviation ( 6.6% ) . these findings reinforced the importance of the indication of nasopharyngoscopy for children with nasal obstruction and normal radiological exams , as this exam allows direct , tridimensional , and dynamic assessment of the cavum area . on the other hand , another study23 that verified the efficacy of lateral cephalometric radiography in the diagnosis of hyperplasia of the pharyngeal tonsils compared with nasal endoscopy , differs from the results of the present study , as the authors substantiated that lateral cephalometric radiography proved to be an effective exam because of its sensitivity , specificity , and high positive and negative predictive values for diagnosis . they concluded that in spite of the superiority of nasopharyngoscopy in assessing the nasopharynx , a cephalometric exam that was negative for hypertrophy of the pharyngeal tonsils would be sufficient to exclude this pathology . based on information that was derived from 3 units that comprise the sus ( sistema nico de sade ) , another study reported that mouth breathing is the most frequently cited complaint and that cavum radiography was the most commonly requested exam by otorhinolaryngologists19 . from the results of the present study , it can be inferred that if only the radiographic exam is used for the detection of hyperplasia of the pharyngeal tonsils , a proper mouth - breather diagnosis and treatment might be compromised , as in some cases the grade of pharyngeal tonsil obstruction was underestimated relative to the nasopharyngoscopic exam . when not treated in childhood , mouth breathing causes many alterations , including craniofacial , dental , and phonoarticulatory changes , as well as alterations in body posture , oral functions , sleep , nutrition , behavior , and others24 . all of these alterations that are presented by mouth breathers require multidisciplinary intervention , including physicians , phonoaudiologists , odontologists , physiotherapists , among others1 . although cephalometry was only regularly correlated with nasopharyngoscopy in diagnosing mouth breathing , that exam was able to complement the endoscopic exam , thus providing information regarding facial growth that could be documented and monitored . this information will be useful for other professionals involved in the care of mouth - breathing patients . cephalometry provides early identification of many alterations that are present in mouth - breathing patients and provides the otorhinolaryngologist with information about the nasopharynx , informs the phonoaudiologist about morphological alterations of the stomatognathic system , and informs the odontologists about facial growth and dental occlusion3 14 23 . therefore , cephalometry is useful not only for the initial diagnosis , but also in cost reduction and promoting collaboration between the multidisciplinary staff members that are involved in the care of mouth - breathing patients . a research study comparing cavum radiography with cephalometry found that in the first exam 61% of the patients were incorrectly positioned25 . importantly , the cephalostat is used in cephalometry to control the positioning of the patient , which explains the superiority of this exam compared to cavum radiography . although nasopharyngoscopy is the gold standard exam for nasopharyngeal assessments , cephalometry is a complementary exam that provides information about craniofacial growth , thus making it possible to document and monitor the complex morphofunctional modifications related to mouth breathing . in this study , cephalometry showed a tendency to underestimate the size of pharyngeal tonsil compared with nasopharyngoscopy ; therefore , we suggest that patients presenting with mouth - breathing symptoms and normal radiography be referred for a complementary assessment with nasopharyngoscopy , which is associated with greater diagnostic accuracy due to its tridimensional and dynamic nature . nasopharyngeal assessment of children can be performed with nasopharyngoscopy and cephalometry , as these exams are associated with a regular and positive correlation . however , we found that cephalometry tends to underestimate the size of the pharyngeal tonsil compared with nasopharyngoscopy . therefore , nasopharyngoscopy is suggested when the cephalometry results for nasopharyngeal obstruction are negative in patients with mouth - breathing symptoms .

summary introduction : hyperplasia of the pharyngeal tonsil is one of the main causes of mouth breathing , and accurate diagnosis of this alteration is important for proper therapeutic planning . therefore , studies have been conducted in order to provide information regarding the procedures that can be used for the diagnosis of pharyngeal obstruction . objective : to verify the correlation between nasopharyngoscopy and cephalometric examinations in the diagnosis of pharyngeal tonsil hyperplasia . method : this was a cross - sectional , clinical , experimental , and quantitative study . fifty - five children took part in this study , 30 girls and 25 boys , aged between 7 and 11 years . the children underwent nasofibropharyngoscopic and cephalometric evaluation to determine the grade of nasopharyngeal obstruction . the spearman 's rank correlation coefficient at the 5% significance level was used to verify the correlation between these exams . results : in the nasopharyngoscopy evaluation , most children showed grade 2 and 3 hyperplasia of the pharyngeal tonsil , which was followed by grade 1 . in the cephalometry assessment , most children showed grade 1 hyperplasia of the pharyngeal tonsil , which was followed by grade 2 . a statistically significant regular positive correlation was observed between the exams . conclusion : it was concluded that the evaluation of the pharyngeal tonsil hyperplasia could be carried out by fiber optic nasopharyngoscopy and cephalometry , as these examinations were regularly correlated . however , it was found that cephalometry tended to underestimate the size of the pharyngeal tonsil relative to nasopharyngoscopy .

pubmed

the incidence of ipsilateral breast tumor relapse ( ibtr ) in patients previously treated for breast carcinoma through breast - conserving surgery , systemic adjuvant therapy and radiation therapy , is estimated to be less than 6% . however , as each case has different clinical relevance ( evolution , prognosis and therapeutic management ) , it is necessary to distinguish in these patients between a true local recurrence ( tr ) of a previous tumor , and the appearance of a new primary tumor ( npt ) [ 1 , 2 , 3 , 4 ] . to do this , we must study the location of the new tumor ( distance from the previous tumor ) , staging ( by imaging and biopsy ) and determine their histological subtype ( through immunohistochemical studies , and the determination of her-2 and hormone receptor expression ) . a 35-year - old premenopausal woman with no relevant medical history of interest , except for a family history of a maternal cousin with breast cancer at 28 years , was referred to the oncologic gynecology department by the palpation of a 1-cm nodule in the right axilla . fine needle aspiration cytology was performed , with the pathological result of suspicion of malignancy . in february 2004 the pathological diagnosis was moderately differentiated invasive ductal carcinoma ( idc ) of the right breast of 0.8 cm in size , with no metastasis detected in 20 lymph nodes removed ( pt1 pn0 m0 ) . immunohistochemical study of the tumor cells showed positive staining for both estrogen receptors ( er ) and progesterone receptors ( pr ) in 50 and 15% , respectively , and showed negative membrane staining of her-2 marker . fluorescence in situ hybridization ( fish ) analysis found no her-2 amplification in the primary tumor . the computer tomography performed after surgical intervention revealed no other organs compromised or distant metastatic extension . preoperative serum levels of the marker ca 15 - 3 were normal . with this diagnosis , the patient was referred to our medical oncology department where she received postoperative adjuvant chemotherapy consisting of 4 cycles of adriamycin 60 mg / m and cyclophosphamide 600 mg / m every 3 weeks . then , adjuvant radiotherapy ( 50 gy ) was performed on right - breast residual tissue , followed by adjuvant hormone therapy with tamoxifen 20 mg daily during the following 4 years . meanwhile , the patient continued routine follow - up visits in our department . in january 2008 ( 4 years after surgery ) , the patient noticed the presence of periareolar thickening in the right breast with redness and heat . in the physical examination a mammography showed the presence of breast architectural distortion with high suspicion of malignancy ( birads-4 ) . the result of the biopsy of the lesion confirmed the presence of an idc consistent with the diagnosis of inflammatory breast cancer ( pt4 nx m0 ) in the same breast where the previous tumor was treated . immunohistochemical study of biopsy sample showed negative staining for both er and pr , and showed positive membrane staining of her-2 marker . then , the patient received treatment with a weekly schedule of paclitaxel ( 80 mg / m ) plus carboplatin ( auc = 2 ) 3 weeks on and 1 week off , in combination with weekly trastuzumab ( initially 4 mg / kg followed by 2 mg / kg every week ) . she received a total of 11 cycles of chemotherapy schedule and 15 cycles of trastuzumab . during that time period , , there was a progressive decrease of inflammatory signs , with disappearance of the redness and heat from the affected area . after surgery , the patient received adjuvant chemotherapy consisting of 8 cycles of weekly paclitaxel ( 80 mg / m ) in combination with weekly trastuzumab ( 2 mg / kg ) , followed by trastuzumab monotherapy ( 6 mg / kg every 3 weeks ) up to 1 year of treatment . later , the patient underwent breast reconstruction surgery , and currently , after 4 years and 8 months of follow - up after the last surgery , the patient remains in complete clinical remission , developing her life normally . however , given the age of the patient at the time of first diagnosis and due to the presence of a second primary tumor in the same breast , a genetic study was performed with negative results for currently known breast cancer - associated genetic mutations . breast cancer is a major public health problem for women throughout the world , and idc is the most frequent form of invasive breast cancer . it accounts for 7080% of all cases of invasive carcinomas , and , in global , is the histologic type of breast cancer with the worst prognosis of all [ 6 , 7 ] . its treatment is based on a multidisciplinary approach consisting in primary tumor surgery , adjuvant chemotherapy and radiotherapy , and hormone therapy if indicated . currently , the treatment of choice for idc is a combination therapy based on neoadjuvant chemotherapy , targeted therapy , surgery , adjuvant chemotherapy , hormone therapy and radiation therapy . numerous randomized prospective studies have shown that breast - conserving treatment in idc is as effective as mastectomy in terms of overall survival , disease - free survival and long - term disease - free survival [ 8 , 9 ] . inflammatory breast carcinoma is a rare and very aggressive form of locally advanced breast carcinomas . it represents 0.55% of primary invasive breast tumors , being more common in caucasian women under 60 years . in these cases , it is important to make a differential diagnosis with locally advanced noninflammatory breast carcinomas that subsequently evolve with inflammation , as well as other non - neoplastic diseases ( mastitis and breast abscess ) , by using biopsies and imaging tests to confirm the differential clinical diagnosis . after breast - conserving treatment , ibtr may represent two distinct types of lesion that it is important to define , a tr or an npt . tr and npt have different natural histories , prognosis , and in turn different implications for therapeutic management [ 1 , 2 , 3 , 4 ] . in a retrospective study , designed by bouchardy et al . , about second primary contralateral breast cancer , it is concluded that women with er - positive first tumors have a decreased risk of second breast cancer occurrence ( standardized incidence ratio ( sir ) : 0.67 ; 95% confidence interval ( ci ) , 0.480.90 ) , whereas patients with er - negative primary tumors have an increased risk limited to er - negative second tumors ( sir : 7.94 ; 95% ci , 3.8114.60 ) . patients with positive family history had a tenfold higher risk of er - negative second tumor which increased to nearly 50-fold when the first tumor was er negative . so the risk of second er - negative breast cancer is really very high after a first er - negative tumor , particularly in women with strong family history . certain studies provide additional evidence on differences between er - positive and er - negative breast cancer , not only in presentation , prognosis , and treatment , but also in etiology and natural history . overall , the risk of developing a second breast cancer among women diagnosed with a first breast cancer of any er status was similar to the risk of developing a first breast cancer in the general population . but it is important to analyze the first breast cancer by means of immunohistochemical markers , because risk of a second tumor depends on er status , period of diagnosis , and family history of breast and/or ovarian cancer ; and consequently , surveillance and prevention of second cancer occurrence should consider these risk factors . the risk of a second breast cancer is the same whether the patient had previously underwent a mastectomy or bct . and with bct , the risk for developing a second primary breast cancer in the preserved breast is similar to the contralateral one . unlike disease recurrence , second primary breast cancer often occurs after the first 36 months , with an average range from 34 to 60 months , requiring a long - term follow - up . secondary malignancies of the breast are rare with a reported frequency of 0.4 to 2.16% . according to the national surgical adjuvant breast and bowel project ( nsabp ) , there is a 14.3% cumulative incidence of ibtr over 20 years since primary operation . ibtr can be defined as the re - emergence of tumor in the previously treated breast . ibtr rate is under 1% per year , but it is about 5 to 10% at 5 years and 10 to 15% at 10 years . when evaluating ibtr , it is important to consider whether it is a tr or an npt . on the one hand , tr are cases consistent with the regrowth of malignant cells not removed by surgery or not killed by radiotherapy , as veronesi et al . defined them . on the other hand , npts are de novo cases of malignancies arising from mammary epithelial cells of the residual breast tissue [ 4 , 8 ] . the complex behavior of ibtr may be related to the fact that the ibtr patient population is composed by these two different entities . each ibtr can be classified as either tr or as npt based on the following criteria : according to pathologic features , npt is designated when it is a distinct histology type ( e.g. from an infiltrating ductal carcinoma to an infiltrating lobular , tubular , or medullar carcinoma ) , or when it has a change from a more invasive to a less invasive carcinoma . by contrast , a change in histology from ductal carcinoma in situ to an infiltrating ductal carcinoma is considered as tr because it is consistent with the natural progression of the disease . depending on whether the relapse occurs at or near the original site of the primary tumor . a tumor is classified as npt if it changes from an aneuploid primary to a diploid relapse ; otherwise , it would be considered as a probable progression representing the natural history . the best way to differentiate npt and tr would be genetic sequencing ( to establish true clonality ) . recently , some molecular techniques such as dna finger printing , loss of heterozygosity pattern or allelic imbalances profile have been used to distinguish npt from tr , but the classification criteria are not standardized yet . patients with npt were significantly younger at initial diagnosis than those who experienced tr . however , there are no age differences at relapse . the rate of np in the ipsilateral breast does not differ significantly from the rate of primary contralateral tumors . currently , salvage mastectomy is the standard treatment for all types of ibtr , which provides locoregional control in 90% of patients . this recommendation is based on an elevated risk of further in - breast relapse with conservative surgeries . but , there is no conclusive evidence of its superiority compared to conservative surgery , the number of repeat lumpectomies is small and the follow - up is not long enough to draw definitive conclusions . further studies are needed to determine the indications . after the surgical management of ibtr , the optimal systemic therapy is also unknown . tr has a poor prognosis in terms of survival rates and development of other metastases , and may benefit from more aggressive adjuvant treatment , with additional radiotherapy , hormone therapy and chemotherapy . by contrast , patients with npt generally have a favorable prognosis , and therapeutic decisions concerning systemic therapy should be similar to those in patients with de novo breast carcinoma , according to the equivalent stage . however , because of the higher risk of developing contralateral breast carcinoma ( genetic predisposition ) there is a need for better chemoprevention strategies . two trials have shown that patients with npt benefit from adjuvant tamoxifen , if indicated , because it reduces contralateral and ipsilateral disease recurrence with minimal side effects . due to declining mortality rates that are , in part , attributable to the use of screening mammography and effective adjuvant therapy , more women are surviving their breast cancer . the care of breast cancer survivors is an important issue that requires an understanding of relapse patterns , establishing appropriate follow - up visits and screening tests . nowadays , there exists a good follow - up among cancer survivors , with higher screening rates . according to pathologic features , npt is designated when it is a distinct histology type ( e.g. from an infiltrating ductal carcinoma to an infiltrating lobular , tubular , or medullar carcinoma ) , or when it has a change from a more invasive to a less invasive carcinoma . by contrast , a change in histology from ductal carcinoma in situ to an infiltrating ductal carcinoma is considered as tr because it is consistent with the natural progression of the disease . depending on whether the relapse occurs at or near the original site of the primary tumor . a tumor is classified as npt if it changes from an aneuploid primary to a diploid relapse ; otherwise , it would be considered as a probable progression representing the natural history . the best way to differentiate npt and tr would be genetic sequencing ( to establish true clonality ) . recently , some molecular techniques such as dna finger printing , loss of heterozygosity pattern or allelic imbalances profile have been used to distinguish npt from tr , but the classification criteria are not standardized yet . patients with npt were significantly younger at initial diagnosis than those who experienced tr . however , there are no age differences at relapse . the rate of np in the ipsilateral breast does not differ significantly from the rate of primary contralateral tumors . currently , salvage mastectomy is the standard treatment for all types of ibtr , which provides locoregional control in 90% of patients . this recommendation is based on an elevated risk of further in - breast relapse with conservative surgeries . but , there is no conclusive evidence of its superiority compared to conservative surgery , the number of repeat lumpectomies is small and the follow - up is not long enough to draw definitive conclusions . further studies are needed to determine the indications . after the surgical management of ibtr , the optimal systemic therapy is also unknown . tr has a poor prognosis in terms of survival rates and development of other metastases , and may benefit from more aggressive adjuvant treatment , with additional radiotherapy , hormone therapy and chemotherapy . by contrast , patients with npt generally have a favorable prognosis , and therapeutic decisions concerning systemic therapy should be similar to those in patients with de novo breast carcinoma , according to the equivalent stage . however , because of the higher risk of developing contralateral breast carcinoma ( genetic predisposition ) there is a need for better chemoprevention strategies . two trials have shown that patients with npt benefit from adjuvant tamoxifen , if indicated , because it reduces contralateral and ipsilateral disease recurrence with minimal side effects . due to declining mortality rates that are , in part , attributable to the use of screening mammography and effective adjuvant therapy , more women are surviving their breast cancer . the care of breast cancer survivors is an important issue that requires an understanding of relapse patterns , establishing appropriate follow - up visits and screening tests . nowadays , there exists a good follow - up among cancer survivors , with higher screening rates .

after breast - conserving treatment , the occurrence of ipsilateral breast tumor relapse raises the concern regarding whether it may represent two distinct types of lesion that it is important to define , a true recurrence ( tr ) or a new primary tumor ( npt ) . tr and npt have different natural histories , prognosis , and in turn different implications for therapeutic management . we report the case of a 35-year - old woman who developed a breast invasive ductal carcinoma , which after receiving breast - conserving treatment with adjuvant chemotherapy , radiotherapy and hormone therapy , developed four years after an inflammatory carcinoma in the same breast , with different expression of immunohistochemical markers than the first breast cancer . the patient was treated with neoadjuvant chemotherapy that allowed the realization of a radical mastectomy with a complete pathological response . we describe the diagnostic and therapeutic approach of ipsilateral breast tumor relapses , along with a review of existing literature .

pubmed

age - related macular degeneration ( amd ) is a leading cause of severe vision loss in people aged 55 years and older in developed countries.1,2 the neovascular form of amd , involving choroidal neovascularization ( cnv ) associated with fluid exudation or bleeding , accounts for the majority of cases of amd - related severe central vision loss.3 upregulation of angiogenic factors , including vascular endothelial growth factor ( vegf ) , has been implicated in the development and progression of amd.4 the management of neovascular amd has been transformed over the past decade with the development and advent of intravitreal anti - vegf treatment , which is significantly more effective in preventing progressive vision loss from neovascular amd than previous alternative treatment modalities , including laser photocoagulation and photodynamic therapy . moreover , anti - vegf therapy has been shown to significantly improve visual acuity ( va ) in many patients with neovascular amd or other retinal diseases.5 current management of neovascular amd involves the use of continuing anti - vegf treatment to control signs of exudation by blocking the growth of abnormal blood vessels to prevent vision loss and potentially improve va . intravitreal anti - vegf agents ranibizumab ( lucentis ; novartis pharmaceuticals canada inc , dorval , qc , canada ) and aflibercept ( eylea ; bayer inc , mississauga , on , canada ) are health canada - approved options for the treatment of neovascular amd . large multicenter clinical trials have demonstrated that monthly ranibizumab , a recombinant vegf - specific monoclonal antibody fragment , prevents vision loss in most patients with neovascular amd , with significant visual gain achieved in some.6,7 the phase 3 randomized view clinical studies demonstrated that intravitreal aflibercept injection every 8 weeks , following 3 initial monthly injections , was noninferior to monthly ranibizumab , which was measured by the primary end point of proportion of subjects who maintained vision ( loss of < 15 letters compared to baseline ) at week 52.8 intravitreal aflibercept injection is a recombinant fusion protein of key domains from human vegf receptors 1 and 2 with the constant region ( fc ) of human immunoglobulin g. aflibercept binds to both vegf - a and proangiogenic placental growth factor . the binding affinity of aflibercept to vegf is substantially higher than that of ranibizumab,9 with a greater potency in vitro and a longer duration of action allowing potentially for less frequent dosing.8 research findings in eyes with neovascular amd demonstrate a mean duration of complete vegf suppression of 36.4 days for ranibizumab compared with ~70 days for aflibercept injection.10,11 switching treatment - resistant neovascular amd patients to aflibercept is an option that may improve visual and anatomic outcomes.12,13 assessment of the overall interaction with the external environment through the visual system is measured using various methods , including va ( 100% contrast , early treatment diabetic retinopathy study [ etdrs ] , and snellen charts ) , contrast sensitivity ( cs ) testing ( 100%0.6% contrast ) , humphrey visual field test , and color vision ( eg , isihara charts , farnsworth dichotomous test ) . the ability to perform everyday visual tasks , such as reading , recognizing faces , driving , telephone use , and using household appliances , depends on visual components beyond high - contrast visual distance acuity , including cs , near vision , color vision , and sensitivity to glare . the snellen eye chart is primarily used to measure va and is depicted as a series of high contrast black - on - white letters in different sizes . relatively small changes in refractive status can be detected by this test , and it is a useful standard for defining vision changes caused by spherical blur . as various types of vision loss are not caused by spherical blur ( eg , cataracts , glaucoma , and irregular astigmatism ) , the snellen va test may not be an adequate or appropriate measure in all cases . cs measures an individual s ability to detect low contrast images and to perceive differences between light and dark , whereas the snellen va chart test measures the level at which an individual can identify fine detail at high contrast using black - on - white letters . using low contrast images , the cs method can detect subtle vision changes that may be concealed by acuity.14 cs could be considered as an adjunct to standard acuity testing , to better assess visual capability , as well as being a useful measure for evaluating a wide range of ocular conditions ( eg , diabetic eye disease , macular degeneration , cataracts , glaucoma , traumatic brain injury , amblyopia , and optic nerve disorders).14 cs is a sensitive and arguably essential outcome end point for a complete assessment of visual function and visual performance , including driving , mobility , reading speed , and general vision - related quality of life.15 additionally , cs has potential applications when measuring the vision of patients with high visual requirements , including athletes and public service personnel . cs charts that utilize letters are familiar to both the patient and the practitioner , in comparison to other nonletter - based cs charts.16 the pelli robson cs test is considered a quick and reliable method in clinical settings.17,18 patients scoring below 1.65 log on the pelli robson cs test are considered to have impaired visual function , with a 6-letter change in cs equating to a 15-letter ( or 3 lines ) change in va.19 both va and cs are associated with the ability to perform daily vision - related activities20 and are considered as significant independent parameters in the determination of visual impairment and quality of life . however , in clinical studies , change in va over time is commonly used as the main primary outcome measure as an effective and reliable indicator of functional deficit or improvement . when measuring va , single optotypes ( usually letters , numbers , or geometric symbols ) with high contrast are presented to the patient ; however , the environment is not always seen in high contrast as presented on a standard acuity chart . therefore , using cs determination to measure the ability to recognize low contrast patterns may detect functional impairment not evident when measuring va only.21 study results have indicated that va and cs do not reliably demonstrate the same parallel progression in visual function loss , although they show a moderate correlation in eyes with neovascular amd.21 both parameters provide important and supplementary information about visual disability,21 supporting the role of multiple visual end point evaluations in interventional studies.21,22 to better assess the overall visual functioning following anti - vegf treatment switch , this study was conducted to determine and evaluate change from baseline to week 12 in pelli robson cs as the primary clinical end point in patients with recalcitrant exudative amd , despite previous ranibizumab treatment who were switched to aflibercept therapy . other outcome measures recorded and assessed were mean change in best corrected visual acuity ( bcva ) , mean change in central retinal thickness ( crt ) , and vision - related quality of life ( or visual functioning ) assessed using the national eye institute 25-item visual function questionnaire ( nei vfq-25 ) single composite score . the latter captures key dimensions of self - reported vision - targeted health status and visual functioning in patients with chronic eye disease . assessment of the overall interaction with the external environment through the visual system is measured using various methods , including va ( 100% contrast , early treatment diabetic retinopathy study [ etdrs ] , and snellen charts ) , contrast sensitivity ( cs ) testing ( 100%0.6% contrast ) , humphrey visual field test , and color vision ( eg , isihara charts , farnsworth dichotomous test ) . the ability to perform everyday visual tasks , such as reading , recognizing faces , driving , telephone use , and using household appliances , depends on visual components beyond high - contrast visual distance acuity , including cs , near vision , color vision , and sensitivity to glare . the snellen eye chart is primarily used to measure va and is depicted as a series of high contrast black - on - white letters in different sizes . relatively small changes in refractive status can be detected by this test , and it is a useful standard for defining vision changes caused by spherical blur . as various types of vision loss are not caused by spherical blur ( eg , cataracts , glaucoma , and irregular astigmatism ) , the snellen va test may not be an adequate or appropriate measure in all cases . cs measures an individual s ability to detect low contrast images and to perceive differences between light and dark , whereas the snellen va chart test measures the level at which an individual can identify fine detail at high contrast using black - on - white letters . using low contrast images , the cs method can detect subtle vision changes that may be concealed by acuity.14 cs could be considered as an adjunct to standard acuity testing , to better assess visual capability , as well as being a useful measure for evaluating a wide range of ocular conditions ( eg , diabetic eye disease , macular degeneration , cataracts , glaucoma , traumatic brain injury , amblyopia , and optic nerve disorders).14 cs is a sensitive and arguably essential outcome end point for a complete assessment of visual function and visual performance , including driving , mobility , reading speed , and general vision - related quality of life.15 additionally , cs has potential applications when measuring the vision of patients with high visual requirements , including athletes and public service personnel . cs charts that utilize letters are familiar to both the patient and the practitioner , in comparison to other nonletter - based cs charts.16 the pelli robson cs test is considered a quick and reliable method in clinical settings.17,18 patients scoring below 1.65 log on the pelli robson cs test are considered to have impaired visual function , with a 6-letter change in cs equating to a 15-letter ( or 3 lines ) change in va.19 both va and cs are associated with the ability to perform daily vision - related activities20 and are considered as significant independent parameters in the determination of visual impairment and quality of life . however , in clinical studies , change in va over time is commonly used as the main primary outcome measure as an effective and reliable indicator of functional deficit or improvement . when measuring va , single optotypes ( usually letters , numbers , or geometric symbols ) with high contrast are presented to the patient ; however , the environment is not always seen in high contrast as presented on a standard acuity chart . therefore , using cs determination to measure the ability to recognize low contrast patterns may detect functional impairment not evident when measuring va only.21 study results have indicated that va and cs do not reliably demonstrate the same parallel progression in visual function loss , although they show a moderate correlation in eyes with neovascular amd.21 both parameters provide important and supplementary information about visual disability,21 supporting the role of multiple visual end point evaluations in interventional studies.21,22 to better assess the overall visual functioning following anti - vegf treatment switch , this study was conducted to determine and evaluate change from baseline to week 12 in pelli robson cs as the primary clinical end point in patients with recalcitrant exudative amd , despite previous ranibizumab treatment who were switched to aflibercept therapy . other outcome measures recorded and assessed were mean change in best corrected visual acuity ( bcva ) , mean change in central retinal thickness ( crt ) , and vision - related quality of life ( or visual functioning ) assessed using the national eye institute 25-item visual function questionnaire ( nei vfq-25 ) single composite score . the latter captures key dimensions of self - reported vision - targeted health status and visual functioning in patients with chronic eye disease . this single - center study was an institutional review board ( irb)-approved , investigator - masked , prospective , interventional , noncontrolled , single - arm study that included 40 randomly selected neovascular amd patients with persistent fluid on spectral domain optical coherence tomography ( sd - oct ) following at least three intravitreal injections of ranibizumab in the previous 6 months . if both eyes of individual subjects met the study inclusion criteria , they were included in the study population dataset ( table 1 ) . subjects were switched to 2 mg aflibercept injection on a dosing protocol that matched the recommended treatment posology for neovascular amd , involving treatment initiation with three doses administered once a month for the first 3 months ( weeks 0 , 4 , and 8) , followed by an injection every 2 months for the first 12 months of treatment . assessments included the pelli robson log cs measurement at 1 m , bcva converted to logarithm of minimum angle of resolution ( logmar ) using standard methods,23 crt using sd - oct ( heidelberg spectralis ; heidelberg engineering inc . , vista , ca , usa ) , and the nei vfq-25 questionnaire with composite score . statistical data analysis was performed once all patients had completed the week 12 visit , using microsoft excel ( microsoft corporation , redmond , wa , usa ) . a two - sided paired t - test was used to compare the means of continuous variables . if not otherwise stated , all values are presented as mean standard deviation ( sd ) . the study was conducted in accordance with the principles of the declaration of helsinki and in compliance with good clinical practice and applicable regulatory requirements . the study procedure and informed consent document were approved by irb services ( aurora , on , canada ) prior to initiating the study . written informed consent was obtained from all participants before study enrollment . a total of 49 eyes of 40 patients ( 27 male and 13 female ) with treatment - resistant neovascular amd were evaluated at baseline and at the week 12 visit following treatment switch to aflibercept from ranibizumab . no patients were lost to follow - up , and there were no treatment discontinuations . there were no ocular or nonocular adverse events in the patient population during the duration of the study . the baseline age was 80.26.8 years ( mean sd , range 6893 years ) . the mean number of ranibizumab injections in the previous 6 months before switch to aflibercept was 5.1 ( range 36 ) . ranibizumab injections ( mean sd 28.222.1 [ range 386 ] ) were administered before initiation of aflibercept treatment . the duration of prior ranibizumab therapy was 36.824.7 months ( mean sd , range 694 ) , with a treatment start date from 2008 to 2015 . the mean cs increased from 1.32 log units at baseline to 1.40 log units at week 12 ( p=0.00079 ) . mean va was stable from baseline to week 12 : mean logmar va was 0.51878 at baseline and 0.53204 at week 12 ( median 0.4 and 0.42 , respectively ) . at week 12 , there was a statistically significant decrease of 22 m in mean crt from 354 m at baseline to 332 m at week 12 ( p=0.004 ) . there was no demonstrated relationship between change in crt and va measurements ( p=0.88 ) . there was however an associated relationship between improvement in cs and mean change in crt ( p=0.000046 ) . of 40 patients , 26 ( 65% ) experienced an overall improvement in nei vfq-25 composite score from baseline to week 12 ; 8 of 40 ( 20% ) patients experienced no change ; and 6 of 40 ( 15% ) experienced an overall decrease in composite score . a total of 49 eyes of 40 patients ( 27 male and 13 female ) with treatment - resistant neovascular amd were evaluated at baseline and at the week 12 visit following treatment switch to aflibercept from ranibizumab . no patients were lost to follow - up , and there were no treatment discontinuations . there were no ocular or nonocular adverse events in the patient population during the duration of the study . the baseline age was 80.26.8 years ( mean sd , range 6893 years ) . the mean number of ranibizumab injections in the previous 6 months before switch to aflibercept was 5.1 ( range 36 ) . ranibizumab injections ( mean sd 28.222.1 [ range 386 ] ) were administered before initiation of aflibercept treatment . the duration of prior ranibizumab therapy was 36.824.7 months ( mean sd , range 694 ) , with a treatment start date from 2008 to 2015 . the mean cs increased from 1.32 log units at baseline to 1.40 log units at week 12 ( p=0.00079 ) . mean va was stable from baseline to week 12 : mean logmar va was 0.51878 at baseline and 0.53204 at week 12 ( median 0.4 and 0.42 , respectively ) . at week 12 , there was a statistically significant decrease of 22 m in mean crt from 354 m at baseline to 332 m at week 12 ( p=0.004 ) . there was no demonstrated relationship between change in crt and va measurements ( p=0.88 ) . there was however an associated relationship between improvement in cs and mean change in crt ( p=0.000046 ) . of 40 patients , 26 ( 65% ) experienced an overall improvement in nei vfq-25 composite score from baseline to week 12 ; 8 of 40 ( 20% ) patients experienced no change ; and 6 of 40 ( 15% ) experienced an overall decrease in composite score . the efficacy evaluation following treatment switch to aflibercept from ranibizumab incorporated both cs and va measurement as independent outcome measures at 12 weeks following an initial treatment - loading phase of 3 consecutive monthly aflibercept injections . overall , the study population received close to the planned 4-weekly aflibercept dosing after switch initiation . adherence to strict inclusion criteria ensured that the study population included only suboptimal responders with persistent fluid , despite previous near monthly ranibizumab treatment . it was shown that in this switch population , there was a statistically significant improvement in the primary outcome end point of change in cs from baseline to week 12 , with an average cs change of two letters broadly comparable to a five - letter ( one line on etdrs chart ) difference in va in terms of vision function performance . it has been reported , for example , that a 6-letter difference in cs had a similar impact on self - reported difficulty with everyday vision - related tasks as a 15-letter ( 3 lines on etdrs chart ) va difference in a population - based sample of individuals aged 65 years and older.19 the evaluation of change in cs may be a clinically meaningful measure of treatment benefit in terms of quality of vision on anti - vegf therapy . published literature has shown that cs is an important measure of visual function in patients with subfoveal cnv due to amd , and the potential benefits of treatment may not be completely characterized by va measurement alone.24 obtaining information on the overall quality of vision that includes va and cs as efficacy outcomes may provide physicians , optometrists , and general ophthalmologists with a better and more complete understanding of their patient s visual status and help improve patient care.20,25 pauleikhoff 26 noted in a review of natural history and treatment outcomes in neovascular amd that reducing the risk of further va and cs loss might allow neovascular amd patients to maintain better vision - related functional abilities . this study demonstrated that there is a general and immediate decreasing trend in mean crt after treatment switch to aflibercept , while no statistically significant change from baseline va was noted . in this neovascular amd population of suboptimal responders to ranibizumab , persistence of fluid is associated with impairment in visual function as measured by cs . the fluid reduction seen following a switch to aflibercept numerous studies have evaluated the effectiveness of switching to aflibercept from another anti - vegf agent in treatment - resistant neovascular amd patients with persistent or recurrent fluid , despite prior multiple anti - vegf injections . wykoff et al27 reported 6-month results from the turf trial providing evidence that aflibercept treatment may be anatomically valuable in some recalcitrant exudative amd eyes , while maintaining prior visual gains attained with ranibizumab treatment . a decrease in the proportion of cnv lesions that were graded as active at 12 months after switch from ranibizumab to aflibercept was noted by barthelmes et al.28 chang et al29 prospectively assessed the efficacy of aflibercept in a cohort of 40 treatment - resistant neovascular amd patients ; at 12 months after switch , mean bcva improved from baseline by 4.7 letters ( 95% ci : 2.17.3 , p<0.001 ) and crt decreased by 97.2 m ( 95% ci : 54.4140.1 , p<0.001 ) . a meta - analysis noted a small but statistically significant improvement in bcva 6 months following treatment switch to aflibercept in patients with treatment - resistant neovascular amd on another anti - vegf agent.12 the nei vfq-25 questionnaire captures patient - reported visual function and was used to assess vision - related quality of life before and after treatment switch . the fact that two - thirds of patients experienced an overall improvement in nei vfq-25 total score at week 12 compared with baseline score is encouraging . however , the study sample is too small to allow evaluation of the statistical relationship between different subgroup responses , although this might usefully be addressed in a future study analysis . in the view studies , involving treatment - naive neovascular amd patients , total nei vfq-25 score was a prespecified secondary efficacy variable and improved scores were observed for both ranibizumab and aflibercept treatment groups , with the greatest improvements observed for mental health and general vision.8,30 yuzawa et al30 reported results evaluating change in composite nei vfq-25 score based on categorical change in va ( worsened , unchanged , and improved ) over 52 weeks in the view studies , comparing the approved dosing for each agent . meaningful improvement in nei vfq-25 composite score was attained only in patients who gained five etdrs letters or more from baseline va to week 52 . when reviewing the results of our study , differences in participant selection , baseline features , and inclusion criteria limit generalizability and direct comparisons between reported anti - vegf switch studies in neovascular amd . the sample size of the switch cohort in this study was relatively small , many patients had received chronic long - term ranibizumab treatment , and follow - up of participants was limited at 12 weeks only . comparative analyses involving a control arm of patients continuing on ranibizumab despite recurrent or persistent fluid might have strengthened the overall analysis . a crossover study design incorporating a reswitch arm from aflibercept back to ranibizumab may be a useful consideration in the development of future anti - vegf treatment switch studies . it would also be of interest to conduct a larger study to evaluate long - term outcomes , including the potential for greater improvement in vision - related functional benefit with continuing treatment , after a treatment switch to aflibercept from ranibizumab . despite limitations , the study results reported herein demonstrate interesting findings with regard to disease progression that may be useful in the design of future switch studies in neovascular amd . va and cs should be considered as significant independent parameters for determining degree of visual impairment and treatment response , as they provide predictive information on visual disability and vision - related quality of life . using the pelli robson chart , cs determination is a standardized technique that is straightforward and reproducible ; therefore , we suggest that cs be more widely considered as a valuable additional outcome end point in future clinical studies of neovascular amd , diabetic macular edema ( dme ) , and retinal vein occlusion . switching of anti - vegf agents in neovascular amd patients is a viable treatment strategy to improve outcomes among initial nonresponders or for patients exhibiting recalcitrant exudative disease activity on continued anti - vegf treatment . the 2016 preferences and trends membership survey by the american society of retina specialists found that more than 80% of respondents considered that persistent or recurrent fluid on oct best determines inadequate response to an anti - vegf treatment in neovascular amd , and more than 75% considered it reasonable to switch due to inadequate response after three to six initial injections . other studies have shown that improvements in anatomic parameters and stable or moderately improved va outcomes may be attained in treatment - resistant neovascular amd patients switched to intravitreal aflibercept from another anti - vegf agent . results of this prospective , interventional investigation reveal a statistically significant improvement in cs and a statistically significant reduction in crt over 12 weeks after treatment switch to aflibercept from ranibizumab in recalcitrant neovascular amd patients , with no meaningful change from baseline va observed . in our study series , a demonstrated correlation was observed between crt decrease and improvement in pelli robson log cs score at 12 weeks after treatment switch to aflibercept . vision functioning , assessed using the nei vfq-25 score , improved from baseline to week 12 in two - thirds of the anti - vegf switch population . the observed positive correlation between improvement in cs and reduction in crt supports the argument that measurement of cs may provide a more complete understanding of the benefits of anti - vegf treatment on vision - related functional performance in patients with neovascular amd . as emphasized earlier , measurement of cs could provide useful early information on visual impairment not identifiable on va assessment alone , as well as provide another method for monitoring global treatment benefit and individual patient responses . overall , cs measurement may provide a better understanding of visual performance challenges faced by individuals with vision impairment . an extended study duration involving a larger patient population would allow for further investigation and potential validation of results with regard to improvement in cs and other visual functioning parameters . further analyses exploring multiple measures of visual function as efficacy end points in treatment studies of dme and other retinal diseases are warranted .

purposethis study evaluated visual function and anatomic and vision - related quality - of - life outcomes in recalcitrant neovascular age - related macular degeneration ( amd ) subjects switched to aflibercept ( eylea ) from ranibizumab ( lucentis).methodsin a single - center study conducted in barrie , on , 40 patients with persistent fluid despite previous ranibizumab treatment were switched to aflibercept with 3 consecutive monthly doses . main outcome measure was mean change from baseline to week 12 in pelli robson contrast sensitivity ( cs ) . secondary outcomes were mean change in best corrected visual acuity ( bcva ) , central retinal thickness ( crt ) , and national eye institute 25-item visual function questionnaire ( nei vfq-25 ) score . a two - sided paired t - test was used in the statistical data analysis to compare the means of continuous variables.resultsforty-nine eyes ( baseline visual acuity [ va ] > 6/120 ) were evaluated . ranibizumab injections ( mean standard deviation [ sd ] 28.222.1 [ range 386 ] ) were administered prior to treatment switch . mean cs improved from 1.32 at baseline to 1.40 log units at week 12 . va was stable throughout . mean crt decreased from 354 m at baseline to 332 m at week 12 ( 22 m , p=0.004 ) . twenty - six ( 65% ) patients experienced an overall improvement in nei vfq-25 score . interestingly , a correlation was observed between improvement in log cs and crt change ( p=0.000046).conclusioncontrast sensitivity improved statistically and significantly , and crt decreased from baseline to week 12 after a switch to aflibercept from ranibizumab . analysis of cs as an independent outcome end point in neovascular amd treatment switch studies may provide a more complete understanding of visual response .

pubmed

multiple sclerosis ( ms ) is a common autoimmune system disease which affects the central nervous system with network vascular inflammation , demyelination and axonal damage symptoms ( 1 , 2 ) . it is a chronic disease of the central nervous system and causes severe cognitive and physical impairments ( 3 , 4 ) . accordingly , it has been documented that white matter lesion may affect the cognition of ms patients ( 5 ) . among the various clinical forms , relapsing - remitting is the most common type , found in approximately 85% to 90% of cases ( 6 ) . although , it is difficult to differentiate primary progressive multiple sclerosis ( ppms ) from relapsing remitting multiple sclerosis ( rrms ) in the early phases , yet previous studies have demonstrated that mrs can be used as a potential way to diagnose ppms and rrms ( 2 ) . in fact , after traumatic and rheumatic diseases , ms is the third factor for asthenic diseases , and has been the subject of extensive studies worldwide ( 7 ) . multiple sclerosis is a disease of young adults and most of patients are diagnosed between the ages of 20 and 40 ( 9 ) . although the exact reason for this disease is unknown , it has been reported that several factors such as genetic and environmental factors ( especially viruses ) are involved in the pathogenesis of ms ( 1 , 10 , 11 ) . accordingly , several investigators are trying to improve symptoms of ms by alteration in the behavior of ms patients including exercise ( 4 ) . in spite of such efforts interestingly , researchers do not limit this disease just to a single gene and candidate several immune and non - immune genes in the pathogenesis of ms . recently , scientists have investigated interleukin-25 ( il-25 ) including its effects on the immune system . this cytokine has a similar structure to il-17 and plays a key role in stimulating and development of t helper 2 ( th2 ) responses ( 12 ) . furthermore , il-25 is involved in the control function of endothelial cell , th1 and th17 , which are the main cells involved in the pathogenic activities of the immune system ( 12 ) . therefore , any factor , which can regulate the expression of the cytokine , can be considered as a candidate for investigation in the ms disease process . it has been reported that there is a known polymorphism ( c424c / a polymorphism ) within exon 2 of the il-25 gene ( 13 ) , which may be associated with immune system - related disease . therefore , according to this explanation , it seems that the polymorphism may be associated with ms . thus , the main aim of this study was to evaluate the association between c424c / a polymorphism within the il-25 gene with ms in an iranian population . in this case - control study , according to the study of arababadi et al . ( 1 ) and using sample - size estimation we compared two binomial independent proportions with the following equation : where significance level was 0.05 , the expected power was considered 90% , p1 = 1% ( frequency of aa genotype in ms patients ) , p2 = 17% ( frequency of aa genotype in healthy controls ) , k = 1 ( sample size was considered equal in both groups ) , and = 15% ( the minimum difference in frequency of aa genotype in the two groups , which was considered clinically important ) . based on the above parameters , sample size was determined as 75 subjects in either group . in this study , 80 patients with ms were recruited for blood sampling yet according to the inclusion and exclusion criteria , 74 patients with relapse and remitting ms ( rrms ) and 75 healthy controls were finally enrolled during august 2013 to september 2014 . " blood samples were collected from consecutively admitted patients to the sina hospital , the main referral hospital for ms patients from all provinces of iran . age and gender matched control samples were also collected from the tehran blood transfusion services . the occurrence of ms was confirmed based on mcdonald s criteria ( diplopia , blurry vision , finger pruritus and disability ) ( 14 ) and brain magnetic resonance imaging ( mri ) results by an expert neurologist , according to clinical and preclinical findings . having at least one central nervous system ( cns ) demyelinating event and being at least 18 years old to give informed consent were considered as the main inclusion criteria . the two groups were matched in terms of age and sex and subjects with genetic diseases , diabetes , autoimmune disease and smokers were excluded from the study . informed consent was filled out by all participants and this study was approved by the ethics committee of rafsanjan university of medical sciences ( rums ) with number 9/1077 on the 15th of july 2013 . regarding ethical considerations , patients data were collected by their nurses and the data and blood samples were trans - located to the laboratory without names . dna was purified from peripheral blood immune cells of participants using a commercial kit ( cinnaclon , tehran , iran ) , according to the manufacturer s instructions . the pcr amplification of exon 2 of the il-25 gene and rflp ( using bsrfi restriction enzyme ) was performed according to the study of buning and et al . numerical variables are presented as mean sd , while categorical variables are summarized by absolute frequencies and percentages . continuous variables were compared using independent two - sample t - test , and categorical variables were compared using the chi - square test across the two study groups . one - sample kolmogorov - smirnov test was applied to test for normal distribution in cases and controls . the logistic regression model was established to estimate the odds ratio ( or ) and 95% confidence interval ( ci ) of ms development in patients with the ac and aa compared to patients with the cc genotype . for the statistical analysis , the spss version 18.0 software for windows ( spss inc . , chicago , il ) was used . all p values were two - tailed with statistical significance defined by p 0.05 . the results also revealed 1 to 2% missing values in the evaluated genotypes and alleles . in this case - control study , according to the study of arababadi et al . ( 1 ) and using sample - size estimation we compared two binomial independent proportions with the following equation : where significance level was 0.05 , the expected power was considered 90% , p1 = 1% ( frequency of aa genotype in ms patients ) , p2 = 17% ( frequency of aa genotype in healthy controls ) , k = 1 ( sample size was considered equal in both groups ) , and = 15% ( the minimum difference in frequency of aa genotype in the two groups , which was considered clinically important ) . based on the above parameters , sample size was determined as 75 subjects in either group . in this study , 80 patients with ms were recruited for blood sampling yet according to the inclusion and exclusion criteria , 74 patients with relapse and remitting ms ( rrms ) and 75 healthy controls were finally enrolled during august 2013 to september 2014 . " blood samples were collected from consecutively admitted patients to the sina hospital , the main referral hospital for ms patients from all provinces of iran . age and gender matched control samples were also collected from the tehran blood transfusion services . the occurrence of ms was confirmed based on mcdonald s criteria ( diplopia , blurry vision , finger pruritus and disability ) ( 14 ) and brain magnetic resonance imaging ( mri ) results by an expert neurologist , according to clinical and preclinical findings . having at least one central nervous system ( cns ) demyelinating event and being at least 18 years old to give informed consent were considered as the main inclusion criteria . the two groups were matched in terms of age and sex and subjects with genetic diseases , diabetes , autoimmune disease and smokers were excluded from the study . informed consent was filled out by all participants and this study was approved by the ethics committee of rafsanjan university of medical sciences ( rums ) with number 9/1077 on the 15th of july 2013 . regarding ethical considerations , patients data were collected by their nurses and the data and blood samples were trans - located to the laboratory without names . dna was purified from peripheral blood immune cells of participants using a commercial kit ( cinnaclon , tehran , iran ) , according to the manufacturer s instructions . the pcr amplification of exon 2 of the il-25 gene and rflp ( using bsrfi restriction enzyme ) was performed according to the study of buning and et al . numerical variables are presented as mean sd , while categorical variables are summarized by absolute frequencies and percentages . continuous variables were compared using independent two - sample t - test , and categorical variables were compared using the chi - square test across the two study groups . one - sample kolmogorov - smirnov test was applied to test for normal distribution in cases and controls . the logistic regression model was established to estimate the odds ratio ( or ) and 95% confidence interval ( ci ) of ms development in patients with the ac and aa compared to patients with the cc genotype . for the statistical analysis , the spss version 18.0 software for windows ( spss inc . , chicago , il ) was used . all p values were two - tailed with statistical significance defined by p 0.05 . the results also revealed 1 to 2% missing values in the evaluated genotypes and alleles . the results revealed that eight ( 11.3% ) out of 74 patients with ms had the aa genotype , while 16 ( 22.5% ) had ac and 47 ( 66.2% ) had cc genotypes ( table 2 ) . the results also showed that 8 ( 11.4% ) , 18 ( 25.7% ) and 44 ( 62.9% ) of the healthy controls had the aa , ac and cc genotypes , respectively . the chi - square test revealed that the frequency distribution of the genotypes was not statistically different between patients with ms and the controls ( p = 0.901 ) ( table 2 ) . according to the logistic regression model , the or of ms development in patients with ac versus patients with cc genotypes was 0.832 ( 95% ic : 0.378 - 1.832 ) ( p = 0.648 ) . the corresponding results for aa versus cc genotypes was 0.936 ( 95% ic : 0.323 - 2.710 ) ( p = 0.903 ) . the results also indicated that the frequencies of a and c alleles were 110 ( 77.5% ) and 32 ( 22.5% ) , respectively , in the patients with ms and were 106 ( 75.7% ) and 34 ( 24.3% ) , respectively , in the healthy controls . the statistical analysis found that the differences in distribution of a and c alleles between groups were not statistically significant ( p = 0.728 ) ( table 2 ) . the or of ms development in patients with the a allele versus patients with the c allele was 0.907 ( 95% ic : 0.522 - 1.574 ) , which did not reach statistical significance level ( p = 0.729 ) . multiple sclerosis is an autoimmune disease , with several immune - related genes involved in its pathogenesis ( 15 ) . it has been confirmed that th1 and th17 responses are the main causes of progression of ms ( 16 ) . therefore , th2 responses , which regulate th1 and th17 , can improve the clinical and laboratory outcome of the disease ( 16 ) . it is believed that il-25 , as a th2 cytokine , can participate in the regulation of immune responses during inflammatory diseases including ms . accordingly , previous studies suggested that serum levels of il-25 were decreased in ms patients when compared with the healthy controls ( 17 ) . it has been proposed that polymorphisms within cytokine genes are associated with their expression ( 18 , 19 ) . the authors of this study hypothesized that the polymorphisms within the il-25 gene may be associated with ms disease ; hence , the c424c / a polymorphism within the il-25 gene was evaluated in this study . the results of our study identified that neither aa , ac and cc genotypes nor a and c alleles were associated with ms in the iranian population . to the best of our knowledge , this is a unique study , which evaluated the c424c / a polymorphism within the il-25 gene in patients with ms , yet there is a study , which evaluated this polymorphism in inflammatory bowel disease ( ibd ) , as an inflammatory disease ( 13 ) . parallel to our results , researchers have reported that the c424c / a polymorphism within the il-25 gene was not associated with ibd ( 13 ) . interestingly , this study documented that the expression level of il-25 decreased in ibd patients ( 13 ) . again , as mentioned previously , it has also been revealed that expression of il-25 decreased in ms patients ( 17 ) . based on our results and the mentioned study it may be concluded that although il-25 may play significant roles in the pathogenesis of inflammatory diseases such as ms and ibd , c424c / a polymorphism is not important in the pathogenesis of these diseases . on the other hand , another study on iranian patients with ms revealed that other polymorphisms , except c424c / a , within il-25 are associated with ms ( 17 ) . thus , it may be concluded that other polymorphisms like 4076a > a , 3672 t > ta , 3712 g > ga , and 3463c > ca , but not the c424c / a polymorphism , may regulate il-25 expression . according to our and previous studies it appears that evaluation of the c424c / a polymorphism , as the first report , and lack of evaluation of other polymorphisms within the il-25 gene as well as lack of evaluation of serum levels of il-25 are the strong and weak points , respectively , of our study . additionally , it can be proposed that using a large sample size may be better for understanding the roles of c424c / a polymorphism in the pathogenesis of ms . according to the earned results of our investigation it can be hypothesized that c424c / a polymorphism within the il-25 gene is not associated with ms and can not be considered as a risk factor for ms development .

backgroundmultiple sclerosis ( ms ) is a common autoimmune system disease which affects the central nervous system . it has been documented that interleukin-25 ( il-25 ) plays key roles in suppressing th1 responses , which is increased during ms.objectivesthe aim of this study was to investigate the c424c / a polymorphism within the il-25 gene in ms patients in comparison to healthy controls.patients and methodsin this case - control study , 74 patients with ms and 75 healthy controls were selected . polymerase chain reaction - restriction fragment length polymorphism ( pcr - rflp ) was used in order to determine c424c / a polymorphism within the il-25 gene.resultsthe results showed that there was no statistical significant difference in distribution of genotype ( aa , ac and cc ) and allele ( a and c ) frequencies between ms patients and healthy controls ( p = 0.901 and p = 0.728 , respectively).conclusionsin conclusion , it appears that the c424c / a polymorphism within the il-25 gene has no significant relationship with ms , and this polymorphism is probably not associated with ms complications , its onset and gender distribution .

pubmed

aortic aneurysm ( aa ) can be life - threatening and aorta size is the best criterion for determining if an intervention is necessary to prevent ruptures , dissections or aneurysm - related deaths.1 ) however , even large aas seldom cause symptoms,2)3 ) although the incidence of aortic disease increases with age.4)5 ) therefore , it is important for clinicians to be cautious in their evaluation of patients at risk . aneurysm of the ascending thoracic aorta ( ata ) most often results from cystic medial degeneration.2 ) atherosclerosis is an infrequent cause of aneurysm of ata , in contrast to that of the descending thoracic aorta ( dta ) and abdominal aorta , where atherosclerosis plays an important role in development of aa.2 ) meanwhile , thoracic aas ( taas ) and abdominal aas ( aaas ) share common risk factors , such as age and hypertension . therefore , elderly male subjects with hypertension might be at high risk for both taa and aaa . efforts to detect high risk populations for coronary heart disease ( chd ) have been widely applied and coronary calcium measurement using non - contrast computed tomography ( ct ) is the method of choice for assessing cardiovascular risk in asymptomatic subjects.6 ) screening for aaa using ultrasound has been recommended in elderly men who are current or past smokers,7 ) but the screening strategy for taa in elderly subjects is not well - established , even though it is a fatal disease.8)9 ) the more widespread applications of cardiac ct and thoracic ct for cardiovascular risk stratification and lung cancer screening have made it possible to measure thoracic aortic diameter using non - contrast ct , resulting in several successful case reports.10)11 ) the purpose of the current study was to use non - contrast ct in a prospective manner to explore the prevalence of aas and determine whether screening for chd and aas is necessary in a population with multiple risk factors for chd in a korean population . five hundreds and two consecutive korean male hypertensive patients without history of aa were prospectively enrolled from june , 2012 to april , 2013 at the outpatient clinic of severance cardiovascular hospital ( seoul , korea ) . subjects were eligible if they were 65 years old and had provided signed informed consent . of these 542 patients who had done non - contrast ct of whole aorta , 15 patients ( 2.8% ) with a history of aortic valve surgery and 30 patients ( 5.5% ) with a suboptimal ct image were excluded . most of the suboptimal images were those with errors in raw ct data storage , which caused problems of analysis in the sagittal and coronal planes using reconstructed images . serum calcium , phosphorus , total cholesterol , low density lipoprotein - cholesterol ( ldl - c ) , high density lipoprotein - cholesterol ( hdl - c ) , triglycerides and serum creatinine levels were measured after a minimum 12-hour fasting period . hypertension was defined as systolic blood pressure 140 mm hg and/or diastolic blood pressure as 90 mm hg or treatment with antihypertensive agents . diabetes was defined as treatment with hypoglycemic agents or insulin , or fasting glucose 126 mg / dl . dyslipidemia was defined as any of the following : total cholesterol 240 mg / dl , ldl - c 130 mg / dl , hdl - c 40 mg / dl , triglyceride 150 mg / dl or treatment with lipid lowering agents . all examinations were performed using an aquilion one 320-row ct system ( toshiba medical systems , otawara , japan ) . dual scanograms were used for planning the examination and determining the anatomical range to be covered . multiple volumes were placed to cover the entire aorta from above the aortic arch to the aortic bifurcation . patients underwent aorta ct with a prospective electrocardiography - gating wide - volume protocol ( from four to six volumes , according to body height ) . the tube voltage was 120 kv and the effective tube current was adjusted using the adaptive iterative dose reduction three dimensional algorithm . the resulting four to six individual volume data sets were automatically stitched together immediately after reconstruction to generate one ct data set of the whole aorta . all data were reconstructed using a standard soft - tissue and lung kernel ( fc43 ) . maximal ascending aorta diameter ( atamax ) was measured in the axial plane from just above the aortic root to the aortic arch perpendicular to the aortic axis . similarly , maximal descending thoracic aorta diameter ( dtamax ) was measured at the dta distal from the aortic arch to the diaphragm level in the same axis ( fig . 1 ) . atamax and dtamax were confirmed in the sagittal and coronal planes using reconstructed images ( fig . maximal thoracic aorta diameter ( tamax ) was defined as the largest diameter among atamax , dtamax and maximal diameter of the aortic arch . maximal abdominal aortic diameter ( aamax ) was defined as the maximal diameter of the abdominal aorta from the diaphragm to the first slice superior to the aortic bifurcation . taa was defined as tamax 40 mm and aaa was defined as aamax 30 mm.7)12 ) distribution of relevant variables was reported either as a percentage or as the meanstandard deviation . a binary logistic regression analysis was used to identify risk factors associated with taa and aaa . variables with a p<0.2 in univariate analysis were included in the multiple logistic regression model . collinearity among explanatory variables was identified using variance inflation factor ( vif ) , and highly correlated variables ( vif>5 ) were excluded . five hundreds and two consecutive korean male hypertensive patients without history of aa were prospectively enrolled from june , 2012 to april , 2013 at the outpatient clinic of severance cardiovascular hospital ( seoul , korea ) . subjects were eligible if they were 65 years old and had provided signed informed consent . of these 542 patients who had done non - contrast ct of whole aorta , 15 patients ( 2.8% ) with a history of aortic valve surgery and 30 patients ( 5.5% ) with a suboptimal ct image were excluded . most of the suboptimal images were those with errors in raw ct data storage , which caused problems of analysis in the sagittal and coronal planes using reconstructed images . height , weight , and blood pressure were measured during their visit . serum calcium , phosphorus , total cholesterol , low density lipoprotein - cholesterol ( ldl - c ) , high density lipoprotein - cholesterol ( hdl - c ) , hypertension was defined as systolic blood pressure 140 mm hg and/or diastolic blood pressure as 90 mm hg or treatment with antihypertensive agents . diabetes was defined as treatment with hypoglycemic agents or insulin , or fasting glucose 126 mg / dl . dyslipidemia was defined as any of the following : total cholesterol 240 mg / dl , ldl - c 130 mg / dl , hdl - c 40 mg / dl , triglyceride 150 mg / dl or treatment with lipid lowering agents . all examinations were performed using an aquilion one 320-row ct system ( toshiba medical systems , otawara , japan ) . dual scanograms were used for planning the examination and determining the anatomical range to be covered . multiple volumes were placed to cover the entire aorta from above the aortic arch to the aortic bifurcation . patients underwent aorta ct with a prospective electrocardiography - gating wide - volume protocol ( from four to six volumes , according to body height ) . the tube voltage was 120 kv and the effective tube current was adjusted using the adaptive iterative dose reduction three dimensional algorithm . the resulting four to six individual volume data sets were automatically stitched together immediately after reconstruction to generate one ct data set of the whole aorta . all data were reconstructed using a standard soft - tissue and lung kernel ( fc43 ) . maximal ascending aorta diameter ( atamax ) was measured in the axial plane from just above the aortic root to the aortic arch perpendicular to the aortic axis . similarly , maximal descending thoracic aorta diameter ( dtamax ) was measured at the dta distal from the aortic arch to the diaphragm level in the same axis ( fig . 1 ) . atamax and dtamax were confirmed in the sagittal and coronal planes using reconstructed images ( fig . maximal thoracic aorta diameter ( tamax ) was defined as the largest diameter among atamax , dtamax and maximal diameter of the aortic arch . maximal abdominal aortic diameter ( aamax ) was defined as the maximal diameter of the abdominal aorta from the diaphragm to the first slice superior to the aortic bifurcation . taa was defined as tamax 40 mm and aaa was defined as aamax 30 mm.7)12 ) distribution of relevant variables was reported either as a percentage or as the meanstandard deviation . a binary logistic regression analysis was used to identify risk factors associated with taa and aaa . variables with a p<0.2 in univariate analysis were included in the multiple logistic regression model . collinearity among explanatory variables was identified using variance inflation factor ( vif ) , and highly correlated variables ( vif>5 ) were excluded . patient demographics and clinical characteristics are shown in table 1 . the mean age was 73 years . diabetes , dyslipidemia and coronary artery disease were observed in 171 patients ( 34.5% ) , 318 patients ( 64.1% ) and 256 patients ( 51.6% ) , respectively . three hundred and sixty - four patients ( 73.4% ) had a history of smoking . mean diameter of atamax , dtamax , tamax , and aamax was 38.44.0 mm , 28.53.0 mm , 38.94.0 , and 23.05.5 mm , respectively . the prevalence of taa and aaa was 36.5% ( 181/496 ) and 6.0% ( 30/496 ) , respectively . there were 16 patients ( 3.2% ) who had taa and aaa simultaneously . however , prevalence of aa suitable for an indication for intervention according to current guidelines ( tamax 55 mm and aamax 60 mm)2 ) was very low in this study . those who were indicated for intervention were seen only in one case of the taa group and one case of the aaa group . prevalence of taa was 64.6% in patients with aaa and 40.0% in patients without aaa . patients with aaa had a higher occurrence of taa compared with those without aaa ( p=0.010 ) ( fig . there was no significant difference in prevalence of taa between nonsmokers and those who had a history of smoking or were current smokers . in contrast , prevalence of aaa was significantly higher in current / past smokers compared to non - smokers ( p=0.046 ) . of the 365 current / past smokers , aaa was found in 27 patients ( 7.4% ) , but of the 124 non - smokers , aaa was observed in only three patients ( 2.4% ) , and there were no nonsmokers with a large aaa ( > 40 mm ) . determinants for taa , aaa and both taa and aaa are shown in table 2 . in the multivariate logistic regression analysis , determinants for taa were age { odds ratio ( or ) 1.059 , 95% confidence interval ( ci ) 1.018 - 1.101 , p=0.005 } , absence of dyslipidemia ( or 0.621 , 95% ci 0.418 - 0.923 , p=0.018 ) , body surface area ( or 11.92 , 95% ci 2.787 - 50.97 , p=0.001 ) , diastolic blood pressure ( or 1.029 , 95% ci 1.009 - 1.049 , p=0.004 ) and presence of aaa ( or 3.070 , 95% ci 1.398 - 6.754 , p=0.005 ) . in contrast , aaa was independently associated with presence of dysplipidemia ( or 2.792 , 95% ci 1.091 - 7.143 , p=0.032 ) , current / past smokers ( or 4.074 , 95% ci 1.160 - 14.31 , p=0.028 ) and presence of taa ( or 3.367 , 95% ci 1.550 - 7.313 , p=0.002 ) . aortic size is critical to key decisions regarding management of aas.12 ) this study evaluated the entire aorta diameter , from the thoracic aorta to the abdomen aorta just above the iliac bifurcation , using axial images of non - contrast ct scans in a population with multiple risk factors for both chd and aa . the study population included high - risk individuals , but blood pressure and serum lipids were well controlled in most patients , as indicated by blood pressure and laboratory test results . nevertheless , prevalence of taa was considerably high and taa was associated with various clinical factors including older age , larger bsa and higher diastolic blood pressure . interestingly , taa was associated with the absence of dyslipidemia , while aaa was significantly associated with the presence of dyslipidemia . one possible explanation might be that the etiology of aas differs between ascending and descending aorta.1 ) that is , above the ligamentumarteriosum the disease is non - atherosclerotic , while below the ligamentumarteriosum arteriosclerosis is abundant.1 ) therefore , taa might not be associated with dyslipidemia . another explanation for the conflicting results might be that the mean values of total cholesterol , ldl - c and hdl - c were almost within normal limits , suggesting the serum lipids were well controlled in most patients . in addition , since the current study was performed at a tertiary referral hospital , many of the patients were taking statins for the primary or secondary prevention for coronary artery disease , not for the treatment of dyslipidemia . therefore , effects of high lipids on atherosclerosis might be minimized in the studied population and result in conflicting outcomes . therefore , further studies are needed to clarify the relationship among taa , aaa , dyslipidemia , serum cholesterol and statin therapy . thoracic aortic aneurysm was more prevalent in patients with aaa compared to those without aaa , even after adjusting for multiple confounding factors . in addition , more than one - third of the study subjects had a taa , and it was remarkably more prevalent than aaa among high - risk subjects in the current study , in contrast to previous reports that aaa is more prevalent than taa.1 ) taa prevalence continuously increases,13 ) and is thought to be related to increased age and hypertension in the general population . however , most taas had a diameter < 50 mm , which is not large enough to indicate a need for immediate intervention . therefore , the clinical implications of the high prevalence of taa need further evaluation , since taas are known to be an indolent process and to grow very slowly , at approximately 0.1 cm per year.9)14 ) future studies should establish a method for aortic screening for taa and strategies for conservative as well as surgical management in elderly hypertensive patients . previous studies consistently demonstrated a relationship between smoking and aaa.11 ) the present study , which involved a high - risk population of asian males , found that aaa was independently associated with smoking history in addition to dyslipidemia . moreover , prevalence of aaa was 2.4% among nonsmokers , which is very low compared to previous studies involving caucasians.15)16 ) in addition , none of the nonsmokers had a large aaa ( > 40 mm ) . in population - based screening studies in japan , aaa was rarely encountered17)18 ) and the prevalence of aaa was also reportedly low in chinese populations , even in patients with severe coronary artery disease.19 ) this suggests that race influences aaa prevalence and that asians generally have a low prevalence of aaa . the present study also reported a low prevalence of aaa in an asian population and reinforced the impact of smoking on aaa . these results high - light the need to clearly define the usefulness of routine screening for aaa among asian non - smokers . the primary limitation of this study is that a ct scan with axial images could not properly evaluate the very proximal portion of the ascending aorta including the aortic root . in addition , even in the sagittal and coronal planes , aortic root diameter could not be accurately measured using non - contrast ct due to the complexity of the structures surrounding the aortic root . therefore , different imaging modalities are needed to accurately detect aortic root aneurysms . this study was performed in a population of elderly asian males with hypertension at high risk for cardiovascular disease . therefore , the results of the current study can not be applied to females , other ethnicities , or groups with a low - risk of cardiovascular disease . the prevalence of aa was significant and taa was more prevalent than aaa in elderly korean males with hypertension who had multiple risk factors for chd and aa . future research should establish distinct screening strategies for taa and aaa according to risk factors and ethnicity . the primary limitation of this study is that a ct scan with axial images could not properly evaluate the very proximal portion of the ascending aorta including the aortic root . in addition , even in the sagittal and coronal planes , aortic root diameter could not be accurately measured using non - contrast ct due to the complexity of the structures surrounding the aortic root . this study was performed in a population of elderly asian males with hypertension at high risk for cardiovascular disease . therefore , the results of the current study can not be applied to females , other ethnicities , or groups with a low - risk of cardiovascular disease . the prevalence of aa was significant and taa was more prevalent than aaa in elderly korean males with hypertension who had multiple risk factors for chd and aa . future research should establish distinct screening strategies for taa and aaa according to risk factors and ethnicity .

background and objectivesscreening strategies for aortic aneurysm ( aa ) according to risk factors and ethnicity are controversial . this study explored the prevalence of aa and determined whether screening is necessary in a population of multiple risk factors.subjects and methodsfrom june , 2012 to april , 2013 , 542 consecutive elderly ( 65 years ) male hypertensive patients without a history of aa were prospectively enrolled . after excluding 15 patients ( 2.8% ) with aortic valve surgery , 30 patients ( 5.5% ) with suboptimal computed tomography ( ct ) images , the remaining 496 patients ( age 735 years ) comprised the study population . maximal diameters of the thoracic and abdominal aorta were measured using non - contrast ct.resultsthe prevalence of thoracic aa ( taa , diameter 40 mm ) and abdominal aa ( aaa , diameter 30 mm ) was 36.5% ( 181/496 ) and 6.0% ( 30/496 ) , respectively . in the multivariate logistic regression analysis , determinants for taa were age { odds ratio ( or ) 1.059 , 95% confidence interval ( ci ) 1.018 - 1.101 , p=0.005 } , dyslipidemia ( or 0.621 , 95% ci 0.418 - 0.923 , p=0.018 ) , body surface area ( or 11.92 , 95% ci 2.787 - 50.97 , p=0.001 ) , diastolic blood pressure ( or 1.029 , 95% ci 1.009 - 1.049 , p=0.004 ) and aaa ( or 3.070 , 95% ci 1.398 - 6.754 , p=0.005 ) . in contrast , aaa was independently associated with dysplipidemia ( or 2.792 , 95% ci 1.091 - 7.143 , p=0.032 ) , current / past smokerfs ( or 4.074 , 95% ci 1.160 - 14.31 , p=0.028 ) , and taa ( or 3.367 , 95% ci 1.550 - 7.313 , p=0.002).conclusionthe prevalence of aa was significant and taa was more prevalent than aaa in elderly korean males with hypertension . future research should establish distinct screening strategies for taa and aaa according to risk factors and ethnicity .

pubmed

in developed countries , endometrial cancer morbidity has exceeded that of cervical cancer to become the most common invasive malignancy of the female genital tract . early screening methods cervical cancer have made major progress because of the improvement of cytologic preparation methods and unified evaluation system in the 1950s . drawing from the successful experience of cervical carcinoma screening , we are pressed for an effective approach to screening endometrial carcinoma and its precursor to reduce new cases and deaths . endometrial complex hyperplasia with atypia and endometrial intraepithelial neoplasia ( ein ) are considered as precursors of type i endometrioid adenocarcinoma , and endometrial glandular dysplasia is the precursor of type ii carcinoma . a certain period of time for these lesions developing to carcinoma traditionally , endometrial samples for histologic analysis can be obtained from d & c or under hysteroscope , and these methods are considered to be reliable for evaluating the endometrial condition . to date , the adequacy of specimens obtained using the sap-1 device compared to specimens obtained by d & c or hysteroscopic biopsy , which is better for evaluating the condition of the endometrium , is unknown . the aim of this study was to investigate whether liquid - based surepath preparation for endometrial cytology test ( ect ) can maintain the three - dimensional structure of the endometrium . histology is the gold standard for evaluating the accuracy , sensitivity , specificity , positive prediction value ( ppv ) , and negative prediction value ( npv ) of ect . this study was performed from november 2011 to may 2014 at the obstetrics and gynecology department of peking university first hospital and it had been approved by the hospital ethics committee . the risk factors included : ( 1 ) age 40 years ; ( 2 ) intrinsically high estrogen including polycystic ovarian syndrome and ovarian tumors with an abnormal level of estrogen such as granulosa cell tumors ; ( 3 ) extrinsically high estrogen including hormone replacement therapy and postoperative breast cancer patients taking tamoxifen ; ( 4 ) high body mass index ( bmi ) 25 kg / m ; ( 5 ) hypertension , hyperlipidemia and diabetes ; ( 6 ) family history of cancer including hereditary nonpolyposis colon cancer , lynch syndrome , and first - degree relatives with gynecologic tumor ; ( 7 ) history of radiation and smoking ; ( 8) abnormal uterus bleeding ( aub ) , especially postmenopausal vaginal bleeding ; ( 9 ) abnormal endometrium assessed by ultrasound as follows : thickness ( postmenopausal women 4 mm or menopausal women 20 mm ) , occupation disease of the uterine cavity or heterogeneous . while patients with following conditions were excluded out of this study : ( 1 ) ultrasound scanning suggested uterus cavity was distorted by multiple uterine myomas or adenomyoma ; ( 2 ) patients with intra - uterine device . they provided written informed consent and underwent an ect using the direct sampler sap-1 device ( saipujiuzhou corporation , beijing , china ) [ figure 1 ] . this device was patented and received permission to use it in our clinic in china . the sap-1 sampling device . it consists of a flexible latex loop with spines on the side and a smooth tip to prevent injury to the myometrium . there is an outer protective sheath outside the loop to prevent contamination from cervical and vaginal cells . it is easy to operate and can be used in an outpatient setting , at health examination centers and in community hospitals . steps for obtaining samples using the sap-1 device the loop with the specimen was then immersed in the surepath cell preservation container ( bd diagnostic , burlington , nc , usa ) to release the cells . the 10 ml specimen was transferred into centrifuge tubes with a density reagent ( bd diagnostic , burlington , nc , usa ) to remove blood and mucus . after a two - stage centrifugation at 1000 rpm for 2 min 15 s and then 2000 rpm for 10 min 15 s ( rotina 46s , hettich corporation , german ) , the centrifuge tube was put into the surepath semi - automated slide processor and stained using papanicolaou . the cytological smears were evaluated by two independent gynecological cytologists who were blinded to the study procedures . based on a previously - published diagnostic system , the cytological results were subdivided into four categories as follows : negative for epithelial lesions , benign endometrium , atypical endometrial cell , and suspected for malignant [ figures 3a d and 4a d ] . endometrial tissue samples were fixed in neutral buffered formalin , embedded in paraffin , and stained with hematoxylin and eosin . two gynecological pathologists independently assessed the slides , based on the world health organization diagnostic criteria and ein . if a normal or benign endometrium was given , d & c or hysteroscopic pathology was regarded as a final result , we occasionally encountered a situation as cell features fall short of the criteria of simple / complex hyperplasia with atypia , we should carefully evaluated these lesions to determine whether or not a diagnosis of ein could be made . ( a ) negative for endometrial lesions : long , straight tube - shaped cell clumps with a small amount of stromal cells on the margin is the most common type of cell clumps in the proliferative endometrium , observed using a low - power microscope ( papanicolaou stain , 20 ) ; ( b ) benign endometrium : dilated and branched cell clumps are always seen . the contour of the cell clumps is smooth and occasionally a few stromal cells can be observed ( papanicolaou stain , 40 ) ; ( c ) atypical endometrial cell : double - layer or folded irregular cell clumps are observed ( papanicolaou stain , 20 ) ; ( d ) suspected endometrial carcinoma : papillo - shaped bordered cell clumps with atypical cells can be observed ( papanicolaou stain , 100 ) . ( a ) negative for endometrial lesion : regularly arranged and mono - layer endometrial cells with an oval or round nucleus . the spaces between nuclei are regular , and the chromatin in endometrial cells is delicate ( papanicolaou stain , 100 ) ; ( b ) benign endometrium : crowded cells arranged into a single layer with delicate chromatin and a small nucleolus ( papanicolaou stain , 100 ) ; ( c ) atypical endometrial cell : the spaces in atypical cells are heterogeneous . the chromatin is coarse ( papanicolaou stain , 100 ) ; ( d ) suspected carcinoma : variable size cells with obviously round nucleoli . the contingency table chi - square test was used to compare the adequacy of specimens collected using sap-1 with those collected via d & c and hysteroscopic biopsy . negative for epithelial lesions and benign endometrium were considered negative , while atypical endometrial cells and suspected for malignant were considered positive . histopathologic results were the gold standard . a four - fold table was created to calculate the accuracy , sensitive , specificity , ppv , and npv . the contingency table chi - square test was used to compare the adequacy of specimens collected using sap-1 with those collected via d & c and hysteroscopic biopsy . negative for epithelial lesions and benign endometrium were considered negative , while atypical endometrial cells and suspected for malignant were considered positive . histopathologic results were the gold standard . a four - fold table was created to calculate the accuracy , sensitive , specificity , ppv , and npv . the current study comprised of 1514 patients with endometrial carcinoma risk factors underwent sap-1 sampling on an outpatient basis , and 375 of these women also underwent d & c or hysteroscopy . characteristics including age distribution , menstrual status , and patients symptoms and signs are presented in table 1 . most of the patients were over 40 years of age in both the cytology and histopathology groups ( 91.2% and 90.7% , respectively ) . the percentage of postmenopausal women was 69% and 63.5% , respectively , in the two groups . among 1514 cases with cytological specimens , 576 ( 38% ) patients had aub , and 910 ( 60.1% ) had 4 mm thickness endometrium measured by ultrasound . 169 ( 45.1% ) out of 375 patients with histopathology had aub , and 245 patients ( 65.3% ) had 4 mm thickness endometrium . patient characteristics aub : abnormal uterus bleeding ; some patients were not examined by ultrasound , and their endometrial thickness is missing . as presented in table 2 , 1458 patients ( 96.3% ) had adequate specimens for cytology out of the 1514 patients sampled using sap-1 , while 285 cases ( 76% ) had adequate specimens for pathology out of the 375 patients who underwent d & c or hysteroscopic biopsy . there were 56 ( 3.7% ) cytology and 90 ( 24% ) biopsies that were inadequate . sap-1 can provide more sufficient materials for cytology than d & c or hysteroscopic biopsy for histology ( p < 0.01 ) . adequate and inadequate specimens obtained using cytology and biopsy of the 469 cytology specimens from premenopausal women , 452 patients ( 96.4% ) had an adequate specimen , and 17 patients ( 3.6% ) had an inadequate specimen . while in 137 cases of premenopausal women obtained biopsy specimens , 132 patients ( 96.4% ) had an adequate biopsy , and only 5 patients ( 3.6% ) had an inadequate biopsy . there was no difference ( p = 0.919 ) between cytology and biopsy in premenopausal women . however , 1006 specimens ( 86.3% ) were adequate , and 37 specimens ( 3.7% ) were inadequate out of the 1045 cytology samples obtained using sap-1 in postmenopausal women . in 238 biopsy specimens obtained from postmenopausal women , 153 biopsy specimens ( 64.3% ) were adequate , and 85 specimens ( 35.7% ) were inadequate . it was easier to collect cytology specimens than histology specimens ( p < 0.05 ) . patients with both cytologic and histologic results n = 254 patients ; negative and benign were served as negative ; atypia and carcinoma were as positive . of the 254 patients who had both of cytology and histology , 205 ( 80.7% ) 11 ( 4.3% ) patients were evaluated as positive by cytology , but they were confirmed as negative or benign lesions using histology . there were 28 patients who were positive for atypical cells , as determined by both cytology and histology . there were 10 ( 3.9% ) patients who were diagnosed as negative or benign using cytology , while their occult lesions were discovered upon subsequent hysterectomy . the accuracy of cytology for detecting endometrial precursor and carcinoma was estimated at 91.7% , sensitivity at 73.6% , specificity at 94.9% , ppv at 71.9% , and npv at 95.3% . in past decades , endometrial carcinoma has become the most prevalent gynecologic malignancy in developed cities such as beijing , shanghai , and guangzhou in china . the increased morbidity of endometrial carcinoma is attributed to changes in lifestyle , such as a lack of exercise and increasing fat intake lead to obesity and high bmi . obesity triggered several pathways including hormonal imbalance and hyperactive proliferative pathways to involve in pathogenesis of endometrial carcinoma . the effective procedure for reducing morbidity and mortality in endometrial carcinoma is to screen certain groups with risk factors and design a reliable tool suitable for a mass screening program . dilation and curettage have been used clinically for many years , and originally it was intended to serve as a screening tool for endometrial carcinoma , but researchers realized that specimen adequacy and diagnosis accuracy were unsatisfactory . yarandi et al . reported that the accuracy of d & c was 40.5% , sensitivity 40.5% , specificity 72.3% , ppv 77.1% and npv 25.1% , and in 52.7% of the patients , d & c failed to detect intrauterine disorders , especially focal lesions of the endometrium . moreover , most patients complained of pain and severe discomfort during the operation , and they had persistent bleeding for several days after the procedure . in mossa 's study , the patients undergoing d & c had higher pain scores compared with those who underwent brush cytology . currently , several sampling devices were used to take endometrium cytology samples from patients ; samplers include endoflower , tao brush , and endocyte . for tao brush sampling , estimated that out of 519 patients , the samples of biopsy obtained using the tao brush was inadequate in 361 patients ( 39% ) , and samples obtained using the endoflower were inadequate in 15 patients ( 2% ) . in addition , buccoliero et al . showed the same results in another study that compared the endoflower to biopsy in patients receiving tamoxifen . sap-1 , a direct endometrial sampler device , is especially designed for minimal pain during sampling , it meets the requirements for endometrial samplers , which include : ( 1 ) avoiding contamination from the endocervix and vagina ; ( 2 ) procuring an adequate representative sample of the entire endometrium to detect focal lesions ; and ( 3 ) the procedure should be safe , easy to use , and well - tolerated by the patients . the device has to be noninvasive or minimally invasive , cost - effective , and user - friendly to be accepted by primary care physicians and patients for repeated tests . a soft loop can be flexible within the endometrial cavity , and the spindles can easily obtain adequate endometrial cells with minimal chance of injuring myometrium and bleeding . we are the first to evaluate the adequacy of its specimens compared with d & c and hysteroscopic biopsy . in the current study , adequate specimens obtained using the sap-1 sampler ( 96.3% ) are superior to those obtained using d & c ( 76% ) . in addition , most patients experienced no pain during sap-1 sampling , claiming that they had the same feeling as the insertion of a cytobrush for cervical sampling . our previously - proposed screening strategy involved women of 40 years of age or older , or postmenopausal women as the target screening population . in buccoliero 's study , of 107 asymptomatic postmenopausal women with a thin endometrium ( < 4 mm ) , a biopsy obtained sufficient material for the diagnosis in only 24% of the cases . our study also shows that there are no differences in specimen adequacy between the sap-1 sampler and d & c in premenopausal women . however , for postmenopausal women , the sap-1 sampler provides more adequate specimens than does d & c or hysteroscopic biopsy . conventional smears have not been widely applied in endometrial carcinoma screening attributes of overlapping cells and a heavy background . in addition , the three - dimensional structure is an important ect reference to evaluate the condition of the endometrium . requirements for a stable fixation system and preparation technique include : ( 1 ) preserving the morphology of endometrial epithelial and stromal cells and the endometrial glandular structure ; and ( 2 ) eliminating obscuring factors such as excess red blood cells , mucus , overlapping cells , and inflammatory cells . the 95% alcohol fixation and conventional cytology can not earn a place in endometrial cancer screen due to regression cells and high background levels , such as excess blood , mucus , overlapping cells , and inflammatory cells . according to previous japanese publications , conventional cytology was initially used to examine the endometrial lesion , with a sensitivity about 78% , specificity of 95% , ppv of 56% , and npv of 98% . transvaginal ultrasonography was previously thought to be an efficient tool because of conventional cytology 's low accuracy . compared with a conventional smear , thin - layer cytology provided more cell clumps and a clearer background , as shown by norimatsu et al . to improve the diagnostic accuracy of endometrial cytology , remondi et al . used the endoflower sampler and the thinprep preparation to evaluate 768 postmenopausal women , and found an accuracy of 93.6% , sensitivity of 92% , specificity of 95% , ppv of 73% , and npv of 99% . the sensitivity , specificity , ppv , and npv for the uterobrush were 88.9% , 100% , 100% and 98.9% , respectively . direct uterine sampling with the tao brush sampler using a liquid - based preparation method for detection of endometrial cancer and atypical hyperplasia , the sensitivity and specificity were 95% and 66% . the results of the current study demonstrate that the sap-1 sampler combined with the liquid - based surepath preparation method is a reliable diagnostic method . the overall accuracy for detecting endometrial cancer , complex hyperplasia with atypia and ein was 92.4% , with a sensitivity of 73% , a specificity of 95.8% , a ppv of 75% , and a npv of 95.3% . almost all patients enrolled into this study received the ect for screening , so women with positive cytologic results or persistent bleeding underwent hysteroscopic biopsy or d & c. in the current study , patients with carcinoma that was subsequently confirmed by histology were initially diagnosed with atypical endometrial cells or suspected carcinoma using cytology . the main reasons that the accuracy of this study was lower than that in previously studies are as follows : patients with positive results including atypical cells underwent hysteroscopic biopsy and d & c , and some of them were subsequently diagnosed as normal or benign . in addition , other methods , including enlarging the sample quantity to collect more information to correctly distinguish focal atypical endometrial lesions from benign lesions , should be investigated using immunohistochemistry and biochemistry to improve the accuracy of endometrial cytology . in conclusion , the sap-1 sampler combined with surepath preparation may become a reliable method for screening endometrial carcinoma and its precursor , especially in postmenopausal and asymptomatic women . if this screening procedure can be used in the high risk - factor women , some unnecessary d & c may be avoided , and asymptomatic women with the precursor may benefit from early detection and management .

background : the aim of this study was to compare specimen adequacy of sap-1 provided for cytology with that of dilation and curettage ( d & c ) or hysteroscopy for histology , and evaluate the accuracy of combining endometrium sampling by sap-1 and liquid - based cytology using surepath preparation for screening endometrial carcinoma and its precursor.methods:endometrial specimens from women ( n = 1514 ) with risk factors were obtained using an sap-1 device for cytological analysis ; histological samples were obtained from 375 of these women who underwent d & c or hysteroscopy . cytological specimens were prepared to liquid - based smear using surepath technology and stained by papanicolaou . histological samples were processed in routine pathology and stained by hematoxylin and eosin.results:adequate specimens for cytology were obtained from 1458/1541 patients ( 96.3% ) , while adequate samples for pathology were obtained from 285/375 patients ( 76% ) . however , for postmenopausal women , 1006 of 1045 cytology ( 86.3% ) were adequate , 153 of 238 histology ( 64.3% ) were adequate , it was easier to collect cytological specimens than histological specimens ( p < 0.05 ) . the accuracy of endometrial cytology for detecting endometrial carcinoma and its precursor was 92.4% ( sensitivity , 73% ; specificity , 95.8% ; positive predictive value , 75% ; and negative predictive value , 95.3%).conclusions : endometrial cytology using sap-1 sampling and surepath preparation may be a reliable approach for screening patients with endometrial carcinoma and its precursor .

pubmed

breast cancer is the most common cancer worldwide among women , with nearly 1.7 million new cases in 2012 . the highest incidence rates are in europe and north america , and by country are highest in belgium , denmark , and france.1 breast cancer mortality has decreased in some parts of the world , with screening mammography associated with an up - to-50% reduction in mortality rate.2 although there has been controversy about mammogram screening , it remains the most commonly performed procedure for breast cancer detection.3,4 the results of the digital mammography imaging screening trial ( dmist ) proved digital mammography ( dm ) was as efficacious as screen - film mammography ( sfm).5 as such , most of the mammography units in the us are now digital . dm is less likely to be lost or damaged , and has slightly higher sensitivity than sfm.5 however , despite evidence from dmist that dm offers some improved benefit in women with dense breast tissue , this remains a limitation of conventional mammography in general.5,6 breast cancers will have the same density as surrounding tissue , and can be obscured by overlapping tissue.7,8 with tumors potentially obscured by overlapping tissue in two - dimensional ( 2-d ) conventional mammography , a 3-d procedure , such as tomosynthesis , may help solve this problem.7 also , there is hope that the use of intravascular contrast material with dm will take advantage of malignant neovascularity to enhance tumor detection . this article reflects on these technologies in mammographic imaging , current uses , and potential patient impact . previous reviews of tomosynthesis913 have summarized technical developments and the promising early results of small digital breast tomosynthesis ( dbt)-reader studies using selected tomosynthesis cases . we add in this review newer prospective population - screening trials from oslo , italy , and australia , which provided a larger amount of data and more definitive results acquired from clinical settings.1417 the newest developments in solving the challenge of radiation exposure in tomosynthesis are presented . furthermore , this review will address a wider audience than radiologists , to inform those of any medical specialty hoping to learn about developments in mammography . sfm has been in use for over 30 years , and in the last 10 years has been replaced in most practices in the us by full - field dm.18 this advance enables viewing and manipulation of dm images on computer workstations . with these steps separated , each can be potentially optimized.19 dm is similar to sfm in that both are performed with two views of each breast in screening mammography , a compromise between keeping radiation levels low yet imaging the majority of the breast tissue . 2-d is a common abbreviation for two - view dm.9 radiation dose is a concern , given that mammography is a screening examination performed on millions of women potentially every year . the dm systems used in the dmist trial were shown to have had a 22% lower mean glandular dose than sfm per acquired view.20 dose parameters reported for dmist were for state - of - the - art equipment used during the trial , and many of the digital systems have been refined since , and performance may have improved.2023 a specific digital system using slot scanning with a photon - counting detector that eliminates stray or scatter radiation and improves noise reduction and image quality has been found to reduce doses by 40%60%,24,25 and has shown the lowest mean glandular dose in clinical screening settings in europe.26 indeed , it was found in a dose survey of the irish breast - screening program that the lower dose resulted from the use of dm systems , and the photon - counting system accounted for 25% of mammography units in ireland.27 clinical performance for this system is comparable to other dm systems in screening,28 and has not been shown to be inferior in radiologist performance.25 one limitation of both sfm and dm is false positives ( recall for additional imaging ) . women have been recalled for additional imaging from the earliest days of mammogram screening . since screening mammography is performed on normal asymptomatic women , minimizing harm is important.3,4 the false - positive rate of mammography is one of the recurrent criticisms of mammography.3,4 in the us , up to 10% of screened women are recalled for further mammogram views , ultrasound imaging , and/or biopsies , and most of these women do not have cancer.4,5,29 screening mammography can not fully assess all abnormalities on the basis of the two routine views . recalls are necessary , because a possible lesion needs to be distinguished from a true lesion , but recalls can also lead to false - positive biopsies . these evaluations are typically time - consuming for the radiologist and patient , and stressful and inconvenient for the patient . nonetheless , recall improves breast cancer detection , and a balance is sought between increased sensitivity and improved specificity.29 recall rates in the us are about twofold higher than in europe , with guidelines in the us at overall < 10% , compared to guidelines in europe of 3%7% ( < 5%).20 if the additional views for evaluation of abnormal screening mammograms could be reduced , this would be advantageous to patients , referring physicians , and radiologists . sensitivity on average is about 70%.5 the problem of false - positive mammogram findings and missed lesions is caused by the overlapping tissue in 2-d images.7,8 abnormalities may be hidden in dense fibroglandular tissue , and the malignant features of cancer may be obscured . the american college of radiology breast imaging reporting and data system describes four breast parenchymal patterns used in mammography reports , with fatty having the least amount of fibroglandular tissue and extremely dense having the most ( figure 1).30 the number of women with dense breast tissue is not insignificant . over half of women younger than 50 years have what is considered dense breast tissue , and a third of women over 50 years.31 in women with the densest breasts , the sensitivity of mammography may be as low as 30%48%,32,33 and has been found to be a major risk factor for interval cancers.32 women with mammographic density in 75% or more of the breast have been shown to have an increased risk of breast cancer , either detected by screening or less than 12 months after a negative screening mammogram ( interval cancer),34 and the risk was found to be greater in younger women.34 it is not known to what extent the increased risk of interval cancer is due to masking of cancer from dense tissue or to rapid growth of tumors in dense tissue.32,35,36 dbt also uses x - rays like conventional mammography , but creates image slices of a tissue volume . the slices are thin , and each is seen as a 1 mm plane in sharp focus , with the tissue above and below a plane appearing out of focus . the thin slices decrease the problem of confusing overlapping tissue.10 dbt is a mammography - based technique using breast compression similar to 2-d dm , but obtains multiple low - dose images using a moving x - ray tube head rotating across the breast in an arc of 15%50% ( figure 2).9,10,37 the time varies from about 5 to 25 seconds depending on manufacturer differences.10 the image data are used to reconstruct thin tissue slices , such as are seen in computed tomography scans.38 typically , the two conventional views ( craniocaudal and mediolateral oblique ) are done of each breast of a single breast compression per view . the same anatomy of conventional mammograms is more clearly defined with dbt , with the breast anatomy separated into different tissue planes ( figure 3 ) . dbt is therefore called quasi-3-d technology because it differentiates tissues in slices.37,38 total radiation exposure varies by breast density and thickness , but overall dbt radiation exposure is approximately the same as that of sfm or dm.9,13,39 since the system is a modified dm unit , dbt images can be obtained in any of the usual mammogram views or planes.10 compression of the breast for tomosynthesis is similar to that of conventional mammography.10 compression maximizes image quality , decreases patient motion , and decreases radiation dose , but is somewhat painful.40 however , since the problem of tissue overlap is improved by dbt , some reduction of compression is possible , improving patient comfort.40 the feasibility of using tomosynthesis was studied over 10 years ago,38 with us food and drug administration ( fda ) approval for a commercial system obtained in 2012.41 dbt can be used for screening ( two views , asymptomatic patients ) and diagnostic ( multiple views in symptomatic patients ) mammography.41,42 it has all the advantages of dm , such as fewer artifacts , consistent quality , and digital image processing.9,10 there is emerging evidence that adding dbt to standard mammography increases mammogram accuracy.12,13,43 limited studies in cancer - enriched groups demonstrated the potential to decrease recall rates and increase cancer detection.9,10,13,43,44 studies were done by acquiring both conventional mammogram views and at the same time dbt views . these were done by exposing the patient twice , and thus resulted in a radiation dose about twice that of mammography alone , but nonetheless below the limits set by the mammography quality standards act ( mqsa).15,39,45 a recent population - based prospective clinical trial ( oslo trial ) studied the 50- to 69-year age group with biennial screening , in a single institution with a single group of radiologists , and 12,631 women participating . as in some of the early small studies , the partial results reported that by integrating 2-d and 3-d ( ie , dm + dbt ) mammography screening , there was a 40% higher invasive cancer - detection rate compared to dm screening alone.15 another population - based prospective study has reported partial results of 7,292 screened women who similarly had dbt added to dm ( storm [ screening with tomosynthesis or mammography ] ) , and reported that cancer detection rose 34% and false - positive recalls could decrease by 17% without missing any cancers.45 the additional cancers detected were found in all breast densities , from fatty to dense . the partial results of these clinical trials were still considered small and not adequate to determine which subgroups would benefit most by adding dbt to dm.46 subsequent additional analyses by subgroups reported that centers with high rates of false positives would benefit most from dbt , and that greater accuracy results from adding dbt to dm than from double - reading by dm alone.14,16,17 although using dm with dbt doubles the radiation dose in screening , it has been considered necessary for detection and characterization of calcifications , a potential sign of ductal carcinoma in situ , and additionally to enable orientation of dbt findings when comparing with prior mammograms.47,48 the research studies are promising , indicating that dbt is addressing some of the long - standing criticisms of conventional mammography . the addition of dbt to dm ( ie , standard 2-d mammography ) improves the accuracy of interpretation of mammograms.13,15 as discussed earlier , findings questioned on conventional mammography , such as possible masses , are subsequently found on additional imaging to be overlapping glandular tissue . these structures are more easily recognized to be normal on dbt , because only one thin plane of tissue is seen , diminishing confusing overlap . common benign masses , such as cysts and fibroadenomas , may be more evident as benign masses , because the margins are better seen.7,37 the dbt data suggest that not only is there a reduction in recall rate with dbt49 but also that dbt can more clearly depict benign findings than routine screening views ( such as lymph nodes or skin calcifications ) , diminishing the need for recall for additional imaging . reducing recall rates diminishes the so - called potential harm of recalling normal patients and decreasing the number of false - positive biopsies . in all of the early and most recent studies in both europe and the us , the recall rate for most radiologists was reduced when adding dbt to dm , despite the already much lower recall rates in europe compared to the us.15,29,37,45,49 dbt appears to increase cancer detection , and the additional cancers detected are invasive malignancies missed by conventional mammography.15 ductal carcinoma in situ , a currently a controversial histologic abnormality versus an early breast cancer , is not more frequent with dbt.15 although dbt will help reveal more cancers in women , it will not detect all cancers.50 dbt shows promise in demonstrating the same or higher accuracy as digital spot - compression mammography.51 therefore , dbt alone can also eliminate the need for additional mammogram images in many cases of abnormal mammograms , as well as send patients directly to ultrasound without further mammography . dbt also may decrease the necessity to perform extra mammogram views to identify the exact location of a breast lesion . radiation dose and patient comfort would benefit if supplementary mammogram views were no longer necessary . also , with the overall need for compression decreased for tomosynthesis , some studies found that dbt can be done with more patient satisfaction , because up to 50% less compression than dm can potentially be applied , with no loss in diagnostic capability.40 for all these reasons , as well as time savings , dbt benefits the patient and radiologist , with improved efficiency in detection and characterizing abnormalities . this also translates into less stress for the patient when there is a decrease in the amount of additional imaging necessary for determining when a lesion is benign versus suspicious . the radiation dose for the combined examination of conventional mammography and dbt is slightly more than twice the dose of mammography alone , because combining both dbt and dm delivers double the radiation dose to the breast . although this is lower than mqsa limits , radiation has to be considered with the possibility of screening millions of women each year with double the radiation dose.39,52 to solve this problem , synthetically reconstructed 2-d images can be made from the dbt data , eliminating the need for double exposures.53 this eliminates the need to do an exposure for a dm , and also drops the radiation dose to that of conventional mammography . the most recent studies of these synthesized images demonstrated similar accuracy as those of conventional dm , and fda approval was obtained in the us for the vendor hologic.48,54 therefore , potentially there will no longer be a need for radiation exposure to obtain 2-d images , and the dose for dbt will fall down to that of conventional mammography.37,54 radiologist - interpretation time is longer for dm + dbt than for dm alone.15 this is because a routine mammogram is four images , whereas a dbt can be 200 images or more per patient . longer interpretation times may be offset by the decreased recall rates and decreased need for additional views.37 it is also uncertain how dbt use would be similar or different among radiologists with varying practice patterns and expertise.37 the cost of a dbt system is substantially greater than that of a dm system , and also has the expense of digital storage for the large file sizes of dbt . however , compared with breast magnetic resonance imaging ( mri ) , dbt is a relatively low - cost innovation . in addition , if dbt reduces recall rates , cost savings may result from fewer biopsies and recalls . at present , dbt is undergoing further technological changes and is a focus of clinical research trials under way or nearing completion . large population studies are in progress , and in addition to those already mentioned , there is the malm trial , a uk trial , and a yale study.12 a clinical trial is planned in north america by the american college of radiology imaging network to compare dm to dbt ( t - mist [ tomosynthesis mammographic imaging screening trial ] ) in a multiple - vendor , multisite study . its true performance may become more obvious when it has been integrated into such additional population screenings . incorporating dbt with other modalities is also being studied , as well as contrast - enhanced tomosynthesis , that could provide an mri - like study while being simpler to perform and of much lower cost . automated breast ultrasound with dbt is also evolving , which may be very promising for screening dense breasts . computer - assisted detection , used routinely in conventional mammography to increase sensitivity , is not yet incorporated into dbt but appears feasible,55 and may assist in improving detection of subtle lesions.37 investigators have evaluated the combination of dm with intravenous contrast enhancement to obtain more functional information from the neovascularity found in malignant tumors . two methods described have been temporal contrast - enhanced dm ( cedm ) and dual - energy cedm . with cedm , a dm unit has been modified to maximize the sensitivity of the unit to low concentrations of iodine contrast by increasing the voltage . a mask image is obtained , and following the injection of contrast , sequential or temporal images are then obtained.56 the examination takes approximately 15 minutes , and the total dose is similar to a conventional single - view mammogram . in dual - energy cedm , the dm unit is adapted to generate high energy exposures for energies above the energy absorption of iodine ( k - edge ) , taking advantage of the differences in x - ray attenuation through materials of different composition.56 contrast is injected with the breast in compression , and two paired exposures are obtained : one set at low energy and one at high energy . this technique tends to limit problems with patient motion , as the patient is not in compression as long as with cedm , and two views of each breast are obtained and combined to enhance contrast - uptake areas.56 the use of contrast medium takes advantage of mapping abnormal blood flow , as does mri . early evaluation of the feasibility of the use of contrast medium with dm revealed 80% of pathologically proven breast carcinomas were enhanced,57,58 with excellent correlation between the size of enhancement and the histologic size of tumors.58 this technique may be particularly useful in patients with dense breast tissue.5759 high sensitivity has also been found with contrast - enhanced dual - energy techniques , with sensitivities between 86%96%,6062 as well as with contrast use with dbt.63,64 promising results evaluating the feasibility of contrast - enhanced dbt has been shown in one small study.64 jochelson et al demonstrated dual - energy cedm was comparable to mri for the depiction of the index tumor in patients with known breast cancer , with each identifying 96% of cancers , and dual - energy cedm depicted additional cancers in the breast with better specificity.62 potential clinical applications of cedm include not only detecting mammographically occult cancers in dense breast tissue , but also like mri help to determine the local extent of disease , or assessment of residual or recurrent disease . the potential advantages over mri are shorter examination time and ability to perform this exam at the same time as dm , image registration allowing easy comparison to conventional mammography , and preoperative guidance for biopsy if only seen with mammography.56 further developments in optimizing imaging parameters and continued improvements in decreasing radiation exposure are ongoing in dm . dbt is proving to address some of the limitations of conventional mammography , with improved cancer detection , fewer false positives , and lower recall rates . the true performance of dbt remains to be determined when the results of more large - population trials have concluded .

despite controversy regarding mammography s efficacy , it continues to be the most commonly used breast cancer - screening modality . with the development of digital mammography , some improved benefit has been shown in women with dense breast tissue . however , the density of breast tissue continues to limit the sensitivity of conventional mammography . we discuss the development of some derivative digital technologies , primarily digital breast tomosynthesis , and their strengths , weaknesses , and potential patient impact .

pubmed

over - the - counter ( otc ) cough and cold medications have long been criticized for lack of evidence supporting their efficacy . this study demonstrates that diphenhydramine indeed inhibits cough reflex sensitivity.diphenhydramine , a commonly - used first - generation antihistamine in the us , is rightfully also classified as an antitussive ( cough suppressant ) in the united states food and drug administration ( fda ) otc monograph . over - the - counter ( otc ) cough and cold medications have long been criticized for lack of evidence supporting their efficacy . diphenhydramine , a commonly - used first - generation antihistamine in the us , is rightfully also classified as an antitussive ( cough suppressant ) in the united states food and drug administration ( fda ) otc monograph . cough is the most common complaint for which patients in the united states seek medical attention . although billions of dollars are spent annually on non - prescription , otc cough preparations , scant evidence exists supporting the efficacy of these drugs as antitussives . three non - narcotic oral agents are listed as antitussives in the united states food and drug administration ( fda ) final monograph for otc antitussive drug products : chlophedianol , dextromethorphan , and the first - generation h1-antihistamine , diphenhydramine . chlophedianol was first introduced as an antitussive in germany in the 1950s , but very little published literature supports its efficacy . dextromethorphan , also available since the 1950s , is the most commonly used otc antitussive in the united states and many other areas of the world . although published studies exist demonstrating the ability of dextromethorphan to inhibit experimentally induced cough in healthy volunteers and to diminish pathological cough , most of these investigations are decades old and lack the more robust objective and subjective end points employed in current antitussive trials . diphenhydramine has been shown to inhibit citric acid - induced cough in healthy volunteers , as well as to diminish pathological chronic cough relative to placebo . furthermore , the guidelines of the american college of chest physicians ( accp ) recommend the combination of a first - generation h1-antihistamine and a decongestant as the treatment of choice for chronic cough due to upper airway cough syndrome ( formerly known as postnasal drip syndrome ) and acute cough due to the common cold , though this recommendation is based largely on a vast body of clinical experience and expert opinion , in the absence of adequately powered , prospective , randomized , controlled clinical trials . acute cough due to viral upper respiratory tract infection ( uri ; common cold ) accounts for the majority of the approximately 27 million cough - related health care provider office visits annually in the united states , as well as the significant cost of otc products purchased to suppress troublesome cough . yet , to the authors knowledge , none of the three fda - approved otc antitussive products ( chlophedianol , dextromethorphan , and diphenhydramine ) has ever been shown to inhibit experimentally induced cough in subjects with acute cough due to uri . capsaicin , the pungent extract of red peppers , has been shown in over three decades of clinical experience , to induce cough in a safe , dose - dependent and reproducible manner . thus , capsaicin cough challenge testing has become an important tool in clinical research , allowing for the accurate measurement of the effect of a pharmacological intervention on the sensitivity of the cough reflex [ 9 , 10 ] . the standard end point measured in capsaicin cough challenge testing is the concentration of capsaicin inducing 5 or more coughs ( c5 ) . in healthy volunteers , this end point has been demonstrated to be highly reproducible , in the short - term ( 20 min to 14 days ) and long term ( months to years ) . recently , it has been demonstrated that cough reflex sensitivity , though transiently enhanced during acute uri , remains stable in the first week of illness . thus , in a clinical trial , any changes in cough reflex sensitivity observed during this period may be attributed to the pharmacological intervention being studied , rather than to spontaneous variation in cough reflex sensitivity . to evaluate the effect of a diphenhydramine - containing , multicomponent syrup , compared with a dextromethorphan - containing syrup and placebo , on cough reflex sensitivity in otherwise healthy subjects with acute uri . the study protocol was approved by the institutional review board of montefiore medical center , bronx , ny . otherwise healthy adult nonsmokers with the onset of symptoms consistent with acute viral upper respiratory tract infection ( uri ) within the previous 72 h were recruited and enrolled after providing informed consent . the study protocol was approved by the institutional review board of montefiore medical center , bronx , ny . subjects did not have a history of asthma or other pulmonary disease , nor history or symptoms suggestive of gastroesophageal reflux disease . subjects presenting with symptoms suggestive of influenza , such as high fever , myalgias , or severe illness , were excluded . individuals with uri who had taken any cough / cold medicines for their illness were excluded , as were those currently receiving any medication known to affect cough reflex sensitivity , or who had received any such medication within a time frame that would leave the possibility of a lingering effect of a previously - ingested medication on the sensitivity of the cough reflex . upon enrollment , subjects were randomized to receive each of the three study formulations within a 35 days period , in random order , 2 h prior to capsaicin challenge testing , in a double - blind fashion . the study drugs consisted of : diphenhydramine ( 25 mg ) and phenylephrine ( 10 mg ) in a natural cocoa formulation ( dr . cocoa , infirst healthcare , london , uk ) ; dextromethorphan ( 30 mg ) syrup with licorice and sugar water among inactive ingredients ( father john s medicine , oakhurst co. , levittown , ny , usa ) ; and , a solution of dextrose in water . a post - ingestion period of 2 h was chosen to coincide with near - peak blood concentrations of diphenhydramine and dextromethorphan [ 2 , 13 ] . study drugs were prepared as 20 ml liquid doses in opaque bottles that each subject was instructed to drink , without commenting on the taste or smell of the liquid . the investigators therefore were unaware of the appearance , smell , and other characteristics of the study drugs administered . briefly , subjects inhaled single , vital - capacity breaths of ascending , doubling concentrations ( range 0.491000 m ) of aerosolized capsaicin solution , administered via a compressed air - driven nebulizer controlled by a dosimeter , with 1-min intervals between inhalations , until 5 or more coughs resulted in the 15 s following an inhalation the end point of capsaicin challenge testing is the concentration of capsaicin inducing 5 or more coughs ( c5 ) . to evaluate the effect of each of the three study formulations , differences in log c5 responses were analyzed by 1-way anova for dependent samples combined with tukey s hsd test for pairwise comparisons . twenty - two subjects [ 16 females ; mean age 39.2 13.3(sd ) year ] were recruited and completed the study . sd ) log c5 values observed after administration of each of the study drugs were : 0.57 0.75 after placebo ; 0.80 0.83 after dextromethorphan ; and , 0.97 0.74 after the diphenhydramine - containing formulation ( fig . 1 ) . differences in log c5 responses were analyzed by 1-way anova for dependent samples combined with tukey s hsd test for pairwise comparisons . a significant difference ( p = 0.0024 ) was established among the groups , with pairwise analysis revealing a significant increase in mean log c5 [ 0.4 0.55(sd ) ; p < 0.01 ] for the diphenhydramine - containing medication versus placebo , but not for dextromethorphan versus placebo.fig . 1values for cough reflex sensitivity to capsaicin ( log c5 ) determined 2 h after ingestion of diphenhydramine ( dph ) , dextromethorphan ( dx ) , and placebo in adult nonsmokers with acute viral upper respiratory tract infection . cough reflex sensitivity was significantly inhibited ( log c5 increased ) after diphenhydramine ( p < 0.01 ) . error bars represent sem values for cough reflex sensitivity to capsaicin ( log c5 ) determined 2 h after ingestion of diphenhydramine ( dph ) , dextromethorphan ( dx ) , and placebo in adult nonsmokers with acute viral upper respiratory tract infection . cough reflex sensitivity was significantly inhibited ( log c5 increased ) after diphenhydramine ( p < 0.01 ) . error bars represent sem in terms of side effects , five subjects reported slight drowsiness after ingesting the diphenhydramine - containing formulation , as did one subject after placebo . five subjects reported side effects after ingesting the dextromethorphan syrup : slight drowsiness in two subjects , slight nausea in two subjects , and dry mouth in one subject . in the united states , the majority of otc cough preparations contain dextromethorphan or diphenhydramine as the pharmacologically active component . significant criticism has been directed at the otc cough and cold market because of the lack of adequately performed clinical trials demonstrating these agents to be effective antitussives [ 2.7 ] . to the authors knowledge , this study represents the first demonstration of the ability of diphenhydramine to suppress cough reflex sensitivity in subjects with acute uri . furthermore , the diphenhydramine - containing formulation proved a more effective antitussive than did dextromethorphan , with both agents administered at standard antitussive doses . however , it should be noted that cough reflex sensitivity was measured 2 h after study drug administration , to coincide with near - peak blood concentrations of the agents under investigation . such timing of the cough challenge may not have allowed demonstration of the maximal antitussive effect of dextromethorphan , as a recent study of healthy volunteers found that maximal inhibition of capsaicin cough sensitivity by dextromethorphan was not observed until 6 h after oral administration . the multicomponent diphenhydramine - containing syrup investigated in this study also contains the decongestant phenylephrine at standard otc dose as well as natural cocoa flavoring . to our knowledge , theobromine , a component of cocoa , has been shown to have antitussive effect in healthy volunteers in one previous study , however , the amount of theobromine contained in one dose of the medication evaluated herein is much smaller than that required for cough reflex inhibition . nevertheless , the thickness and cocoa flavor of the diphenhydramine - containing formulation may be contributing to the overall efficacy of the medication by creating a demulcent effect that has been proposed as an important component of the perceived therapeutic effect of cough syrups . the diphenhydramine - containing syrup contained a natural cocoa flavoring ; the dextromethorphan - containing syrup contained licorice and sugar water ; and , the placebo was a dextrose solution . however , we do not feel that the lack of perfect blinding affected our results . subjects knew that during the three study days , they would be receiving two active medications and a placebo . they were unaware , of course , of which flavorings the active and placebo formulations would have . had subjective end points been examined , especially soon after drug administration , then certainly the possibility of a demulcent effect of the various liquids may have contributed to subject perception and experience . however , our study measured only the objective end point of cough reflex sensitivity to capsaicin , 2 h after study drug administration , by which time any local throat sensations and demulcent effects would have dissipated . it is noteworthy that a recent study demonstrated that sweet substances can affect cough reflex sensitivity to capsaicin . thus , our placebo preparation was also sweetened so as to present subjects with sweet liquids on each of the 3 days of testing . although the first - generation antihistamine , diphenhydramine , is classified as an antitussive by the fda and is a component of numerous otc cough and cold preparations , the present study , to our knowledge , contributes the initial evidence demonstrating the ability of this agent to inhibit cough reflex sensitivity in acute pathological cough . further clinical trials are needed to adequately evaluate this and other otc cough and cold products , so as to allow physicians and consumers alike to make informed treatment decisions based on proper scientific data .

background : currently available over - the - counter cough remedies historically have been criticized for lack of scientific evidence supporting their efficacy . although the first - generation antihistamine diphenhydramine is classified as an antitussive by the united states food and drug administration , to the authors knowledge it has never been shown to inhibit cough reflex sensitivity in subjects with pathological cough . objective : to evaluate the effect of diphenhydramine on cough reflex sensitivity . setting : montefiore medical center , an academic medical center in new york city . methods : twenty two subjects with acute viral upper respiratory tract infection ( common cold ) underwent cough reflex sensitivity measurement employing capsaicin challenge on 3 separate days , 2 h after ingesting single doses of study drug ( to coincide with peak blood concentrations ) , administered in randomized , double - blind manner : a multicomponent syrup containing diphenhydramine ( 25 mg ) , phenylephrine ( 10 mg ) , in a natural cocoa formulation ; dextromethorphan ( 30 mg ) syrup ; and , placebo syrup . the standard endpoint of cough challenge was used : concentration of capsaicin inducing 5 coughs ( c5 ) . main outcome measure : effect on cough reflex sensitivity ( c5 ) . results : a significant difference ( p = 0.0024 ) was established among groups , with pairwise analysis revealing a significant increase in mean log c5 ( 0.4 0.55 ( sd ) ; p < 0.01 ) for the diphenhydramine - containing medication versus placebo , but not for dextromethorphan versus placebo . conclusions : our results provide the initial evidence of the ability of diphenhydramine to inhibit cough reflex sensitivity in subjects with acute pathological cough . timing of cough reflex sensitivity measurement may not have allowed demonstration of maximal antitussive effect of dextromethorphan .

pubmed

the global population , especially those in developed countries , is getting older and this trend is predicted to continue in the coming decades [ 1 , 2 ] . some have defined aging as a decreased ability to resist cellular stresses or insults [ 3 , 4 ] , and in fact , aging is one of the most important cardiovascular risk factors for predisposing conditions such as diabetes , hypertension , and hypercholesterolemia . accordingly , the incidence and prevalence of clinical and subclinical cardiovascular diseases increase dramatically with age , making cardiovascular disease the most common cause of death among the elderly . the endothelium has a primary role in adjusting vascular function by the production of nitric oxide ( no ) and other biologically active vasodilator materials that decrease vascular resistance , inhibit platelet adhesion and aggregation , and decrease vascular smooth muscle cell proliferation . alterations in the control of these processes , a feature of endothelial dysfunction , often leads to atherosclerosis and other vascular disorders that are accompanied by a proinflammatory , proliferative , and procoagulatory state . the endothelium is ideally placed to bear the brunt of hemodynamic stresses , oxidized lipids , and oxidative radicals , all of which increase their vulnerability to aging . this is true not only in healthy subjects without underlying risk factors , but also in older people and those with cardiovascular risk factors . indeed , those with cardiovascular risk factor / disease will benefit more . for instance , eight weeks of exercise significantly improve endothelial function , as measured by flow - mediated dilation , in diabetic patients but not in healthy subjects . although there are clear health benefits of exercise in the elderly , a detailed understanding of the molecular basis underlying these improvements remains incomplete . in this minireview , we discuss some mechanisms thought to be involved in endothelial aging . we focus on the role of oxidative stress and subsequent inflammation and the role of exercise in boosting antioxidative and anti - inflammatory mechanisms . the endothelial cells form a monolayer that lines blood vessels to form an interface between circulating blood and the smooth muscle layer . in addition to its barrier function , the endothelium modulates coagulation , growth , and inflammation throughout the circulatory system . it also contributes to adjusting tissue perfusion by secreting several vasoactive substances , which can be vasoconstrictors ( endothelin-1 , angiotensin ii , thromboxane a2 , etc . ) or vasodilators ( no , prostacyclin , and endothelium - derived hyperpolarizing factor ( edhf ) , etc . ) . among the many vasoactive agents released by the endothelium , no has been characterized in greatest detail . it is released in response to a variety of chemical and physical stimuli to cause vasodilation , such that one of the most common methods for assessing endothelial health is by determining vascular capacity to produce no . no is produced by the catalytic activity of no synthase ( nos ) , which transforms l - arginine to l - citrulline . all isoforms of nos require five cofactors / prosthetic groups : flavin adenine dinucleotide ( fad ) , flavin mononucleotide ( fmn ) , heme , tetrahydrobiopterin ( bh4 ) , and ca / calmodulin . calcium is required for the activation of neuronal nos ( nnos ) and endothelial nos ( enos ) but not for the activity of inducible nos ( inos ) . enos binds to caveolin-1 in endothelial cell caveolae , a subset of specialized lipid domains that form invaginations and so increase intracellular microdomains where organelles and anchoring proteins aggregate . caveolin-1 inhibits enos activity , and this interaction is regulated by ca / calmodulin . upon agonist activation , the increases in intracellular calcium results in ca / calmodulin binding , which then displaces caveolin and reverses its inhibitory interaction with enos . mechanical stimuli such as shear stress and vascular smooth muscle stretch also raise intracellular calcium concentrations . several chemical events such as interaction with ca / calmodulin , heat shock protein 90 , and subsequent association of akt results in enos phosphorylation at ser1177 are involved in no production . released no causes vasorelaxation , which in turn results in increased blood flow and reduced blood pressure , inhibition of platelet adhesion and aggregation , inhibition of leukocyte adhesion , reduction in smooth muscle proliferation , and retardation of atherogenesis . endothelial dysfunction is defined as functional alterations in endothelial physiology characterized by reduction of vasodilator substance output ( in particular no ) and augmentation in endothelium - derived contracting factors . this imbalance leads to a vasoconstrictive , hypercoagulative , proliferative , and proinflammatory state , so favoring atherosclerosis . over time , most humans are exposed to a variety of modifiable cardiovascular risk factors , such as hyperglycemia and insulin resistance , obesity , altered lipid profile , hypertension , and glomerulosclerosis . . bed rest of about 48 hours induces vascular dysfunction , which is then followed by insulin resistance , dyslipidemia , and increased blood pressure . thus , shortening of hospital stays and bed confinement periods are highly beneficial in the elderly . bed rest also increases circulating endothelial cells , possibly due to increased endothelial cell apoptosis resulting from reduced shear stress during bed rest . , as long as the damage is minimal or maintenance systems work properly as in young subjects , the functional capacity of the endothelium is preserved . in cases of severe damage , injured cells are omitted by a poorly described mechanism and replaced by dividing neighboring cells . circulating progenitor endothelial cells also play a role in the repair mechanisms of injured endothelial cells . however , as part of the aging processes , the cumulative effects of stresses coupled with the inevitable metabolic changes that occur with time , there is a decline in the function and repair capacity of the endothelium . since endothelial cells can only undergo a limited number of divisions , they eventually enter a state of senescence , which is an endogenous and hereditary process of biological aging , in which cells are still metabolically active , but express a pro - inflammatory , prooxidative , and proatherogenic phenotype . the cumulative effects of these parameters strongly promote a decline in the functional capacity of endothelium . all forms of cardiovascular disease have an increased prevalence in the elderly , even in those free of cardiovascular risk factors . since there are no changes in endothelium - independent vasodilation in older humans and animals , it is reasonable to suggest that vascular age - dependent endothelial changes largely reflect the release no , although there are also some changes in the production and release of other endothelial derived vasodilators such as prostacyclin and edhf , along with increases in vasoconstrictor prostanoids . there are also several age - related structural changes in endothelium such as increases in the expression of adhesion molecules , permeability , sensitivity to apoptotic stimuli , with decreases in angiogenic and regenerative capacities [ 43 , 44 ] . oxidative stress is an imbalance between production of oxidizing agents , such as free radicals , and opposing antioxidant systems which scavenge or metabolize those reactive agents . free radicals are reactive chemical molecules having a single unpaired electron in an outer orbit . this unstable configuration provides energy which is released through reactions with adjacent molecules such as proteins , lipids , carbohydrates , and nucleic acids . oxygen - free radicals or , more generally , reactive oxygen species ( ros ) , as well as reactive nitrogen species ( rns ) , are products of normal cellular metabolism . oxidative stress interferes with endothelial function in different ways , but the most prominent mechanism is via reduction of no bioavailability , which is the net product of the rate of no production and its degradation by superoxide . in the case of no generation , reduced vasoconstrictive responses to the nos inhibitor n(g)-monomethyl - l - arginine ( l - nmma ) in older patients and reduced shear stress - induced no release and vasodilation in older animals suggest decreased production of no in aged endothelium . in spite of these , direct measurements of enos in aged animals were inconclusive , as raised , fallen , or unchanged levels have been reported [ 4952 ] . the increased production of superoxide anions in the aging vascular wall rapidly inactivates no [ 53 , 54 ] . cyclooxygenase ( cox ) and nadph - oxidase have central roles in ros production [ 47 , 5558 ] . removal of the endothelium or inhibition of nadph - oxidase reduces vascular superoxide generation in the aorta of aged wistar - kyoto rats . superoxide rapidly reacts with no to generate cytotoxic peroxynitrite ( onoo ) , a reaction with several consequences . first , onoo alters the function of biomolecules by protein nitration as well as by causing lipid peroxidation . for example , potassium channels , which hyperpolarize vascular cells and mediate regulate vasorelaxation , are inhibited by nitration [ 60 , 61 ] . second , onoo causes single - strand dna breakage , which in turn activates nuclear enzyme poly(adp - ribose ) polymerase ( parp ) ( a nuclear dna - repair enzyme ) . third , it decreases no bioavailability causing impaired relaxation and inhibition of the antiproliferative effects of no . furthermore , onoo oxidizes bh4 , an important cofactor for nos , leading to the uncoupling of enos and causing it to produce superoxide instead of no . ros - induced peroxidation of membrane lipids alters the structure and the fluidity of biological membranes , so having global detrimental effects on vascular function . the role of oxidative stress in aged endothelial dysfunction is shown by the ability of vitamin c to restore the impaired vasodilatory response to acetylcholine only in subjects aged 60 years or older . this indicates that oxidative stress is a critical mechanism for endothelial dysfunction only in older subjects . administration of bh4 also improves flow - mediated dilatation ( fmd ) in older sedentary subjects , while having no beneficial effects in young or older trained people . all cells have evolved highly complex enzymatic and nonenzymatic antioxidant systems that act synergistically to defend the body from free radical - induced damage . the most efficient enzymatic antioxidants are glutathione peroxidase , catalase , superoxide dismutase , heme oxygenase-1 ( ho-1 ) , nad(p)h quinone oxidoreductase-1 ( nqo-1 ) , and thioredoxin . nonenzymatic antioxidants include vitamins e and c , thiol antioxidants ( glutathione , thioredoxin ) . attenuation of antioxidant defense mechanisms during the aging process has been proposed in some studies . for instance , reduced concentration of plasma sod , but not cellular sod , occurs in rats . higher production of peroxynitrite causes antioxidant enzyme deactivation , as is the case for manganese sod ( mnsod ) in mitochondria . levels of protein expression and enzymatic activity of glutathione peroxidase 1 ( gpx-1 ) are lower in proangiogenic endothelial progenitor cells ( derived from cultured blood mononuclear cells ) from older subjects . atherosclerosis is an inflammatory disease that is mediated by monocyte - derived macrophages which accumulate in arterial plaques and become activated to release cytokines that cause tissue damage . as evidence accumulates favoring the role of inflammation during the different phases of atherosclerosis , it is likely that markers of inflammation such as high sensitivity c - reactive protein ( hs - crp ) could be increasingly used to provide additional insights on the biological status of atherosclerotic lesions . elevations of crp are considered independent predictors of cardiovascular events and of the outcome of acute coronary syndromes . besides their roles markers of systemic inflammation and as predictors of cardiovascular risk , crp and other inflammatory cytokines also directly trigger vascular dysfunction , possibly by altering calcium channel expression and activity , upregulating of rho - kinase expression and function , increasing ros production , and/or enhancing cox expression . in turn , cox - derived constrictor prostanoid(s ) products cause vascular hypercontractility [ 74 , 75 ] and increased formation of ros . increases in cox - induced ros production and the expression of proinflammatory mediators , such as il-1b , il-6 , tnf- , cox-2 , and inos , occur during aging . the nf-b pathway is a critical component of inflammatory processes activated by oxidative stress [ 33 , 78 ] . nf-b is an ubiquitous transcription factor with multiple roles such as mediating inflammatory responses to a variety of signals , immune function , endothelial cell activation , and control of cell growth [ 7981 ] . nf-b is normally located in the cytoplasm in an inactive form by virtue of binding to a family of inhibitory nf-b ( ib ) proteins . upon cell stimulation by a wide variety of stimuli , signals responsive ikk- and ikk- ( also known as ikk-1 and ikk-2 ) are activated , which results in the phosphorylation of ib and its proteasomal degradation . ib degradation liberates nf-b , allowing it to translocate to the nucleus and induce gene expression of a number of proinflammatory cytokines , such as il-1 , il-6 , tnf- , cox-2 , lipoxygenase , inos , and adhesion molecules ( vcam-1 , icam-1 , pcam , e - selectin ) . aging increases nf-b levels due to activation of ikk/ and degradation of ib . under usual conditions , the activation of nf-b during inflammation is temporary and limited . in aging , however , a chronic and self - perpetuating condition exists . proteins such as tnf- , il-1 , il-6 , and cox-2 that are nf-b induced also activate nf-b production , thus creating a vicious cycle . aging increases plasma levels of tnf- , il-6 , il-1 , crp , and inflammatory blood cells [ 8486 ] . the plasma concentration of il-6 correlates with senile neural atrophy and inflammatory diseases , such as type 2 diabetes and atherosclerosis . plasma levels of tnf- and il-6 are also predictors of disability and mortality among elderly . the significance of exercise as a modifiable risk factor for cardiovascular disease is widely acknowledged . physical inactivity and poor diet , preceded only by tobacco , are the leading causes of death . the american college of sports medicine , defines exercise as any and all activity involving generation of force by the activated muscle(s ) that results in disruption of a homeostatic state . endurance exercise is characterized by prolonged and continuous periods of contractile activity ( high repetition ) against low resistance . resistance exercise ( also termed strength training ) involves short periods of contractile activity ( low repetition ) against a high opposing resistance . sprint exercise consists of short periods of maximal ( intense ) repetitive contractile activity with a low interval and against a low resistance , for example , running 100 m sprint race . however , sprint training can also be performed against high resistance resulting in a combination of resistance and endurance modalities , for example , running with added weights . increased physical activity and fitness , of both men and women , some studies even suggest greater benefits ( up to 50% risk reduction ) for exercise in terms of all - cause mortality and death from cardiovascular disease . follow - up study evaluated the relationship between physical activity and risk of all - cause mortality in a large number of elderly ( 7080 women aged 7075 and 11668 men aged 6583 ) . they found an inverse dose - response relationship between exercise and all - cause mortality . risk reductions were 3050% higher in females than in males in every category of exercise intensity . this study clearly shows that there are clear health benefits from all levels of physical activity . regular aerobic exercise can slow down the age - related losses in endothelial function supposedly by restoration of no availability consequent to prevention of ros production . aging is associated with a limited capacity of the vasculature to release no , as older subjects show reduced levels of plasma nitrite in response to exercise . the difference in plasma nitrate / nitrite ratio between older and young sedentary subjects heat - stimulated hand and foot skin increased blood flow are higher in trained older subjects compared to sedentary matched controls and are correlated with nitrate / nitrite ratios , suggesting better endothelial function secondary to greater no bioavailability . trained elderly subjects also exhibit higher flow - mediated brachial artery dilation compared to sedentary counterparts . table 1 summarizes the findings of recent clinical studies on the endothelial benefits of exercise in the elderly . exercise training upregulates antioxidant defense mechanisms in several tissues , presumably due to increased levels of oxidative stress that occurs during exercise . exercise - induced production of ros is proposed to evoke specific adaptations such as increasing repair mechanisms for oxidative damage , increasing resistance to oxidative stress , and lowering levels of oxidative damage . on the other hand , excessive production of ros can have detrimental effects . boosting levels of intrinsic antioxidant potential and reduction in lipid peroxidation occur in healthy elderly men after habitual physical activity . a critical role has recently been described for a transcription factor nuclear factor ( erythroid - derived 2)-like 2 ( nrf2 ) against oxidative stress . normally , nrf2 is located in the cytoplasm and kept dormant by the cytoplasmic repressor kelch - like ech - associated protein 1 ( keap1 ) . a variety of activators , including oxidative free radicals , release , and translocate nrf2 into the nucleus where it regulates the expression of antioxidant enzymes such as nqo-1 , glutathione - s - transferase , glutathione peroxidase , and ho-1 . diminished nrf2 activity contributes to increased oxidative stress and mitochondrial dysfunction leading to endothelial dysfunction , insulin resistance , and abnormal angiogenesis as observed in diabetics . ho-1 , which is mainly induced through the nrf2-keap1 signaling pathway ( also known as heat shock protein 32 ) , is the inducible isoform of heme oxygenase that catalyzes nadph - dependent decomposition of heme to carbon monoxide ( co ) , ferrous iron , and biliverdin . three isoforms of ho have been identified : both ho-2 and ho-3 are 33-kda constitutively expressed isoforms . an important role of ho-1 in the antioxidant defense system arises from an induction of ferritin synthesis that diminishes the cellular pool of free iron and also from the enhancement of bilirubin levels , which are potent antioxidants . carbon monoxide activates soluble guanylate cyclase , a key enzyme in the cell signaling cascade leading to relaxation of smooth muscle , and thrombocyte disaggregation . ho-1 is a sensitive and reliable marker of oxidative stress and cytoplasmic expression levels of ho-1 increase in leukocytes of endurance - trained male subjects after a half - marathon run . there is a paradoxical increased expression of ho-1 in a control group of untrained men at rest , suggesting that the downregulation of the baseline expression of ho-1 in athletes reflects an adaptation mechanism to regular exercise training . the direct effect of exercise on nrf2 expression has received much less attention except for a report that exercise increases nuclear levels of nrf2 in the proximal renal tubules of old rats . the increased expression of enos after exercise both in animals and human beings [ 111114 ] also occurs in patients with stable coronary artery disease and chronic heart failure [ 115 , 116 ] . exercise - induced upregulation of vascular enos expression is closely related to the frequency and the intensity of physical forces within the vasculature , especially shear stress . shear stress is the product of all the perpendicular and parallel flow - mediated forces on endothelial cells . the types of these hemodynamic forces , either laminar or oscillatory , greatly impact the function and properties of endothelial cells and also determine the signal transduction pathways that are activated . laminar flow , which is augmented during moderate and intense physical activities , upregulates enos expression while oscillatory forces , which are associated with hypertension , leads to increased nadph - oxidase activity and augments oxidative stress . the mechanotransduction mechanisms that sense physical forces to cause altered gene expression are not completely described . some reports suggest that activation of inward rectifying k cannels , followed by stimulation of outwardly rectifying cl channels , plays a major role in this process . membrane hyperpolarization , due to inward kcurrents , drives extracellular ca into the cells through two shear stress - dependent ion channels ( p2x purinoceptors and transient receptor potential channels ) . raised intracellular calcium levels lead to a dissociation of caveolae - bound enos and increased production of no . other intracellular events are also thought to mediate increased no production in response to shear stress ; however , the relative importance of these mechanisms is not clear ( table 2 ) . increased no synthesis secondary to amplified shear stress induces extracellular superoxide dismutase ( sod ) expression in a positive feedback manner so as to inhibit the degradation of no by ros . another parallel mechanism that participates in this harmony is the upregulation of enos through exercise - induced ros production , since exercise - induced increases in shear stress stimulates vascular production of ros by an endothelium dependent pathway . superoxides are rapidly converted to h2o2 by sod ; hydrogen peroxide then diffuses through the vascular wall and increases the expression and activity of enos [ 120 , 121 ] . thus , increased expression of sod1 and sod3 ( which facilitate the generation of h2o2 from superoxide ) augments the effect of h2o2 on exercise - induced enos expression . on the other hand , enos expression another putative mechanism is exercise - induced increases in arterial compliance which is mediated by reduction of plasma et-1 concentration as well as the elimination of et-1 mediated vascular tone . twelve weeks of aerobic exercise training increase arterial compliance , while decreasing plasma et-1 levels . moreover , the increase in central arterial compliance observed with et - receptor blockade before the exercise intervention was eliminated after exercise training . these results indicate that endogenous et-1 participates in the beneficial influence of regular aerobic exercise on central arterial compliance . exercise produces a short - term inflammatory response that is accompanied by leukocytosis , increases in oxidative stress and plasma levels of crp . this proinflammatory response is followed by a long - term anti - inflammatory adaptive response . regular exercise reduces crp , il-6 , and tnf- levels while increasing anti - inflammatory substances such as il-4 and il-10 [ 125 , 126 ] . controlling the release and activity of at least two cytokines , tnf- and il-6 , could contribute to the natural protective effects of physical activity . interleukine-6 ( il-6 ) is the first cytokine to be released into the circulation during exercise , and its levels increase in an exponential fashion in response to exercise . contracting skeletal muscles upregulate levels of il-6 mrna and the transcriptional rate of the il-6 gene is also markedly enhanced by exercise . il-6 acts as both a proinflammatory and anti - inflammatory cytokine : when secreted by t cells and macrophages , il-6 stimulates the immune response and boosts inflammatory reactions , while muscle - produced il-6 exerts anti - inflammatory effects through its inhibitory effects on tnf- and il-1 , and activation of interleukin-1 receptor antagonist ( il-1ra ) and il-10 . il-10 in turn reduces the production of several proinflammatory cytokines , such as tnf- and il-1 . exercise - induced increases in plasma il-6 correlate with the muscle mass involved in exercise activity and the mode , duration , and intensity of exercise , and this is especially the case in older individuals . in addition , starkie et al . report that infusion of recombinant human il-6 ( rhil-6 ) into human subjects simulated exercise induced an il-6 response in the prevention of endotoxin - induced increase in plasma tnf- . exercise also suppresses tnf- production by an il-6 independent pathway , as there are modest decreases in plasma tnf- after exercise in il-6 knockout mice . in addition , il-6 enhances lipid turnover and stimulates lipolysis as well as fat oxidation via activation of amp - activated protein kinase . mice deficient in il-6 ( il6/ ) develop mature onset obesity and have disturbed carbohydrate and lipid metabolism that is partly reversed by il-6 replacement . the lipolytic effect of il-6 on fat metabolism was confirmed in two clinical studies of healthy and diabetic subjects [ 137 , 139 ] . visceral fat is potentially a cause of low - grade systemic inflammation , which in turn leads to insulin resistance , type 2 diabetes , and atherosclerosis . during exercise , il-6 also increases hepatic glucose production . glucose ingestion during exercise reduces il-6 production by muscles , suggesting that il-6 is released by a reduction in glycogen levels during endurance exercise and the consequent adrenergic stimulation of il-6 gene transcription via protein kinase a activation . this cytokine exerts anabolic effects in muscles by inducing protein synthesis and inhibiting protein degradation [ 130 , 142 ] . in some animal studies , this cytokine prevents muscle wasting by attenuating apoptotic dna fragmentation and downregulating tnf - driven apoptotic pathways . in agreement with this , four weeks of exercise reduce the extent of tnf - triggered myocyte apoptosis in old rats [ 144 , 145 ] aging is an important independent risk factor for the development of cardiovascular disease , which is manifest as endothelial dysfunction . a large body of evidence underlines the importance of oxidative stress and inflammation as prominent features of the aging process . reduced no bioavailability and decreased responsiveness to other endothelial - derived vasodilators promote a vasoconstrictive , hypercoagulative , and proliferative state which favors the development of atherosclerosis . vascular oxidative stress is the product of increased reactive oxygen species , such as superoxides , and reduced antioxidant defense . oxidative stress - induced damage promotes a chronic inflammatory state which perpetuates a vicious cycle of endothelial dysfunction . exercise training prevents and restores age - related impairment of endothelial function , possibly by the restoration of no availability consequent to prevention of oxidative stress and alleviating inflammatory processes .

the endothelium plays a critical role in the maintenance of cardiovascular health by producing nitric oxide and other vasoactive materials . aging is associated with a gradual decline in this functional aspect of endothelial regulation of cardiovascular homeostasis . indeed , age is an independent risk factor for cardiovascular diseases and is in part an important factor in the increased exponential mortality rates from vascular disease such as myocardial infarction and stroke that occurs in the ageing population . there are a number of mechanisms suggested to explain age - related endothelial dysfunction . however , recent scientific studies have advanced the notion of oxidative stress and inflammation as the two major risk factors underlying aging and age - related diseases . regular physical activity , known to have a favorable effect on cardiovascular health , can also improve the function of the ageing endothelium by modulating oxidative stress and inflammatory processes , as we discuss in this paper .

pubmed

axillary lymph node status is the most important prognostic factor in breast carcinoma and prognosis worsens with increasing number of metastatic lymph nodes [ 1 , 2 ] . according to the american joint committee on cancer ( ajcc)/international union against cancer ( uicc ) tumor ( t)-node ( n)-metastasis ( m ) classification , nodal disease is classified in three groups based on the number of axillary metastatic lymph nodes : n1 , 13 metastatic lymph node(s ) , n2 , 49 metastatic lymph nodes and n3 , 10 or more metastatic lymph nodes . however , the number of metastatic lymph nodes depends on the number of removed lymph nodes that are dissected by the surgeon and examined by the pathologist . various studies have shown that the number of metastatic lymph nodes is greater with increasing number of removed lymph nodes [ 412 ] . it is difficult to assess the axillary lymph node status reliably without removing sufficient numbers of lymph nodes depending on the surgeon and/or pathologist . studies conducted in recent years indicate that the ratio of the number of metastatic lymph nodes to the number of removed lymph nodes denoted as lymph node ratio ( lnr ) provide a more useful prognostic information compared to nodal disease classification according to the number of metastatic lymph nodes [ 9 , 1316 ] . a review on the prognostic value of lnr indicated that the threshold value of clinically significant lnr varies in different studies and emphasized that these studies vary by sampling size and tumor stage . until now , analysis was made mostly in all lymph node - positive patients , without subdivision in n1 , n2 , and n3 disease groups in the lnr studies [ 9 , 13 , 14 , 16 , 1823 ] . lnr threshold value separating the whole series in two prognostic groups with significantly different survival was given as 0.20 [ 18 , 24 ] and 0.25 . in three series including patients with n2 and n3 disease receiving adjuvant high - dose chemotherapy with stem - cell support , lnr threshold value of prognostic significance was determined as 0.80 by nieto et al . and schneeweiss et al . , and 0.70 by bolwell et al . . our group determined the lnr threshold value as 0.80 in a previous study including patients with t1,2,3n3m0 disease . in all of the above - mentioned studies patients with higher lnr had significantly worse prognosis compared to those with lower lnr . a striking result from these studies was that the lnr threshold value of prognostic significance was greater in series excluding n1 disease , compared to those including it . based on this , we proposed that identifying a separate lnr threshold value for each n disease group may be useful . fortin et al . recommended axillary radiotherapy for patients with a lnr 0.40 or above in the group with 13 metastatic lymph node(s ) and for patients with a lnr 0.50 or above in the group with 4 or more metastatic lymph nodes among t1-t2 node - positive patients . there is limited number of studies investigating the prognostic value of lnr in patients with only n1 disease [ 2931 ] . in this study , we evaluated the prognostic significance of the number of removed and metastatic lymph nodes and lnr in breast carcinoma patients with 13 axillary metastatic lymph node(s ) . we retrospectively reviewed the file records of women who underwent surgery for breast carcinoma between january 1993 and december 2001 and who were then followed up in sb okmeydan training and research hospital . inclusion criteria for the patients were a histological diagnosis of unilateral invasive breast carcinoma , no previous or concomitant malignant disease , known pathological tumor size ( patients with t4 tumor were not included ) , axillary 13 lymph node(s ) metastasis , no metastasis in ipsilateral internal mammary or supraclavicular lymph nodes and distant site at the time of diagnosis , microscopically tumor - free surgical margins , completion of adjuvant therapy planned according to standard therapy protocols , and a follow - up period of at least five years . a total of 924 patients ( including 174 patients who underwent surgery at the study hospital ) who met these criteria were enrolled in the current study . follow - up data were obtained from file records and , in some patients , through telephone calls . locoregional recurrence was defined as the first site of recurrence involving the chest wall ( local ) or / and ipsilateral axillary , supraclavicular , and internal mammary lymph nodes ( regional ) . first disease recurrence was recorded as distant metastasis if it was either distant metastasis or concurrent distant metastasis and locoregional recurrence . disease - free survival ( dfs ) , locoregional recurrence - free survival ( lrfs ) , and distant metastasis - free survival ( dmfs ) times were defined as the time interval between tumor excision and detection of first disease recurrence , locoregional recurrence or distant metastasis respectively or the date of last follow - up . in 21 patients who developed a second malignancy ( excluding basal cell carcinoma ) , the diagnosis date of the second malignancy was considered as the last followup date . in 9 patients whose death was unrelated to cancer , fifty - five patients developed locoregional recurrence ( including 16 patients who developed axillary recurrence ) , 243 patients developed distant metastasis , and 9 patients developed concomitant locoregional recurrence and distant metastasis ; in patients without disease recurrence median follow - up time was 108.5 months . the fisher exact test was used to compare the axillary recurrence rates of the patient groups . patients were grouped according to the number of removed and metastatic lymph nodes and lnr threshold value . kaplan - meier method was used for calculation and plotting of the dfs , lrfs , and dmfs curves of the patient groups , and the log - rank test was used for the comparison of the survival curves . survival analyses ( for dfs , lrfs , and dmfs ) were performed separately for the whole series and for the patient group having at least 10 lymph nodes removed from the axilla . patients were grouped in two different ways according to the number of lymph nodes removed ( grouping a : 15 , 69 , 1015 and 16 and more removed lymph nodes ; grouping b : 15 and 6 and more removed lymph nodes ) . to determine the lnr threshold value that will separate patients in two prognostic groups of low and high disease recurrence risk with significantly different survival rates , survival analyses were conducted with lnr threshold values between 0.10 and 0.40 for the whole series , and between 0.10 and 0.25 for the group with at least 10 lymph nodes removed , at increments of 0.05 . the lnr that produced the significant survival difference between the groups and gave the highest log - rank x value was considered as the most significant threshold value . in grouping a , dfs was significantly worse in patients with 15 lymph node(s ) removed compared to patients with 69 ( p = 0.016 ) , 1015 ( p = 0.024 ) , and 16 and more ( p = 0.015 ) lymph nodes removed ; there was no significant difference between the dfs of the three groups which had 6 or more lymph nodes removed ( figure 1 ) . in grouping b constructed based on this data , dfs of patients with 15 lymph node(s ) removed was significantly worse than those with 6 or more lymph nodes removed ( p = 0.008 ) . in multivariate cox analysis , grouping b had independent prognostic value ( table 2 ) , whereas grouping a had not . in grouping a , lrfs was significantly worse in patients with 15 lymph node(s ) removed compared to patients with 69 ( p = 0.008 ) , 1015 ( p = 0.002 ) and 16 and more ( p = 0.009 ) lymph nodes removed ; there was no significant difference between the lrfs of the three groups which had 6 or more lymph nodes removed . according to grouping b , lrfs of patients with 15 lymph node(s ) removed was significantly worse than patients with 6 or more lymph nodes removed ( p = 0.001 ) . both groupings had independent prognostic value in cox analysis ( p = 0.005 and p < 0.001 , resp . ) . in terms of dmfs there was no significant difference between patient groups according to either grouping a or b. axillary recurrence was 9.4% ( 8/85 patients ) in patients who had 15 lymph node(s ) removed , while 0.9% ( 8/839 patients ) in patients who had 6 or more lymph nodes removed , and the difference was significant ( p < 0.001 ) . the median value of lnr was 0.143 ( range 0.0261.00 ) for the whole series . the most significant lnr threshold value separating patients in low- and high - risk groups in terms of dfs was 0.20 ( p < 0.001 ) ( figure 2 ) ; this ratio had independent prognostic significance in cox analysis ( p < 0.001 ) . when grouping b , which is based on the number of lymph nodes removed and has independent prognostic significance , was added to this analysis , its prognostic significance was lost ( p = 0.527 ) , while the significance of lnr persisted ( p < 0.001 ) . the most significant lnr threshold value separating patients in two risk groups in terms of lrfs was 0.30 ( p < 0.001 ) ( figure 3 ) ; this ratio had independent prognostic significance in cox analysis ( p < 0.001 ) ( table 3 ) . when groupings a and b were added to this analysis , their prognostic significance was lost ( p = 0.325 and p = 0.190 , resp . ) , while the significance of lnr persisted ( p = 0.004 and p = 0.006 , resp . ) . the most significant threshold value separating patients in two prognostic groups in terms of dmfs was 0.15 ( p < 0.001 ) ( figure 4 ) ; this ratio had independent prognostic significance in cox analysis ( p < 0.001 ) ( table 4 ) . as mentioned above , there was no significant relationship between the number of removed lymph nodes and survival based on any patient grouping for all three types of survival endpoint . dfs was significantly better in patients with 1 positive node compared to those with 3 positive nodes ; there was no significant difference between those with 1 and 2 positive node(s ) or 2 and 3 positive nodes . based on this , when patients were grouped as those with 1 positive node versus 2 - 3 positive nodes , dfs was significantly better for patients with 1 positive node ( p = 0.016 ) ; this grouping had independent prognostic significance in cox analysis ( p = 0.007 ) . there was no significant relationship between the number of metastatic lymph nodes and lrfs . dmfs of patients with 1 positive node was significantly better than those with 3 positive nodes ; there was no significant difference between those with 1 and 2 positive node(s ) or 2 and 3 positive nodes . based on this , when patients were grouped as those with 1 positive node versus 2 - 3 positive nodes , dmfs was significantly better for patients with 1 positive node ( p = 0.022 ) ( figure 5 ) ; this grouping had independent prognostic significance in cox analysis ( p = 0.017 ) . the most significant lnr threshold value to separate patients in low- and high - risk groups in terms of dfs was 0.15 ( p = 0.005 ) ; this ratio had independent prognostic significance in cox analysis ( p = 0.001 ) . when the grouping based on metastatic lymph node number ( 1 positive versus 2 - 3 positives ) was added to this analysis , its prognostic significance was lost ( p = 0.294 ) , while the prognostic value of lnr moved slightly outside of the limit of significance ( p = 0.064 ) . there was no significant lnr threshold value to separate patients in two prognostic groups in terms of lrfs . the most significant lnr threshold value to separate patients in two prognostic groups in terms of dmfs was 0.15 ( p < 0.001 ) ( figure 6 ) ; this ratio had independent prognostic significance in cox analysis ( p < 0.001 ) . when the grouping based on metastatic lymph node number ( 1 positive versus 2 - 3 positives ) was added to this analysis , its prognostic significance was lost ( p = 0.803 ) , while lnr continued to be a significant prognostic factor ( p = 0.010 ) . in our series composed of patients with breast carcinoma having 13 metastatic axillary lymph node(s ) , dfs and lrfs of patients with 15 lymph node(s ) removed from their axilla were significantly worse than those with 6 or more lymph nodes removed , and this grouping had independent prognostic significance for both types of survival outcomes . in patient groups with more than 5 lymph nodes removed , there was no significant relationship between the increased number of removed lymph nodes and dfs or lrfs . studies involving patients with 13 axillary lymph node metastasis indicate significantly better overall survival and disease - free survival with increasing number of lymph nodes removed . karlsson et al . determined that in node - positive patients who did not receive radiotherapy , locoregional recurrence decreased significantly with increasing number of nonmetastatic lymph nodes removed and indicated that patients with 13 positive nodes and less than 10 nonmetastatic lymph nodes removed are candidates for postmastectomy radiotherapy , since their 10 year cumulative locoregional recurrence incidence is over 20% . in a study by schaapveld et al . , overall survival was found to be significantly worse in patients with less than 10 lymph nodes removed compared to patients with 10 or more nodes removed ; however , since overall survival can be deceptive as it includes all deaths along with nonbreast cancer related deaths , relative survival ( the ratio of the overall survival and the expected survival ) was analyzed and the number of removed lymph nodes was shown to be nonsignificant . truong et al . did not observe a significant relationship between the number of lymph nodes removed and locoregional recurrence , distant metastasis , and overall survival rates . in our series , axillary recurrence rate was significantly higher in patients with 15 lymph node(s ) removed ( 9.4% ) compared to those with 6 or more nodes removed ( 0.9% ) . it is generally accepted that a sufficient axillary dissection to avoid leaving probable metastatic lymph nodes behind is necessary for surgical control of the disease in the axilla and for correct evaluation of the axillary status in node - positive patients . our study results confirm this approach : high rate of axillary recurrence in patients with few lymph nodes removed indicates that actually there may be more than three metastatic lymph nodes in the axilla ( n2 maybe n3 disease ) and that these may have been left in the axilla . in our study , the most significant lnr threshold value separating patients in low- and high - risk groups with significant survival difference was 0.20 for dfs , 0.30 for lrfs , and 0.15 for dmfs . when grouping b ( with patients grouped according to 15 lymph node(s ) removed versus 6 or more nodes removed ) which had independent prognostic significance was added to these cox analyses conducted for dfs and lrfs , its significance was lost , while the prognostic significance of lnr threshold values persisted . since lnr is a derivative of the number of metastatic lymph nodes and the number of lymph nodes removed , it should not be included in the cox analysis with one of these two parameters . the prognostic significance of lnr is superior to the number of lymph nodes removed for dfs and lrfs , and lnr has independent prognostic significance for dmfs while the number of removed lymph nodes has not ; thus , lnr can be used to separate patients with n1 disease in low- and high - risk groups regardless of the total number of lymph nodes removed . series of patients having 13 positive lymph node(s ) showed that patients with high lnr have worse survival compared to those with low lnr : truong et al . , in their series of patients having 139 node(s ) removed and not receiving radiotherapy , determined the most significant lnr threshold value for locoregional recurrence , distant metastasis , and overall survival as 0.25 , and recommended radiotherapy following mastectomy for patients with lnr > 0.25 . also , in another series of patients having 141 lymph node(s ) removed and not receiving radiotherapy , truong et al . indicated that in terms of locoregional recurrence prediction , lnr was more valuable than the number of metastatic lymph nodes and recommended postmastectomy radiotherapy for patients with lnr > 0.20 , since their 10-year locoregional recurrence risk was above 20% . yildirim and berberoglu , in their series of patients having at least 10 lymph nodes removed and not receiving radiotherapy , determined the most significant lnr threshold value for prediction of locoregional recurrence risk as 0.15 . in our study , different threshold values for most significant lnr for dfs , lrfs , and dmfs were identified . if the survival analyses of this study were performed for dfs only , the threshold value of 0.20 would have been higher than the most significant threshold value for dmfs ( 0.15 ) and 167 patients with lnr > 0.150.20 would have been classified as with low risk despite their high risk for distant metastasis . although in daily practice , it may be difficult to determine a different threshold value for each type of disease recurrence , its potential use in planning a more appropriate adjuvant therapy should be taken into consideration . yildirim and berberoglu in their series of all node - positive patients with at least 10 lymph nodes removed from the axilla , identified the optimum lnr threshold value as 0.15 for locoregional recurrence and 0.20 for distant metastasis and determined significantly higher disease recurrence rates in patients having a lnr above these thresholds . in our study , it is generally accepted that at least 10 lymph nodes need to be removed in order to classify nodal disease based on the number of metastatic lymph nodes according to tnm classification [ 8 , 12 , 35 , 36 ] . nodal disease classification can not be done in patients having less than 10 lymph nodes removed , while that was the case with some of the patients in our series . when at least 10 lymph nodes are removed from the axilla and metastasis is found in 13 of them , these patients are classified as with n1 disease by tnm classification , and thus are accepted as a homogenous group . however , our study results suggest that n1 disease does not constitute a homogenous prognostic group : when patients were grouped according to the number of metastatic lymph nodes , dfs and dmfs were significantly better in patients with 1 positive node compared to 2 - 3 positive nodes and this grouping had prognostic significance independent of age , menopausal status , tumor size , histological type , adjuvant chemotherapy , radiotherapy , and hormonal therapy . patients with lnr > 0.15 had significantly worse dfs and dmfs , and lnr had independent prognostic significance according to this threshold value in cox analysis ; when a grouping based on the number of metastatic lymph nodes was added to these analyses , its prognostic significance was lost , while the prognostic significance of lnr persisted though it moved a little outside of the limit of significance for dfs ( p = 0.064 ) . moreover , as also mentioned above , lnr should be included in the cox regression analysis alone , as it is derived from the number of removed and metastatic lymph nodes . according to these results , lnr , which is a more powerful prognostic factor than the number of metastatic lymph nodes , can be used to separate n1 disease patients having at least 10 lymph nodes removed into low- and high - risk prognostic groups for distant metastasis development , thus providing more detailed and useful prognostic information than tnm nodal disease classification . in patients having 10 or more lymph nodes removed , number of removed and metastatic lymph nodes and lnr did not have prognostic significance for lrfs . this result indirectly suggests that in n1 disease , removal of at least 10 lymph nodes may be sufficient to obtain locoregional control and removal of more lymph nodes may not be related to locoregional recurrence , and hence , n1 disease may be homogeneous in terms of locoregional control . irrespective of the number of lymph nodes removed from the axilla , lnr is a useful tool in separating breast carcinoma patients with 13 metastatic lymph node(s ) into low- and high - risk prognostic groups . thus , lnr may be useful to standardize adjuvant therapy for patients operated in hospitals that use different axillary dissection width and have different median number of removed lymph nodes as well as to draw reliably comparisons between treatment results coming from such different hospitals .

we evaluated the prognostic significance of lymph node ratio ( lnr ) , number of metastatic lymph nodes divided by number of removed nodes in 924 breast carcinoma patients with 13 metastatic axillary lymph node(s ) . the most significant lnr threshold value separating patients in low- and high - risk groups with significant survival difference was 0.20 for disease - free survival ( p < 0.001 ) , 0.30 for locoregional recurrence - free survival ( p < 0.001 ) , and 0.15 for distant metastasis - free survival ( p < 0.001 ) , and the patients with lower lnr had better survival . all three lnr threshold values had independent prognostic significance in cox analysis ( p < 0.001 for all three of them ) . in conclusion , lnr is a useful tool in separating breast carcinoma patients with 13 metastatic lymph node(s ) into low- and high - risk prognostic groups .

pubmed

the oh functionality is commonly activated by attachment of a good leaving group , e.g. , by conversion into an ester derivative of a strong acid ( tosylate , mesylate , triflate ) , usually employing mitsunobu or appel conditions.1 this classic methodology suffers from poor atom economy and creates large amounts of waste , thus rendering these processes inefficient and unattractive , although remarkable progress towards catalytic processes was made in the past decade.2 in nature , activation of hydroxy groups often proceeds through enzymatic phosphorylation by kinases using activated phosphate ( adenosine triphosphate , atp ) or sulfation catalyzed by mammalian sulfotransferases in conjunction with the respective cofactor , i.e. , 3phosphoadenosine5phosphosulfate ( paps).3 nonspecific bacterial arylsulfotransferases are also able to sulfate hydroxy groups by using organic sulfate donors.4 the requirement for molar equivalents of these cofactors in synthetic applications is problematic from a scaleup perspective . although enzymatic atp recycling is feasible with reasonable efficiency ( up to several hundred ttns),4a,5 paps can not be recycled efficiently so far.4 most importantly , atpdependent kinases and papsdependent sulfotransferases are typically involved in biosynthetic pathways and signal messaging and thus are often highly substratespecific and hence do not allow broad synthetic applications . an efficient alternative to kinases is the use of atpindependent phosphatases using cheap inorganic polyphosphates ( pyrophosphate , ppi ; triphosphate , pppi ; polyphosphoric acid , polyp ) as activated phosphate donor ( pdonor ) , which circumvents the need for cofactor recycling.6 additionally , due to their occurrence in biodegradation pathways , phosphatases usually display a broad substrate spectrum , and inorganic phosphate formed as byproduct is environmentally benign . a major drawback resides in the reversibility of phosphate transfer , causing the undesired hydrolysis of the newly formed phosphate ester , which requires a kinetically tightly controlled system ( figure 1 ) . mechanism of ( a ) competing hydrolysis and ( b ) transphosphorylation involving a covalent phosphoenzyme intermediate catalyzed by phonse.26 the use of phosphatases for synthetic applications dates back to the late 1940s , when axelrod and appleyard achieved transphosphorylation using phosphataseenriched extracts and pnitrophenyl phosphate ( pnpp ) or phenolphthalein phosphate as donor.7 later , nordlie identified glucosepyrophosphotransferase activity of e. coli alkaline phosphatase;8 however , the method was applied on a preparativescale only decades later.9 asano et al . applied acid phosphatases from enteric bacteria to the phosphorylation of nucleosides and designed enzyme mutants with reduced hydrolytic activity.10 acid phosphatases from shigella flexneri ( phonsf ) and salmonella enterica ser . typhimurium lt2 ( phonse ) were found to have a broad substrate spectrum11 and were applied in combination with aldolases to synthesize natural and unnatural sugar molecules on a gramscale.12 in drug discovery , phosphorylation is a common prodrug strategy to enhance the accessibility of drug candidates within the body by enhancing their polarity and solubility.13 in some cases , the phosphorylated form may be the active species.14 in synthetic chemistry , phosphate di and triesters are widely applied as brnsted acid catalysts for various asymmetric transformations15 and as leaving groups in transitionmetalcatalyzed coupling reactions16 as well as in organolithium17 and alkene synthesis.18 in contrast , phosphate monoesters have attracted so far little attention as tools for functionalgroup modification in classic chemical processes , most likely as a consequence of their insolubility in organic solvents resulting from their negative charge . current chemical routes to phosphate monoesters often proceed via the corresponding phosphate di or triesters and require harsh reagents and conditions leading to hardly separable product mixtures . in this study , we aimed to evaluate a set of phosphatases as potential candidates for synthetic applications . various techniques were investigated to enhance product titers of phosphorylated alcohols and to minimize the thermodynamically favored hydrolytic reaction . enzyme candidates were selected from the family of acid phosphatases , which have been shown to transfer efficiently a phosphate moiety from a highenergy inorganic di / tri / polyphosphate onto various hydroxylated compounds . nonspecific acid phosphatase phonse from salmonella typhimurium and acid phosphatase phonsf from shigella flexneri have been reported to convert various primary and secondary alcohols and sugars11 and were implemented in cascade reactions involving glucose6phosphate19 and dihydroxyacetone phosphate as intermediates.12 acid phosphatase phytase catalyzes the hydrolysis of phytate , a phosphorylated natural cyclitol by liberating inorganic phosphate and is therefore commonly used as animal feedstock additive.20 acid phosphatase aphast from salmonella typhimurium lt221 was selected due to its phosphotransferase activity on alcohols using pnpp as donor.22 piacp from prevotella intermedia 23 was selected based on high sequence similarity with robust phocmm developed for nucleoside phosphorylation.24 all enzymes except phytase were overexpressed in e. coli and subsequently purified to homogeneity by using histag or streptag techniques ( figure s1 ) . a common mechanistic feature of phosphatetransferring phosphatases is the formation of a covalent phosphoenzyme intermediate , which undergoes either nucleophilic attack by water ( hydrolysis ) or accepts a suitable nucleophile ( transphosphorylation ) ( figure 1 ) . to this end , most enzymes use histidine as catalytic residue ( phonsf , phonse , piacp and phytase ) except aphast which uses aspartic acid.25 due to the competition between hydrolysis and transphosphorylation in aqueous medium , high substrate loads are required to achieve high product titers , while kinetic control is crucial to keep the phosphorylation / hydrolysis ratio as high as possible . in order to verify the activity of all recombinant proteins , a spectrophotometric assay based on the hydrolysis of pnpp was used . a range of alcohols was investigated , including primary and secondary mono and dihydroxy compounds , as well as cyclic substrates ( table 1 ) . although phytase has been successfully employed in a cascade reaction to convert glycerol into glycerol1phosphate in a nonstereoselective fashion,27 under our assay conditions , the enzyme showed no transphosphorylation activity at ph = 2.5 and 4.2 on any substrate . strong hydrolytic activity on inorganic phosphate donors was still observed , confirming active protein ( data not shown ) . aphast also contains mg ( in addition to the catalytically active nucleophile aspartate21 ) , whose activity is affected by inorganic polyphosphates by metal chelation . although the addition of 10 mm mgcl2 could partly restore the hydrolytic activity on pnpp in the presence of 250 mm ppi ( figure s2 ) , only traces of product and marginal hydrolytic activity were obtained in the transphosphorylation of 1,4butanediol ( 500 mm ) by using 100 mm ppi as donor at ph = 4.2 and 5.5 , independent of the mgcl2 concentration ( 10 , 50 , 100 mm ) ( data not shown ) . this suggests that inorganic oligophosphates ( as opposed to pnpp ) are not ideal phosphate donors for metalcontaining phosphatases , such as aphast . on the contrary , phonsf , phonse and piacp all proved to be efficient catalysts in the phosphorylation of various alcohols ( table 1 ) . substrate scope of phonsf , phonse and piacp in transphosphorylation.[a ] , [ b ] , [ c ] , [ d ] , [ e ] , [ f ] , [ g ] reaction conditions : ph = 4.2 , 50 g ml enzyme , 1 ml of volume , 1 % dmso as internal standard , 30 c , 600 rpm shaking . product levels were determined by consumption of substrate using hplc with ri detector ( control experiments used as reference ) . phosphorylation of primdiols proceeded to various degrees depending on the chain length : while shortchain ethylene glycol ( 1 ) was selectively monophosphorylated , 1,4butanediol ( 2 ) and the unsaturated ( z ) analog 4 yielded the corresponding mono / bisphosphorylated products in a ratio of 90:10 . extending the chain length even further ( 1,6hexanediol , 3 ) gave 85:15 of mono / diphosphate as determined by lcri chromatography ( see supporting information ) . it is obvious , that the presence of a ( negatively charged ) phosphate group in the proximity of the reacting hydroxy moiety determines whether an ,diol undergoes bisphosphorylation : while shortchain ethylene glycol is exclusively monophosphorylated , longchain 1,6hexanediol yields the bisphosphate in up to 15 % . haloalcohol 5 was also converted : phonse tolerated higher substrate concentrations than phonsf and piacp ( 500 mm vs. 300 mm ) resulting in the formation of 113 mm product in 60 min . to probe the chemoselectivity of the enzymes for o vs. nnucleophiles , 6amino1hexanol ( 6 ) the three enzymes exclusively furnished the ophosphate monoester at very similar product levels ( ca . this was proven by p nmr spectroscopy resulting in a chemical shift typical for signals of phosphorylated primary hydroxy groups ( figure s5).28 in line with this observation , 1,6diaminohexane ( 12 ) was not converted , only pdonor hydrolysis was observed ( data not shown ) , proving that these enzymes do not accept amines in place of alcohols . sterically demanding secondary alcohol 7 was transformed only with phonse , which is in line with the previously observed preference of this enzyme for secalcohols.11 similar observations were made with 1,2propanediol ( 8) , which was exclusively phosphorylated at the primalcohol by phonsf and piacp , while phonse showed reduced selectivity ( ca . 90:10 prim / secphosphate , determined by p nmr spectroscopy ; figure s6 ) . in order to elucidate the enantioselectivity of phosphatases , the two enantiomers of 8 were tested separately . in case of phonsf and piacp , similar activities on both enantiomers were observed indicating nonenantioselective phosphorylation . with phonse , a marginal enantiopreference for ( r)8 was detected ( figure s7 ) . to further investigate the enantioselectivity of phonse , both enantiomers of 2butanol ( 9 ) were subjected to screening conditions ; however , no significant difference in the reaction course was observed . furthermore , racemic cyclic transdiol 10 was tested , and beside a moderate amount of monophosphorylated product , no stereoselectivity could be observed ( as determined by measurement of the ee value of remaining 10 by gc after derivatization of both oh functionalities ; see supporting information ) . substrates containing n or snucleophiles or charged groups were not converted ( figure 2 ) , while ppi was still hydrolyzed indicating that the enzymes were still active ( data not shown ) . the absolute chemoselectivity of ophosphorylation vs. n and snucleophiles was corroborated by the nonacceptance of substrates 11 and 12 . mevalonolactone ( rac13 ) and charged substrates , like lactic ( rac14 ) and tartaric acid ( 15 ) ( both enantiomers tested separately ) were not converted . although ophosphod , lserine could be stereoselectively hydrolyzed by a mutant of phonse,29 the wildtype enzyme was not active in the phosphorylation of racserine ( 16 ) . substrates not converted by phonsf , phonse , piacp and phytase [ 300 mm substrate concentration except for rac14 ( 200 mm ) and 17 ( 15 mm ) ] . due to the reversibility of the reaction ( figure 1 ) , kinetic control of the reaction is necessary to ensure optimal product titers by avoiding product depletion by ( reverse ) hydrolysis and to ensure optimal product titers . this may be achieved by adjustment of reaction parameters such as ph or selection of oligophosphate donor.30 therefore , 1,4butanediol ( 2 ) was selected as a model substrate , and enzymes were tested with tri and polyphosphate as alternative to ppi ( table 1 and figures s810 ) . the amount of 4hydroxybutyl phosphate formed could be significantly increased by replacing ppi by high amounts of pppi and polyp in the reaction catalyzed by phonsf and piacp , allowing up to 61 % conversion ( 305 mm product ) with respect to the theoretical maximum . the difference in the slope in product disappearance clearly shows that product hydrolysis is decreased in the presence of tri and polyphosphate compared to ppi . in contrast , these donors had a negative influence on the performance of phonse , where a product maximum was obtained with ppi . moreover , hplc monitoring of the reaction indicated incomplete consumption of polyphosphoric acid . this suggests that this enzyme accepts only short chain oligophosphates as reported for calf intestine alkaline phosphatase.9 since phonsf generally delivered the highest product titers with primary alcohols as acceptors , the enzyme was selected for further investigations with 1,4butanediol concerning the effect of ph ( range 3.84.8 ) by using different pdonors ( figure 3 ) . on the other hand , pppi and polyp usually yielded higher product concentrations at lower ph , due to slower product hydrolysis . however , the poorly defined oligomeric composition of the polyphosphate donor renders precise analysis as well as product isolation challenging , and polyp was not investigated further . effect of pdonor type and ph on the phosphorylation of 2 ( product formation over time ) . conditions : 500 mm 2 , 250 mm ppi or pppi or 100 g l polyp , 50 g ml phonsf , 1 ml volume , 1 % dmso as internal standard , 30 c , 600 rpm shaking . the results depicted in figure 3 using phonsf suggest that a high number of transferable highenergy phosphate moieties as well as low ph values are crucial to achieve and maintain high product titers . inspired by this observation , reactions were conducted at variuos ph values using high amounts of ppi and pppi overnight ( table 2 ) . it should be noted that within the incubation time of 16.5 h both transphosphorylation and hydrolysis of product and phosphate donor will occur , and the balance between the two processes is determined by the activity of the enzymes and the ph . although identical amounts of transferable phosphate moieties are available from 500 mm ppi and 250 mm pppi , significant differences in product formation were obtained after 16.5 h reaction time . using 500 mm ppi as a donor , product yields of 2 for phonsf and piacp were similar at the three ph values . using ppi , up to 292 mm product ( up to 58 % conversion ) could be obtained accompanied by nearly total consumption of ppi ( > 90 % ) , and the product / pi ratio was ca . 1:2 ( the ideal ratio is 1:1 in the absence of product hydrolysis and complete phosphate transfer ) . however , under these conditions phonse furnished only inorganic monophosphate , indicating total hydrolysis of ppi and product . in the presence of 250 mm pppi the picture changed : the transphosporylation activity at ph = 3.5 of phonsf dropped considerably , that of piacp also dropped , but surprisingly considerable phosphorylating activity of phonse was maintained ( 164 mm , 33 % conversion and 54 % pdonor consumption ) . at higher ph values this enzyme was still active , but only hydrolysis was observed , while phonsf and piacp delivered moderate to good amounts of product at full consumption of the pdonor . at 400 mm pppi and ph = 4.2 both phonsf and piacp showed the highest phosphorylating activity resulting in up to 393 mm product ( 79 % conversion , product / pi ratio ca . 4:5 ) at 75 % pdonor consumption . amount of product and pi formed in acid phosphatase catalyzed phosphorylation of 2 with ppi or pppi as donor , at three ph values with phonsf , piacp and phons . reaction conditions : 500 mm 2 , phosphate donor as indicated , 50 g ml phonsf , ph as indicated , 1 ml volume , 1 % dmso as internal standard , 30 c , 600 rpm shaking , 16.5 h. ideal ( maximum obtainable ) product / pi ratio is 1:1 with ppi and 2:1 with pppi . cons.donor : consumption of pdonor calculated as follows : ( c prod + c pi)/c theoretical max . , higher pppi concentrations translate into higher product concentration and lower pi concentration , indicating more efficient phosphate transfer due to reduced product hydrolysis . from the data it is clear that using these phosphorylating enzymes in synthesis effectively requires a careful study of the donor concentrations , the type of donor , and the ph ; in batch processes also the time of incubation should be carefully chosen . a long incubation time will result in low yields or near complete hydrolysis of the phosphorylated product . consequently , kinetic control of the phosphorylation is very important , and physical separation of the phosphatase by employing a continuous flowreactor has proven to be an efficient tool to maintain high product levels ( by minimizing hydrolysis ) and scaleup the reaction.12c,31 under optimized conditions , the contact time between enzyme and substrate(s ) is controlled by the flow rate to ensure that ( rapid ) phosphorylation is complete , but ( slow ) product hydrolysis can not take place after the product has left the enzymecontaining column reactor . we evaluated several epoxy and aminofunctionalized polymeric beads for the covalent immobilization of phonsf and piacp ( table s3 ) . relizyme ha403/m , an aminofunctionalized resin , resulted in the highest specific activity for both enzymes ( 159 and 215 u g dry beads for phonsf and piacp , respectively ) . phonsf ( 300 u ) immobilized on relizyme ha ( 300 mg ) was applied to the preparativescale phosphorylation of 2 , yielding 6.86 g ( 22.5 mmol ) of the corresponding barium salt of 4hydroxybutyl phosphate ( 41 % isolated yield , for details see supporting information ) . the enzymatic phosphorylation of primary and secondary alcohols in aqueous medium using cheap inorganic di , tri and polyphosphate as pdonors catalyzed by three acid phosphatases proceeded with absolute chemoselectivity for o vs. n and snucleophiles and with high regioselectivity for prim over sechydroxy groups . the use of mild reaction conditions allowed the formation of phosphate monoesters as sole products , yielding environmentally innocuous inorganic monophosphate and unreacted phosphate donor as byproducts . the thermodynamically favored hydrolysis of phosphate esters could be minimized by controlling various reaction parameters , thus allowing the use of phosphatases in the synthetic direction . scaleup was demonstrated by the synthesis of 4hydroxybutyl phosphate ( 6.86 g , 41 % yield ) using an immobilized enzyme in flow mode .

abstracta set of phosphatases was evaluated for their potential to catalyze the regio and stereoselective phosphorylation of alcohols using a highenergy inorganic phosphate donor , such as di , tri and polyphosphate . parameters such as type and amount of phosphate donor and ph of the reaction were investigated in order to minimize the thermodynamically favored hydrolysis of the phosphate donor and the formed phosphate ester . diols were monophosphorylated with high selectivities . this biocatalytic phosphorylation method provides selectively activated and/or protected synthetic intermediates for further chemical and/or enzymatic transformations and is applicable to a large scale ( 6.86 g ) in a flow setup with immobilized phosphatase .

pubmed

various non - native biological invaders have been noted for centuries , such as the phylloxera ( a homopteran ) that destroyed grapevines and devastated the wine industry in europe in the 1860s and the chestnut blight that entered north america in the early 20th century and virtually eliminated a dominant tree species . by 1957 , charles elton produced a series of bbc radio broadcasts about the general phenomenon of non - native invasions and transformed the series into a book for a lay audience the next year . although particular invasions continued to attract great attention , elton 's book did not inspire a broad research thrust on invasions or a widespread public and policy response to their growing number . rather , in the mid-1980s , an international project of the scientific committee on problems of the environment ( scope ) engaged over 100 prominent scientists worldwide , produced several widely read edited volumes ( for example , ) , and triggered an explosion of research on invasions , which soon came to be recognized as one of the great ongoing global environmental changes , along with climate , nutrient cycle enhancement , and land - use changes . this surge of research included many studies of how particular non - native invaders eat native species ( for example , the brown tree snake in guam and the hemlock woolly adelgid in the eastern us ) , compete with them ( for example , the gray squirrel in great britain , crown vetch and tall fescue in the us midwest , and the killer alga in the mediterranean ) , or infect them ( for example , whirling disease of rainbow trout in the us ) and sometimes compete with them while vectoring diseases to them ( for example , signal crayfish in europe and the gray squirrel in great britain ) . however , two new lines of research have become increasingly prominent , both indicating that impacts of introduced species are more varied and widespread than had previously been thought . first , in contributions to the scope program , peter vitousek described a different sort of impact . rather than focusing on the population level ( for example , as when the gray squirrel competes with the red squirrel in great britain and transmits squirrel pox ) or the community level ( affecting many species in the community , as when the brown tree snake eats all of the forest bird species in guam ) , he depicted impacts at the ecosystem level : invasions that affected nutrient cycles and other ecosystem - level processes and features ( such as fire cycles or hydrology ) . research on such impacts in a variety of ecosystems has increased greatly , particularly in the new century , showing how a single introduced species can change processes or the physical structure of an ecosystem , or both , sometimes subtly and slowly , but in such a way as to affect a large fraction of the resident species , as in the study by fukami et al . on impacts the other major new direction of invasion biology is a greatly increased focus on genetics and evolution as increasingly sophisticated and accessible molecular genetic techniques become available . molecular genetic research has increasingly revealed a great number of cases of the stealth impact of invaders , hybridization , and introgression with native species ( for example , ) . the phenomenon had previously been detected almost exclusively phenotypically , but many phenotypic responses are sufficiently subtle that they are hidden amidst the normal variation of many natural populations . it is now recognized that substantial introgression can occur even between native species that never hybridize when a newly introduced species constitutes a genetic bridge between them . introgression can also cause substantial evolution of native species , as in the european wildcat , which hybridizes with feral domestic housecats . hybridization between native and introduced species can even produce new , more invasive species ; the best - known example is common cordgrass , spartina anglica , an invader that arose in england from hybridization between native s. maritima and north american s. alterniflora . in addition to showing the great extent of hybridization between introduced and native species , molecular genetic features such as microsatellites , mitochondrial dna , and single - nucleotide polymorphisms have increasingly been used to trace sources and routes of invasions ( for example , ) . a major finding is that introduced populations often combine genes derived from multiple source populations , thus producing novel genetic combinations ( for example , ) . in fact , some introduced populations arising from multiple sources have more genetic variation than any single source population ( for example , ) . also , environmental dna ( edna ) is increasingly used to detect invaders and played a key role in showing the great advance of asian carp toward the great lakes . the burst of research in the wake of the scope project also revealed an increasing number of cases of invasional meltdown , in which two or more introduced species facilitate one another 's establishment or enhance one another 's impact on native species , as when the honeydew from introduced scale insects increases introduced ant populations or when introduced birds disperse seeds of introduced plants . in addition , an increasing number of lag effects have been detected , in which full - fledged widespread establishment and impact of an introduced species lags for years behind initial arrival . the reasons for such lags are various , and some remain mysterious , but the commonness of the phenomenon can not be doubted . the many threads of 25 years of increasingly intensive research on biological invasions all lead to the conclusion that problems caused by the phenomenon are numerous , often tremendously important ecologically or economically worldwide , and growing apace with globalization . amidst this plethora of publications , a small but persistent group of critics has assailed the field of invasion biology and much of the invasion management effort on a variety of grounds , eliciting a spirited response . some critics go so far as to call for an end to invasion biology as a scientific discipline [ 3739 ] . the objections fall primarily into five categories : ( 1 ) the problems are overblown , and similar impacts arise from native species ; most introduced species are not problematic . ( 2 ) in some instances , introduced species increase native biodiversity . ( 3 ) the antipathy toward introduced species is xenophobic . ( 4 ) in the face of globalization , we can not substantially slow the spread of invasions ; it is a hopeless battle . ( 5 ) for sentient animals , such as mammals , the methods used to combat invasive species are inhumane ; in any event , individuals of the targeted species have a right to exist . criticism 5 , which is part of a philosophical dispute that is not near resolution , revolves around the issue of the relative weight of rights of individuals and rights of collective entities such as species . consideration of criticism 2which is based on the fact that , in some local biotas , the number of established introduced species exceeds the number of native species locally eliminated ( whether because of introduced species or not)should be tempered by the fact that many complete species extinctions have been wholly or partly caused by introduced species , especially but not exclusively on islands . in addition , in many instances , species populations , though not eliminated , are reduced to a small fraction of their previous numbers . biodiversity in terms of species numbers is not lost , but there is a major functional and demographic change . criticism 3 is an assertion , generally leveled as part of a social construction of the science of invasion biology and management with scant or no regard for the impacts of invasions that are the stated purpose of the disputed activities . the key criticisms from an ecological standpoint are 1 and 4 : which introduced species really cause harm , and can we really do anything about it if they do ? however , modern invasion biology is very new , and the great majority of introduced species have not been studied in their introduced ranges , and so it is not possible to say that they have no significant impact . perhaps the most comprehensive study of this matter found , for europe , that for only ca . 10% of some 11,000 introduced species has impact been documented , but that this is likely to underestimate the number actually causing impacts because of the dearth of research . one of the major recent research thrusts of invasion biology , as noted above , is unraveling various ecosystem impacts , some of which are initially subtle and take a long time to be manifested ( for example , shifts in nutrient cycling ) . . also found that , of introduced terrestrial vertebrates and freshwater plants and animals , about 30% are known to have harmful impacts . in addition , the lag effect noted above suggests that it would be foolhardy to wait for an impact to be recognized before acting . by then , the invader may well have spread beyond the point at which eradication or containment is possible . furthermore , although some native species in certain circumstances ( almost always associated with some other anthropogenic environmental change ) do cause impacts of the sort frequently associated with introduced invaders , the relative frequency of such impacts is far less than for non - native species . meanwhile , there is growing recognition of the great extent and variety of impacts of introduced species . the lag effect , the subtlety of some impacts that have been occurring from the outset of the introduction , and the greater propensity for non - native species to cause harm mean that the suggestion to manage invasions only after impact is evident is ill advised . attempts to identify which species , if introduced , would be likely to become invasive have had some success ( for example , ) , inspiring the construction of several risk assessment tools , especially for plants . the most prominent is the australian weed risk assessment ( awra ) , which has been refined and adapted for use in permitting introductions in many other regions . none of these tools is foolproof ( for example , the awra fails to reject about 10% of known invasive plants in australia ) , and the multiplicity of factors ( including chance ) that affect whether an introduced population survives , spreads , and becomes invasive will certainly limit their accuracy . nevertheless , they have been valuable in preventing some planned introductions that would likely have led to invasions , and the same tools can be used to identify recently introduced , restricted populations that should be prioritized for rapid attempts at eradication or containment . the effort to prevent invasions or to reverse or mitigate their impacts is not hopeless . biosecurity can be very effective , as evidenced by great improvements in new zealand as interdiction procedures became more stringent ( for example , ) . similarly , the technology exists for greatly improved early detection and rapid response ; employing them is a matter of political will . once a population is established , it is possible in some cases to eradicate it , and the technology to do so has improved greatly . increasingly ambitious eradication projects are being undertaken , and they are marked by striking successes ; the current frontiers for vertebrate eradications are attempts to eradicate multiple species simultaneously , attempts to eradicate invasive species on larger islands and even continents , and attempts to eradicate invasive species on islands inhabited by humans ( unpublished data ) . terrestrial plants , insects , and aquatic organisms have been the most refractory targets for eradication . however , there have been striking eradication successes even for these taxa when populations have not yet spread widely beyond the initial introduction site ( for example , ) . when eradication fails or is not attempted , many technologies can be employed to maintain populations of invaders at low densities . the main , widely applied approaches are physical control ( such as pulling weeds by hand ) , mechanical control ( use of machinery ) , chemical control ( use of herbicides and pesticides ) , and biological control ( introduction of natural enemies of a target invader , from its native range ) . each of these methods has succeeded in many instances and failed in others , and sometimes these methods have had undesirable non - target impacts . however , incremental improvements in techniques for all of these methods are leading to higher success rates and fewer non - target impacts . for instance , sterile males have been used in many maintenance management and even eradication programs for insects , especially flies , and pheromones have been used to disrupt lepidopteran mating and to trap sea lampreys . efficacy of the judas - animal technique , widely used in mammal control and eradication programs , has recently been greatly improved by the use of hormone injections to prolong estrus . autocidal methods using genetic manipulation were proposed in the 1960s and 1970s but not implemented then . today , with the advent of various genetic manipulation techniques , including transgenes , several such projects have advanced to the proof - of - concept stage , particularly for dipterans and fishes , and a similar approach has been proposed for plants . in sum , it is simply untrue that it is futile to fight invasions . a simultaneous and related call for the end of traditional restoration ecology [ 6569 ] as part of a new world order accompanied the calls for an end to invasion biology . this is perhaps unsurprising , as a very large component of restoration ecology consists of eliminating or managing non - native invasive species , and the underlying argument of the advocates of abandoning traditional restoration ecology in favor of novel ecosystems has been that the overwhelming forces of invasions by non - native species and climate change have rendered obsolete the entire notion of returning an ecosystem to some historical state . among prominent advocates of promoting novel ecosystems and abandoning traditional restoration ecology are several of the critics of invasion biology and management , and authors of articles for lay audiences , such as emma marris and fred pearce , have vigorously promoted both agendas . the contention that novel ecosystems are so inevitable as to constitute the new normal is incorrect on several grounds . for one thing , novel ecosystem is undefined , at least in operational terms . in no instance is it stated what specific criteria show that an ecosystem is now novel or what thresholds it has passed to achieve this status . all ecosystems are constantly changing , even without anthropogenic influences [ 7781 ] , leading pickett et al . to propose the metaphor of the flux of nature to replace balance of nature . contrary to claims by advocates of fostering and molding novel ecosystems , modern restoration ecology does not attempt to recreate the past ; rather , the goal is to re - establish the historical trajectory of an ecosystem before anthropogenic influences derailed it . this goal has been endorsed by the united nations convention on biological diversity , the european commission , and major non - governmental organizations such as the international union for the conservation of nature . another problem with the rush to embrace novel ecosystems as a goal is that the sense that much of the earth is rapidly changing to some sort of novel condition is based partly on maps , such as the human global footprint , drawn at far too coarse a scale . the field of restoration ecology is very young and has learned much in a short time from past mistakes . meta - analyses show considerable progress in achieving restoration goals in a variety of ecosystem types , and impressive specific cases abound . there is no evidence that any particular ecosystem can not be restored in the sense of modern restoration ecology ; the impediments are economic and political , not scientific and technological . this does not imply that society should undertake to restore every ecosystem ; each case should be considered by all stakeholders in terms of desirability , cost , and resource availability . policymakers and the general public whose interests they should represent can easily be misled by what they perceive to be the existence of a scientific controversy , and policymakers have used such the drumbeat of claims that the entire enterprise of modern ecological restoration is futile can perniciously influence policy at a time when many nations are undertaking ambitious restoration projects , and the united nations , the three rio conventions , and many governments and large non - governmental organizations have all called for greatly expanded restoration efforts . at the least , the contention that novel ecosystems are inevitable and perhaps desirable encourages any tendency to delay prevention and redress of various harmful environmental impacts rather than to undertake new approaches and to devote new resources to long - term solutions to environmental problems . the same inimical influences on policy are exerted by critics of invasion biology and management . for instance , larson and palmer , in a policy - oriented journal , not only cite emerging thinking about novel ecosystems without mention of any of the weaknesses of the concept but cite davis et al . and schlaepfer et al . as calling into question the damage caused by invasive species without mentioning that these authors represent a small minority of scientists or citing specific rebuttals of those articles . critics of invasion biology and restoration ecology are not likely to keep these vibrant growing fields from progressing scientifically , but they will be able to slow the transformation of scientific knowledge into actions on the ground .

invasions by non - native species have caused many extinctions and greatly modified many ecosystems and are among the major anthropogenic global changes transforming the earth . beginning in the mid-1980s , a dramatic burst of research in invasion biology has revealed a plethora of previously unrecognized impacts and laid bare the scope of the phenomenon . similarly , research on various methods of managing invasions has expanded enormously , yielding incremental improvements in traditional methods and the advent of several new approaches , including the use of species - specific genetic and pheromonal methods . this research has advanced the field of restoration ecology , of which invasion management is a key component . amidst this research progress , a group of critics has attempted to cast doubt on the extent of damaging impacts caused by non - native invasive species , the feasibility of counteracting them and restoring ecosystems , and the motives of scientists engaged in such endeavors . the criticisms are misguided but can potentially impede management of this pressing problem .

pubmed

schwannomas are rare tumors of the nerve sheath that arise from the primary glial cells of the peripheral nervous system . as such although benign and slow growing , schwannomas can cause significant morbidity due to direct involvement of nerves or by local mass effect that can pose particular challenges for treatment . approximately 25 to 45% of all schwannomas are found in the head and neck , where they can originate from virtually any cranial nerve.1 the most common schwannoma is the vestibular schwannoma that arises from the vestibular branch of the eighth cranial nerve and accounts for 51% of all nerve sheath tumors found anywhere in the body.2 this is followed by schwannomas of the trigeminal nerve that comprise up to 8% of all schwannomas.3 by contrast , schwannomas in the nasal cavity or paranasal sinuses and vidian canal schwannomas in particular are much rarer . the vidian canal , also referred to as the pterygoid canal , extends from the pterygopalatine fossa to the foramen lacerum in the floor of the sphenoid sinus4 and contains the vidian artery as well as the vidian nerve . the vidian nerve arises from the joining of the greater superficial petrosal nerve and the deep petrosal nerve . the greater superficial petrosal nerve consists of preganglionic parasympathetic fibers from the geniculate ganglion of the facial nerve . the deep petrosal nerve consists of postganglionic sympathetic fibers from the carotid sympathetic plexus.5 6 7 8 the parasympathetic fibers of the vidian nerve synapse in the pterygopalatine ganglion , whereas the sympathetic fibers innervate the lacrimal gland and secretory pathways of the nasopalatine glands.5 6 schwannomas of the vidian canal can present with a variety of clinical symptoms . damage to the vidian nerve itself can result in decreased or absent lacrimation , corneal desiccation as well as dryness of the nasal mucosa , and cluster headaches.5 additionally , vidian schwannomas have been reported to compress the adjacent internal carotid artery or bulge through the lateral wall of the sphenoid sinus to expand into the cavernous sinus.6 9 to date , only 4 cases of vidian nerve schwannomas and 11 cases of histologically unconfirmed peri - vidian tumors have been reported in the literature.10 11 12 13 14 15 here we present a case of biopsy - proven schwannoma of the vidian canal . we discuss the patient 's initial presentation , imaging characteristics , histologic findings , and treatment for this unusual tumor and review the other reported cases of vidian canal schwannomas in the literature . the patient is a 49-year - old woman with a 24-year history of multiple sclerosis ( ms ) who presented for follow - up evaluation after an acute flare of ms . in the past , her ms flares had been relatively infrequent , with motor and/or peripheral sensory deficits that were well controlled with short - term courses of steroids . during her most recent flare , she experienced numbness of her left head , arm , and leg , with full resolution of symptoms after steroid treatment . a routine surveillance brain magnetic resonance imaging ( mri ) was performed to evaluate for ms progression . mri showed minimal progression of disease compared with imaging from 8 years prior , with extensive t2 , fluid - attenuated inversion recovery , and proton - density prolonged signal in the periventricular , pericallosal , and subcortical white matter consistent with ms . incidentally , the mri also revealed a 19 13 10 mm ovoid mass along the inferior margin of the right cavernous sinus that was not present on previous mris . the lesion was well circumscribed and homogeneously enhancing , with t1 hypointense and t2 hyperintense signal . a repeat dedicated mri was performed that demonstrated the lesion originated within an expanded vidian canal ( fig . a sinus computed tomography scan also showed bony erosion of an expanded vidian canal ( fig . the patient noted some mild nasal congestion in the mornings but was otherwise asymptomatic without any facial pain or paresthesias , abnormal lacrimation , or dry eye symptoms . physical examination was similarly unremarkable with a normal nasal endoscopy and no focal neurologic deficits . the mass was easily identified within a noticeably expanded vidian canal along the lateral floor of the sphenoid sinus ( fig . the canal was opened along the floor of the sphenoid sinus to uncover the mass and a biopsy was obtained . the patient 's recovery was unremarkable , and she had no problems with dry eye or nasal irritation postoperatively . two weeks after surgery , her endoscopic examination showed a well - healed sinus cavity . an ovoid 14 20 13-mm mass ( white arrows ) centered in an enlarged right vidian canal and extending along the inferior margin of the right cavernous sinus . the mass has ( a ) bright t1 signal post contrast , coronal view , and ( b ) bright t2 signal with fat saturation . preoperative sinus computed tomography scan without contrast demonstrating bony erosion of the right vidian canal ( red arrow ) . the mass ( black arrow ) is visible protruding from the right vidian canal , located at the floor of the sphenoid sinus . options of endoscopic surgical resection , single - fraction stereotactic radiosurgery ( srs ) , and conventional fractionated radiotherapy using stereotactic radiotherapy ( srt ) were reviewed with the patient , and she decided to pursue treatment with radiation . given the benign histology , a background of ms that could confer additional sensitivity to radiation , and the patient 's young age , the patient decided to pursue srt to minimize late normal tissue injury risks . schwannomas of the vidian canal are an extremely rare type of intracranial tumor with variable presentation . symptoms of vidian schwannomas can result from compression of surrounding structures or extension into surrounding areas that can cause headaches , facial pain and paresthesias , and facial muscle paralysis . dysfunction of the vidian nerve itself may cause nasal dryness , decreased lacrimation , and corneal desiccation . here we present an unusual case of a vidian nerve schwannoma incidentally identified in a patient who was asymptomatic from the lesion . to date , four documented cases of biopsy - proven vidian nerve schwannomas have appeared in the literature . reported cases include both male and female patients , ranging in age from 13 to 78 years , with both unilateral and bilateral lesions described.10 11 12 13 14 15 presenting symptoms included headache and facial palsy , oculomotor palsy and cerebrospinal fluid leak , unilateral hearing loss secondary to obstructive serous otitis media , and facial paresthesias . complete surgical resection was pursued for all four cases through both open and endonasal approaches , although one patient with bilateral vidian nerve schwannomas had only partial resection of one lesion due to difficulties with surgical access.13 all patients had significantly improved or stable symptoms , and the two patients with the most severe symptoms one with oculomotor palsy and ptosis and the other with headache and facial nerve palsy demonstrated the most dramatic postoperative improvement with near to complete resolution of symptoms.10 13 consistent with these cases , the traditional and curative management of peripheral nerve tumors is surgical ; recent advancements in transnasal endoscopic skull base approaches frequently enable complete resection . radiation therapy by either srs or multiple fraction radiotherapy is also an alternative option for treatment . conservative management through observation with serial imaging is also reasonable when surgical resection is either not feasible or involves a disproportionate risk of postsurgical morbidity and/or mortality . the choice of treatment modality depends on patient choice , functional status , presenting symptoms , tumor location , tumor volume , and in rare instances malignant potential . the ultimate goal of both surgery and radiotherapy is to maximize tumor control while also optimizing preservation of surrounding structures . in this case , the patient decided to pursue treatment with radiation therapy . conservative management was not considered , given the significant increase in tumor volume during the eight - year interval of mri surveillance and the presence of bone erosion . given the rare presentation of a mass located in the vidian canal , the decision was made to confirm the diagnosis with a biopsy prior to treatment . the choice between single - fraction srs or multiple - fraction radiation was made based on size and proximity of the tumor to potential radiation - sensitive structures . minimizing the risk of adverse effects from radiation was the determinant for pursuing fractionated radiation . in recent years , radiation therapy has become a common alternative to surgical resection of intracranial schwannomas.16 17 18 19 20 21 options include either single - fraction srs or conventional daily low - dose fractionation over approximately five weeks . srs is a well - tolerated procedure , typically completed during a single session of irradiation and far more convenient for the patient . srs has become a widely accepted treatment option for vestibular schwannomas and has a high success rate of durable tumor control with often partial reduction in tumor volume , with progression - free survival of 93% at 5 years and 92% at 10 years.22 fractionated radiation has similar results and is chosen over srs when there is a concern that the size of target exceeds safe tolerance to srs , when there are critical radiation - sensitive structures in adjacent areas that may not be able to tolerate the single high - dose collateral exposure , or when there are underlying concerns of the patient to suggest a higher risk of an adverse event . compared with surgery , radiation therapy has been reported in several studies to result in decreased mortality and fewer treatment - related complications for vestibular schwannoma including hearing loss and facial weakness.22 23 24 25 recent advances in imaging and radiation delivery offer more conformal treatment options such as srt which combines both the superior accuracy of target localization of a stereotactic system and reduction in the size of the radiation treatment fields and fractionated treatment , a radiobiologically safer means of delivering radiation when there are concerns for late effects of radiation decades after treatment completion . schwannomas of the vidian canal are extremely rare tumors and may present with symptoms of headaches , facial pain , facial muscle paralysis , decreased lacrimation , and nasal dryness or as an incidental finding . when a vidian canal tumor is identified , endoscopic biopsy can be used to confirm a histopathologically benign diagnosis before considering treatment options .

background schwannomas of the vidian canal are an extremely rare type of intracranial tumor that can have variable clinical presentations including headache , facial pain , facial muscle paralysis , decreased lacrimation , or nasal dryness . we present an atypical case of an incidentally identified asymptomatic vidian canal schwannoma . case description a 49-year - old woman with a history of multiple sclerosis presented for routine surveillance magnetic resonance imaging that detected an ovoid mass originating in the vidian canal . given the unusual location of the lesion , an endoscopic endonasal biopsy was performed and confirmed the diagnosis of a vidian canal schwannoma , for which the patient chose to receive fractionated radiation therapy . conclusion when a vidian canal tumor is identified , endoscopic endonasal biopsy can be used to confirm the diagnosis before pursuing either surgical or radiotherapy treatment . in particular , fractionated radiation therapy offers a radiobiologically safe means of delivering radiation when there is concern for late radiation - related side effects following treatment completion .

pubmed

posterior transpedicular pedicle screw fixation is widely used for obtaining internal fixation for management of the unstable spine mainly caused by trauma , and burst fractures comprise 1020% of such injuries.1 the aims of surgery in these fracture cases include decompression of the neural components , fracture reduction , and providing a stable fixation until arthrodesis is achieved . transpedicular short - segment fixation became popular after the introduction of transpedicular screws by roy - camille23 and the internal fixator by dick.4 this approach includes pedicle screw fixation one vertebra above and one vertebra below the fracture . short - segment spinal instrumentation has been beneficial in the management of thoracolumbar spinal fractures for better correction of kyphotic deformity , greater initial stability , early painless mobilization , and indirect decompression of the spinal canal.57 despite the advantages of this approach , it is also associated with loss of reduction and instrumentation failure in some cases.49 in an attempt to achieve stiffer short - segment constructs , some surgeons add pedicle screws at the fractured vertebra . these screws can be termed intermediate screws . according to several biomechanical and clinical studies , monoaxial or polyaxial pedicle screws inserted at the level of fractured vertebrae , along with the segmental fixation of burst fractures , improved biomechanical stability of the construct.1012 in the past 20 years , the design and implantation techniques of the pedicle screw systems have been modified to reduce the rate of pedicle screw breakage and to facilitate easy application of the connecting rod without undue stress on the construct.1316 the polyaxial head coupling of the pedicle screw was found to reduce the compression bending strength at the screw rod mount , in comparison with a monoaxial screw design.1516 based on research findings , use of the intermediate screw in conjunction with short - segment fixation for fracture reduction and kyphosis correction carries the additional advantages of construct stabilization , lordosis restoration , and preventing screw breakage.10 however , there have been few reports regarding short - segment fixation , which have compared the biomechanical performance of polyaxial versus monoaxial pedicle screws with or without an intermediate screw at the fractured vertebra . this study utilized a calf experimental model of an unstable burst fracture to compare the stiffness of a short - segment pedicle fixation using either monoaxial or polyaxial pedicle screws placed at the level of fracture . twelve fresh - frozen calf lumbar specimens ( l1l5 ) were obtained from a regional slaughterhouse . all specimens were wrapped in doubled plastic bags and stored frozen at 20c . before biomechanical testing , the specimens were thawed at room temperature in a humidity - controlled environment for 8 h. the soft tissues were removed by dissection , leaving the ligaments , facet joint capsules , and intervertebral discs intact . the end vertebrae were trimmed , fixed with plain screws , and embedded in liquid self - curing denture base material and denture base resin ( type ii ) . each pot included a 10-mm screw secured to a metal plate that was incorporated in the resin cast . this screw was located at the level of the middle column of the vertebrae and was used as an anchor for the testing machine . the resin was left to cure for 30 min . to avoid the influence of air exposure on biomechanical behavior , handling experimental material routinely used in in vitro biomechanical investigation in this manner , does not alter the material characteristics of the bone and soft tissues . after the intact spine was tested for flexibility , an unstable burst fracture was created in the l3 vertebra using the pre - injury and the dropped - mass technique . a 2-mm drill bit was used to create holes in the l3 vertebral body with v - shaped corpectomy [ figure 1 ] . after the pre - injury model had been created , the specimen was mounted through its pots on a ferric base . the specimen was flexed to approximately 10 and a 10-kg weight was dropped from a height of 0.5 m on a vertical rail , and then from 0.6 , 0.7 , 0.8 m , and so on , until the v - shaped cut closed . after the creation of the l3 vertebral fracture , it was ensured that the pedicle remained intact for further experimentation . ( a ) the v - shaped corpectomy of the l3 vertebra ; ( b ) the dropped - mass technique used after osteotomy to simulate an unstable burst fracture of the l3 vertebra ; ( c ) short - segment pedicle screw construct involving transpedicular fixation of vertebrae one above and one below the fracture site ; ( d ) use of an intermediate screw at the level of the fractured vertebra to test improvement in stability of the construct the specimens were randomly divided into monoaxial pedicle screw ( mps ) group and polyaxial pedicle screw ( pps ) group by random digits table , and six calves were allocated in each group . two kinds of spinal fixations ( i.e. , with or without intermediate screw placement ) were studied in each group [ figure 1 ] . after a fracture was created , a short - segment posterior pedicle screw construct was used to realign and stabilize the spine . the specimens in the mps group were fixated with monoaxial pedicle screws , and polyaxial pedicle screws were used for those in the pps group . the specimens in which an intermediate screw was not applied received a 4-screw ( monoaxial or polyaxial ) construct incorporating one vertebra above and one vertebra below the fracture . the remaining specimens ( mpsi and ppsi ) underwent a 6-screw construct in which two intermediate screws were added to the fractured vertebrae in addition to the 4-screw construct described above [ figure 2 ] . the intermediate screws were of the same length and were inserted to the same depth as the other four screws . we reused the same specimens in mps group and mpsi group , pps group and ppsi group in order to standardize the procedure and avoid any difference amongst the specimens in the same group . x - ray film and photographs . ( a ) the film showing the burst fracture of vertebral body and the preservation of the anterior longitudinal ligament ; ( b ) mechanical testing for lateral bending ( intact specimen ) ; ( c ) mechanical testing for axial rotation ( intact specimen ) ; ( d ) mechanical testing for flexion and extension ( fracture specimen ) ; ( e ) mechanical testing for flexion and extension ( short - segment fixation specimen ) ; ( f ) mechanical testing for flexion and extension ( intermediate screw fixation specimen ) flow chart showing the sequence of events for each specimen . bt = biomechanical testing ; mps = monoaxial pedicle screw ; pps = polyaxial pedicle screw ; mpsi = monoaxial pedicle screws with intermediate pedicle screws fixation ; ppsi = polyaxial pedicle screws with intermediate pedicle screws fixation each specimen was tested for flexion , extension , lateral bending , and axial rotation as an intact spine , as a fractured spine , and as a fractured spine fixed by either a 4-screw or 6-screw construct . all specimens were subjected to 0500 n flexion and axial compression in a displacement - controlled mode at a rate of 5 mm / min on an mts858 ( material testing system , mts ) testing machine . the mts858 testing machine is used to make the specimen move in the mode of flexion , extension , lateral bending , and axial rotation . the ranges of motion ( roms ) of the relative intervertebral motions were automatically recorded by the eagle-4 and motion analysis position capture and measurement system ( micron company , california , usa ) . the camera system recorded the movements of infrared led markers attached to each of the levels , l2l4 . a digitizing probe was used to locate landmarks on each vertebra from which local coordinate systems were established . the local coordinate systems were aligned to the primary anatomic planes ( sagittal , coronal , and axial ) . the relations of the superior vertebral local coordinate system with respect to the inferior vertebral coordinate system were quantified with euler angle transformations . the roms calculated for the 4- and 6-screw constructs were then compared with the values obtained for the intact and fractured specimens . because the 4- and 6-screw construct results in the mps or pps group were compared for the same specimen , paired student 's t - test was used for statistical analyses , using spss 15.0 statistical software ( spss / pc , chicago , il , usa ) . biomechanical data were evaluated using a general linear model procedure for analysis of variance ( anova ) and the student newman the differences in rom between the two groups were evaluated with independent t - tests . the differences in rom within the same group were evaluated with paired t - tests . twelve fresh - frozen calf lumbar specimens ( l1l5 ) were obtained from a regional slaughterhouse . all specimens were wrapped in doubled plastic bags and stored frozen at 20c . before biomechanical testing , the specimens were thawed at room temperature in a humidity - controlled environment for 8 h. the soft tissues were removed by dissection , leaving the ligaments , facet joint capsules , and intervertebral discs intact . the end vertebrae were trimmed , fixed with plain screws , and embedded in liquid self - curing denture base material and denture base resin ( type ii ) . each pot included a 10-mm screw secured to a metal plate that was incorporated in the resin cast . this screw was located at the level of the middle column of the vertebrae and was used as an anchor for the testing machine . the resin was left to cure for 30 min . to avoid the influence of air exposure on biomechanical behavior , handling experimental material routinely used in in vitro biomechanical investigation in this manner , does not alter the material characteristics of the bone and soft tissues . after the intact spine was tested for flexibility , an unstable burst fracture was created in the l3 vertebra using the pre - injury and the dropped - mass technique . a 2-mm drill bit was used to create holes in the l3 vertebral body with v - shaped corpectomy [ figure 1 ] . after the pre - injury model had been created , the specimen was mounted through its pots on a ferric base . the specimen was flexed to approximately 10 and a 10-kg weight was dropped from a height of 0.5 m on a vertical rail , and then from 0.6 , 0.7 , 0.8 m , and so on , until the v - shaped cut closed . after the creation of the l3 vertebral fracture , it was ensured that the pedicle remained intact for further experimentation . ( a ) the v - shaped corpectomy of the l3 vertebra ; ( b ) the dropped - mass technique used after osteotomy to simulate an unstable burst fracture of the l3 vertebra ; ( c ) short - segment pedicle screw construct involving transpedicular fixation of vertebrae one above and one below the fracture site ; ( d ) use of an intermediate screw at the level of the fractured vertebra to test improvement in stability of the construct the specimens were randomly divided into monoaxial pedicle screw ( mps ) group and polyaxial pedicle screw ( pps ) group by random digits table , and six calves were allocated in each group . two kinds of spinal fixations ( i.e. , with or without intermediate screw placement ) were studied in each group [ figure 1 ] . after a fracture was created , a short - segment posterior pedicle screw construct was used to realign and stabilize the spine . the specimens in the mps group were fixated with monoaxial pedicle screws , and polyaxial pedicle screws were used for those in the pps group . the specimens in which an intermediate screw was not applied received a 4-screw ( monoaxial or polyaxial ) construct incorporating one vertebra above and one vertebra below the fracture . the remaining specimens ( mpsi and ppsi ) underwent a 6-screw construct in which two intermediate screws were added to the fractured vertebrae in addition to the 4-screw construct described above [ figure 2 ] . the intermediate screws were of the same length and were inserted to the same depth as the other four screws . we reused the same specimens in mps group and mpsi group , pps group and ppsi group in order to standardize the procedure and avoid any difference amongst the specimens in the same group . a ) the film showing the burst fracture of vertebral body and the preservation of the anterior longitudinal ligament ; ( b ) mechanical testing for lateral bending ( intact specimen ) ; ( c ) mechanical testing for axial rotation ( intact specimen ) ; ( d ) mechanical testing for flexion and extension ( fracture specimen ) ; ( e ) mechanical testing for flexion and extension ( short - segment fixation specimen ) ; ( f ) mechanical testing for flexion and extension ( intermediate screw fixation specimen ) flow chart showing the sequence of events for each specimen . bt = biomechanical testing ; mps = monoaxial pedicle screw ; pps = polyaxial pedicle screw ; mpsi = monoaxial pedicle screws with intermediate pedicle screws fixation ; ppsi = polyaxial pedicle screws with intermediate pedicle screws fixation each specimen was tested for flexion , extension , lateral bending , and axial rotation as an intact spine , as a fractured spine , and as a fractured spine fixed by either a 4-screw or 6-screw construct . all specimens were subjected to 0500 n flexion and axial compression in a displacement - controlled mode at a rate of 5 mm / min on an mts858 ( material testing system , mts ) testing machine . the mts858 testing machine is used to make the specimen move in the mode of flexion , extension , lateral bending , and axial rotation . the ranges of motion ( roms ) of the relative intervertebral motions were automatically recorded by the eagle-4 and motion analysis position capture and measurement system ( micron company , california , usa ) . the camera system recorded the movements of infrared led markers attached to each of the levels , l2l4 . a digitizing probe was used to locate landmarks on each vertebra from which local coordinate systems were established . the local coordinate systems were aligned to the primary anatomic planes ( sagittal , coronal , and axial ) . the relations of the superior vertebral local coordinate system with respect to the inferior vertebral coordinate system were quantified with euler angle transformations . the roms calculated for the 4- and 6-screw constructs were then compared with the values obtained for the intact and fractured specimens . because the 4- and 6-screw construct results in the mps or pps group were compared for the same specimen , paired student 's t - test was used for statistical analyses , using spss 15.0 statistical software ( spss / pc , chicago , il , usa ) . biomechanical data were evaluated using a general linear model procedure for analysis of variance ( anova ) and the student newman keuls post hoc test in each group ( mps or pps ) . the differences in rom between the two groups were evaluated with independent t - tests . the differences in rom within the same group were evaluated with paired t - tests . the rom results for l2l4 in all directions for the mps and pps groups are summarized in table 1 . the addition of an intermediate screw decreased the rom of the l2l4 segments in both the mps and pps groups . a smaller rom due to the use of an intermediate screw represents enhanced stability of the construct , in contrast to a larger rom that was observed in the standard short - segment construct . compared to the short - segment transpedicular fixation group , the addition of intermediate screws provided a smaller rom in flexion , extension , lateral bending , and torsion in the mps group ( p = 0.001 , 0.006 , 0.077 , and 0.000 , respectively ) , and in flexion , extension , lateral bending , and torsion in the pps group ( p = 0.000 , 0.000 , 0.017 , 0.000 , respectively ) . there was neither a difference in the rom in the spines of the two groups before injury , nor a difference in rom between the mpsi and ppsi groups ( p > 0.05 ) . however , when using the intermediate screw in injured spines , the stiffness in the mpsi and ppsi groups increased significantly as compared with the intact spine ( p < 0.05 ) . there was a significant difference between mps and pps in flexion ( p < 0.01 ) and extension ( p < 0.05 ) [ figure 4 ] . rom in various planes of the intact , fracture model , and postinternal fixation spine within the same group ( n = 6 ; ; mean standard deviation ) bar graphs showing the rom compared with intact spine for ( a ) flexion , ( b ) extension , ( c ) lateral bending , and ( d ) axial rotation loading . mps = monoaxial pedicle screw ; pps = polyaxial pedicle screw ; mpsi = monoaxial pedicle screws with intermediate pedicle screws fixation ; ppsi = polyaxial pedicle screws with intermediate pedicle screws fixation . * p , 0.05 for significant difference between groups management options for unstable burst fractures include anterior , posterior , or combined ( circumferential ) fixation . many surgeons believe that anterior column reconstruction is critical in correcting kyphotic deformity and reestablishing the anterior load bearing capacity of the vertebral component . short - segment posterior spinal instrumentation using pedicle screws remains the standard method for the fixation of thoracic and lumbar fractures , with acceptable results . the advantages of this technique include less surgical dissection , blood loss , and time in surgery , and as a result , decreased perioperative morbidity . however , loss of reduction and instrumentation failure associated with this technique is well described in the literature.159 these failures have been attributed to poor bone quality , inadequate anterior column support , and insufficient points of fixation . the biomechanical properties of monoaxial pedicle screws have been widely reported.1720 segmental fixation with additional monoaxial pedicle screws at the level of the fracture increases construct stiffness and shields the fractured vertebral body from anterior loads . furthermore , this additional point of fixation allows for a 3-point reduction maneuver , analogous to that used for reduction of long bone fractures . in a recent prospective randomized study , the efficacy of the fracture - level screw combination in achieving and maintaining correction for the treatment of unstable thoracolumbar burst fractures was evaluated.21 the authors of that study concluded that reinforcement with a fracture - level screw could help provide and maintain improved kyphosis correction . another mid - term clinical study demonstrated superior results with the fracture - level screw combination , supporting the evidence of other reports.22 interestingly , in most of these studies , the pedicle screws used were monoaxial , despite the current trend toward using polyaxial screws in short - segment fracture fixation.101221 on review of the literature , we found that there was a paucity of data , both clinical and biomechanical , on the use of polyaxial screw as an intermediate in unstable burst fractures , and thus this biomechanical study was instituted . the current study showed that when short - segment constructs contained intermediate screws , flexion , extension , and torsion decreased in both the mpsi and ppsi groups , compared to the respective constructs ( mps and pps ) without this reinforcement . the short - segment construct in the pps group had significantly greater rom in flexion and extension when compared with the mps group . the addition of intermediate screws at the level of a burst fracture significantly increased the stiffness of the short - segment pedicle fixation , irrespective of whether monoaxial or polyaxial pedicle screws were used . however , this increase was more significant for the mpsi construct , as the ppsi already had significantly increased stiffness as compared to the mpsi . these results may be because there is a better coupling between the polyaxial screw heads and the connecting rod . thus , there is less torsion at the coupling site and consequently less stress on the entire construct . the intermediate screw provides a 3-point fixation of the fractured segment . when using monoaxial screws , the addition of an intermediate forces the surgeon to bend the connecting rod to accommodate the additional screw . polyaxial screws , on the other hand , facilitate the installation of the connecting rod , and their biomechanical properties have been reported in several studies.141623 stanford et al.15 suggested that the rod screw link design of the polyaxial screw reduces its static compressive bending yield strength as compared with the fixed screw designs . liu et al.24 pointed out that there were significant differences between the monoaxial screws and polyaxial screws in the bending stiffness , yield load , yield torque , and torsional stiffness in static tests ( p < 0.05 ) . shepard et al.16 suggested that polyaxial screws do not significantly decrease the stiffness of the construct . on the contrary , the polyaxial constructs create more security by permitting better contact and holding strength between the screw head and the rod . when there is the combined effect of bending loads and shear force on the rods , there would be higher resistance to rotational slippage between the rod and the screw head . the limitations of study are that the data were pertinent to results immediately after surgery , but do not take into account early bone resorption or the cyclical loading effect which have long - term impacts on outcome . the flexibility of the construct was tested on a burst fracture model that was created in a controlled and reproducible manner by the pre - injury and dropped - mass technique . nevertheless , the pattern and nature of a spinal burst fracture in clinic is more variable and unpredictable . anekstein et al.12 hypothesized that the true mechanical effect of the intermediate screws is less predictable in in vivo settings . only a prospective clinical study can show the true practical significance of the addition of intermediate screws . although not human , calf spine has been used before as a valid biomechanical model.2527 calf is a tetrapod ; its anatomical characteristics and common fracture site are different from those of the human , and because the calf lumbar spines ( l1l5 ) can be easily dissected from the other vertebrae of calf , we chose the l1l5 calf lumbar spines and made the l3 vertebral fracture instead of the t12 or l1 which are the most common fracture sites in humans . calf spine segments are presumed to have higher bone mineral density than human spines , and therefore have different biomechanical behavior . nevertheless , the use of an animal model allows for smaller inter - specimen differences , as the human spine has been shown to have significant variability in bone mineral density that might potentially affect the test results.2829 as this study is comparative , the authors favored the animal model to that of the human cadaver spine . it can be argued that the number of cases in each group was less in the present study to reach a meaningful conclusion . however , in the previous biomechanical studies,183031 the number of the cases used in the experiment was usually six or seven . in the present study , we used 12 cases and 6 cases in each group and we believe that these numbers have brought forward meaningful results . another disadvantage of this study is that there was no comparison with long - segment fixations and application of the cross link , which have been shown to have better biomechanical stability than the short segment in managing unstable burst fractures . in conclusion , the addition of intermediate screws at the level of a burst fracture significantly increases the stability of a short - segment pedicle fixation , using either monoaxial or polyaxial pedicle screws . however , the short - segment mps fixation group was significantly more stable in flexion and extension than the short - segment pps fixation group . we believe that this study provides a biomechanical rationale for using an intermediate screw at the level of fractured vertebrae with posterior short - segment fixation , especially when using monoaxial screws for fixing the unstable burst fractures of the lumbar region .

background : use of a pedicle screw at the level of fracture , also known as an intermediate screw , has been shown to improve clinical results in managing lumbar fracture , but there is a paucity of biomechanical studies to support the claim . the aim of this study was to evaluate the effect of adding intermediate pedicle screws at the level of a fracture on the stiffness of a short - segment pedicle fixation using monoaxial or polyaxial screws and to compare the strength of monoaxial and polyaxial screws in the calf spine fracture model.materials and methods : flexibility of 12 fresh - frozen calf lumbar spine specimens was evaluated in all planes . an unstable burst fracture model was created at the level of l3 by the pre - injury and dropped - mass technique . the specimens were randomly divided into monoaxial pedicle screw ( mps ) and polyaxial pedicle screw ( pps ) groups . flexibility was retested without and with intermediate screws ( mpsi and ppsi ) placed at the level of fracture in addition to standard screws placed at l2 and l4.results:the addition of intermediate screws significantly increased the stability of the constructs , as measured by a decreased range of motion ( rom ) in flexion , extension , and lateral bending in both mps and pps groups ( p < 0.05 ) . there was neither any significant difference in the rom in the spines of the two groups before injury , nor a difference in the rom between the mpsi and ppsi groups ( p > 0.05 ) , but there was a significant difference between mps and pps in flexion and extension in the short - segment fixation group ( p < 0.05).conclusions : the addition of intermediate screws at the level of a burst fracture significantly increased the stability of short - segment pedicle screw fixation in both the mps and pps groups . however , in short - segment fixation group , monoaxial pedicle screw exhibited more stability in flexion and extension than the polyaxial pedicle screw .

pubmed

the discovery of electrochemical self - assembly ( esa ) of nanostructured hybrid thin films of inorganic semiconductors and organic dyes has opened a new synthetic route for obtaining photoanode material for dye - sensitized solar cells ( dsscs ) . this method bypasses the high - temperature sintering that is typically needed for nanoparticulate tio2 electrodes , which prevents the use of conductive plastic substrates . we have previously demonstrated the use of esa with the zno structures for the construction of photoanodes . minor addition of water - soluble dye molecules with substituents for anchoring to zno surface brought about a significant impact on the crystal growth for evolution of various nanostructures and alteration of the crystallographic orientation of the film . importantly , the coprecipitation of dye molecules does not necessarily deteriorate the crystallinity of zno . in the case of zno / eosin y ( ey ) hybrid thin films , vertically aligned interconnected and crystalline nanowires of zno are formed . the space between the zno nanowire is filled with aggregates of ey . the solid ey can be completely removed by soaking the hybrid film in a mild alkaline such as dilute koh aq . thus , prepared zno thin films , consisting of large single - crystalline grains with internal porous structure , to be called porous crystal zno , have large surface area for adsorption of photosensitizer dye in a large amount and high crystallinity for efficient transport of electrons ; therefore , it can achieve high incident photon to current conversion efficiency ( ipce ) of around 90% . with the dssc application in mind , it would be highly interesting to find a compatible system for fabricating sensitized photocathodes . recently , powar et al . have succeeded in achieving 1.3% conversion efficiency for a dssc in an inverted structure employing a porous p - nio electrode sensitized with a specially designed dye to achieve a high rectification with kinetically reversible co(ii / iii ) ethylene diamine complex electrolyte . we have been seeking to perform cathodic electrodeposition of cuscn , a p - type , wide - bandgap semiconductor , into highly crystallized thin films at low temperature . cuscn has been frequently used as a hole conductor in nanostructured solar cells and also as a sensitized photocathode in combination with hole - injecting dyes . therefore , esa of cuscn - based hybrid thin films could serve nicely as a counterpart to the well - established zno - based hybrid systems . our first attempt for esa of cuscn hybrid system was to employ dye molecules having soft lewis basic ncs groups . on the basis of the hard and soft , acid and base ( hsab ) principle , choice of dyes equipped with hard lewis basic carboxylate is suited for binding to hard lewis acidic ti(iv ) sites of tio2 or zn(ii ) sites of zno . because cu(i ) of cuscn is a typical soft lewis acid , the soft sulfur atom of the ncs should be a good match . as such , dyes having ncs groups , the popular n3 dye ( ru(dcbpy)2(ncs)2 , dcbpy = 2,2-bipyridine-4,4-dicarboxylic acid ) and fltc ( fluorescein isothiocyanate ) were added to the bath for electrodeposition of cuscn . as expected , colored cuscn thin films incorporating n3 and fltc were obtained , while ey added for comparison did not cause any change for the formation of cuscn , which is of course normally colorless . the addition of n3 and fltc also brought about the evolution of characteristic nanostructure of cuscn , clearly indicating their action as structure directing agents ( sdas ) . the electrodeposited cuscn / dye hybrid thin films exhibited dye - sensitized photocathodic properties , evincing the intimate chemistry between cuscn and dyes having soft basic anchors , which appear to be the key for hole injection from the dye excited state . following the success with the above - mentioned strategy , the idea further developed to try substituting cu(i ) of cuscn with cationic dyes instead of substituting ncs with dyes having ncs group . among commercially readily available dyes , we have employed rhodamine b ( rb ) , which is cationic because of the presence of an ammonium group . this strategy indeed showed positive results as we have seen a clear effect of rb as an sda for formation of nanostructure and as a cause of changes in crystallographic orientation of cuscn . however , it was not possible to extract the loaded rb molecules by any kind of post treatment to obtain highly porous structure as in the case of the zno / ey hybrid system . because the mechanism of cuscn electrodeposition was poorly understood at the time of the previous work , the conditions were not properly chosen to achieve hybrid thin films with composition similar to that of zno / ey , for which ey could occupy close to half of the volume of the precipitated solid . in this work , we present a method for esa of hybrid cuscn / rb nanoporous films in which the rb sda may be nearly completely removed . by varying the relative concentration of cu and scn , we can change the crystal orientation of the resulting nanostructure with respect to the substrate . we explore several preliminary dyes and show photoinduced hole transfer to the cuscn ; in addition , we tested the best systems in photoelectrochemical studies to check the dye - sensitized photocathodic properties . at this stage , we still do not have a good combination of dye and electrolyte for cuscn . however , the recent progress with the photocathodic dssc presents a new opportunity to improve the efficiency of dye - sensitized cathode systems that can eventually be combined with existing sensitized anodes for realization of a tandem solar cell to achieve high efficiencies in very simple structures . inorganic chemicals such as lithium thiocyanate dihydrate ( liscn2h2o , kishida ) , copper(ii ) perchlorate hexahydrate ( cu(clo4)26h2o , sigma - aldrich ) , and lithium perchlorate ( liclo4 , sigma - aldrich ) and organic solvents such as n , n - dimethylacetamide ( dma , wako ) and ethanol ( wako ) were of special grade and used as purchased . fluorine - doped tin oxide ( fto ) coated transparent conductive glass ( sheet resistance , 78 /sq , 1.1 mm thick soda lime glass , asahi - du , asahi glass ) was used as the substrate to electrodeposit the cuscn thin films . milli - q ultrapure water ( > 18 m ) was used throughout the experiments . the fto glass was cut into 25 25 mm , degreased by ultrasonically cleaning in detergent and acetone , and finally rinsed with water . it was mounted into a specially designed electrode holder for a homemade rotating disc electrode ( rde ) system . the active area of the fto glass electrode was regulated to a 22 mm diameter circle concentric to the rotating electrode holder by applying a masking tape ( nitto denko n-300 ) . the use of a rde system to introduce laminar flow is essential for obtaining cuscn thin films with perfect homogeneity and with a high reproducibility because the reaction occurs under the mass transport limited regime . the fto glass rde that serves as a working electrode is placed with the active surface down and in the center of the electrochemical cell of a cylindrical shape , equipped with a water - jacket for temperature control ( 25 c ) , together with auxiliary electrodes , a pt wire counter and a ag / agcl reference . a mixture of water and ethanol ( 1:3 in volume ratio ) was used for preparation of the deposition bath . cu(clo4)2 and liscn were dissolved at 9 and 3 or 3 and 9 mm for cu rich or scn rich bath , respectively , while 0.1 m liclo4 was also added as the supporting electrolyte . a small amount of rb up to 1.0 mm was added to obtain cuscn / rb hybrid thin films . a hokuto - denko hsv-100 electrochemical system was used for potential control and current monitoring . cathodic electrolysis at + 0.2 v ( vs ag / agcl ) for 5 min and with a rotation speed of 500 rpm resulted in cuscn thin films which were washed with water , dried under air at room temperature , and subjected to characterization . vis absorption spectra of the films were measured in transmission on a hitachi u-4000 or a perkinelmer lambda 1050 spectrophotometer . fluorescence spectra of the film samples were measured on a photon technology international quantamaster40 equipped with two double - monochromators and a cooled pmt detector . aside from the cuscn / rb hybrid thin film , cuscn hybrid with fluorescein isothiocyanate ( fltc ) , nile blue ( nb ) , cresyl violet ( cv ) , 3,4,9,10-perylenetetracarboxylic acid diimide ( ptcdi ) , and quinacridone ( qd ) , as well as thin solid films of only these chromophores , were prepared for comparison in the fluorescence study . those of rb , fltc , nb , and cv were prepared by hybrid electrodeposition and spin coating their 1 mm solutions in ethanol on a slide glass , while those of ptcdi and qd were prepared by vacuum evaporation either onto an electrodeposited porous cuscn or on a slide glass . surface morphology of the films was observed by field emission scanning electron microscopy ( fe - sem , hitachi s-4800 or jeol jsm-6700f ) . crystallographic characterization of the film was conducted by measuring x - ray diffraction ( xrd ) patterns on a rigaku rint ultima iii instrument . the amount of rb loaded into the film was determined by dissolving a known area of the film into a known volume of ammonia solution and measuring its absorption spectrum . the amount of electrodeposited cuscn was determined by measuring the amount of cu in the ammonia solution dissolving the film according to the classical colorimetric analysis method , employing sodium n , n - diethyldithiocarbamate trihydrate ( ddtc , wako ) to chelate copper ion for extraction with ethyl acetate which exhibits a strong absorption at 436 nm ( = 1.3 10 m cm ) . comparison with the amount of consumed charge yields faradaic efficiency for the electrodeposition of cuscn . a part of the loaded rb molecules could also be extracted by soaking the film in dma . from comparison of the absorption spectra before and after the dma treatment , the fraction of the extractable rb could be determined . several thin - film samples after the dma treatment were scratched off the substrate . the surface area of powder samples prepared in this manner was determined by brunauer teller ( bet ) analysis of the kr gas sorption measurement conducted on a micromeritics tristar ii 3020 system . photoelectrochemical measurements on cuscn thin film electrodes have been performed in a three - electrode setup with a pt wire counter and a saturated calomel reference electrode ( sce ) . porous cuscn thin films were sensitized by soaking them in a 1.0 mm rb aqueous solution for 1 h. a 0.1 m aqueous solution of methylviologen dichloride ( mv , sigma - aldrich ) was used as the electrolyte . the electrode was illuminated from its front side with a visible light generated by a 500 w xe lamp ( ushio ) filtered by uv ( < 420 nm ) and ir ( > 800 nm ) cutoff filters ( intensity , 100 mw cm ) . direct crystallization of -cuscn occurs as a consequence of cathodic reduction of cu in the presence of scn both from aqueous and ethanolic solutions . rhodamine b is commercially provided as chloride and is well - soluble both to water and ethanol . 30 m when 0.1 m liclo4 was added to water as a supporting electrolyte because of the salting - out effect , making it difficult to achieve a high loading of rb to cuscn . on the other hand , ethanol dissolved rb too well to hinder its loading to cuscn . therefore , a mixture of water and ethanol ( 25:75 volume ratio ) was chosen as the solvent that provided good solubility of all chemicals and achieved an efficient loading of rb molecules during electrochemical growth of cuscn . our previous studies on electrodeposition of cuscn employing a rotating disk electrode revealed that the growth of cuscn is typically limited by transport of complexes of cu and scn that form in solution , achieving practically 100% faradaic efficiency for the formation of cuscn . in aqueous solutions , only a 1-to-1 complex is formed , while a neutral 1-to-2 complex is also formed and participates in the electrochemical reaction in ethanolic solutions containing excess of scn . thus , the overall reaction is expressed by eq 1 for the aqueous solution and both eqs 1 and 2 operate in the ethanolic solution.12the chronoamperograms measured during the electrodeposition can be seen in figure 1 . following the initial rise of the current , diffusion - limited steady - state current is reached both for the cu rich and scn rich solutions . however , this should reflect the formation of a bis - coordinated cu(ii ) thiocyanato complex which has a diffusion coefficient that is somewhat higher than that of the monocoordinated complex in ethanol as revealed in our electrochemical analysis . the addition of rb to the bath causes a slight decrease of the steady - state current in the scn rich bath , whereas a slight increase of the current was observed in the cu rich bath , which could be caused by the chemical interaction of the complexes with rb molecules . even though the addition of rb caused a slight decrease of faradaic efficiency for the precipitation of cuscn ( vide infra ) , the film thickness increased linearly with the consumed charge ( see figure 1s of supporting information ) . as the current is almost constant , the film thickness can simply be controlled by the time of the electrolysis . however , it should be mentioned that the high addition of rb at 1.0 mm brings about a characteristic change in the initial profile of the current . in such baths , the current stays small in the beginning of the electrolysis then abruptly increases to achieve the steady - state current . the surface of the fto glass substrates was observed by sem before and after the jump of the current ( figure 2 ) . there is apparently no change before the jump as the bare surface of the fto layer is seen . after the jump , tiny cuscn particles are deposited , especially at the bottom of the valleys and along the ridges of the pyramidal fto grains . although the reason for the incubation time is unclear , it is probable that rb molecules are adsorbed on the fto surface , hindering the nucleation of cuscn . chronoamperograms measured during electrodeposition of cuscn and cuscn / rb hybrid thin films onto an fto coated glass rde ( = 500 rpm ) at + 0.2 v ( vs ag / agcl ) in ethanol / water ( 75/25 , v / v ) mixed solutions containing cu(clo4)2 and liscn in cu rich ( 9 and 3 mm , respectively ) and scn rich ( 3 and 9 mm , respectively ) compositions ; 0.1 m liclo4 as a supporting electrolyte ; and 0 , 0.1 , or 1.0 mm rb . sem pictures of the surface of the fto glass substrates after electrolysis for 20 s ( a ) and 30 s ( b ) in an ethanol / water ( 75/25 , v / v ) mixed solution containing 3 mm cu(clo4)2 , 9 mm liscn , 0.1 m liclo4 , and 1.0 mm rb . cuscn / rb hybrid thin films were electrodeposited for 5 min both from scn rich and cu rich baths while changing the concentration of rb . the morphology of the pure cuscn film looks totally different for those deposited from scn rich and cu rich baths ( panels a1 and b1 of figure 3 , respectively ) . the former consists of large rounded hexagonal columnar particles , whereas the latter shows assemblies of spiky particles . rhombohedral -cuscn has an elongated hexagonal unit cell with lattice constants of a = b = 3.857 and c = 16.449 . as discussed below , the xrd patterns of the films indicate that films of a1 and b1 are oriented for the c - axis perpendicular with the substrate . the hexagonal faces of the deposits in a1 should thus correspond to the ( 003 ) planes of cuscn , while those faces creating the spikes should probably be of the ( 101 ) planes . such differences can be caused by the change of the stability of the crystal faces by the change of the chemical composition of the bath influencing the dissolution and recrystallization of cuscn during the electrodeposition ( eq 3).3when rb is added to the bath , purple - colored cuscn / rb hybrid thin films were obtained . as the concentration of rb increased , the color of the film got deeper , indicating the increased loading of rb into the film . at the same time , the morphology of the film drastically changed ( panels a2 , a3 , b2 , and b3 of figure 3 ) . as discussed later , the loaded rb molecules can almost completely be extracted by dipping the film shown in a3 in dimethylacetamine ( dma ) , making it highly porous . the shape of the particles differs from sample to sample , especially as recognized from the cross sections , indicating anisotropic crystal growth promoted by rb addition . sem photographs of cuscn and cuscn / rb hybrid thin films electrodeposited from scn rich ( a ) and cu rich ( b ) baths containing 0 ( a1 and b1 ) , 0.3 ( a2 and b2 ) and 1.0 mm ( a3 and b3 ) rb . a1a3 and b1b3 correspond to the cross section of a1a3 and b1 b3 , respectively . changes of xrd patterns on rb addition are shown in figure 4 for scn rich and cu rich baths . all these patterns indicate diffraction peaks assigned to -cuscn aside from those originating from sno2 of the fto substrate . the relative intensity of the diffraction peaks of the film samples , however , are different from those of the powder diffraction standard . it is also changes for different rb concentrations and for scn rich ( a ) and cu rich ( b ) baths , indicating changes of their crystallographic orientation . two representative diffraction peaks arising from the ( 003 ) and the ( 101 ) planes are taken to evaluate the orientation change . while the ( 003 ) planes are perpendicular to the c - axis , the ( 101 ) planes are nearly parallel to the c - axis , crossing with the ( 003 ) planes at 78.5 angle because of the elongated unit cell structure . therefore , the relative change of the ( 003 ) and ( 101 ) peak intensities is a good measure for examining how the c - axis of cuscn is oriented with respect to the substrate plane . according to the method described in the literature , the orientation indices ( oi ) are calculated as follows . the intensity factor ( if ) of the standard powder sample is calculated for the respective crystal planes as45taking the intensities indicated in ref ( 23 ) . the if of the film samples are calculated from the ( 003 ) and ( 101 ) peak counts of each measured xrd pattern.67the ratio of the if of the film with respect to that of the powder standard is defined as the oi.89when oi(hkl ) is larger than 1 , the film has a tendency to orient ( hkl ) planes in parallel with the substrate ; when smaller than 1 , just the opposite . pure cuscn thin films electrodeposited without rb show high oi(003 ) , indicating their preferential orientation of the c - axis perpendicular with the substrate , irrespective of the scn rich and cu rich bath compositions . such preferential orientation nicely matches with the arguments for their morphological features described above , namely , the hexagonal facet of the columnar grain in figure 3a1 and the bevels of the spiky grains in figure 3b1 corresponding to the ( 003 ) and ( 101 ) planes , respectively . minor addition of rb to the bath then drastically increases the oi(101 ) upon decrease of oi(003 ) , indicating the change of the crystallographic orientation to lay down the c - axis in parallel with the substrate . such preference is most prominent when [ rb ] = 0.3 mm , for both the scn rich and cu rich baths . however , the higher addition of rb abruptly changes the orientation back to the one in which the c - axis is perpendicular with the substrate in the case of the scn rich bath , whereas the oi(101 ) value only moderately decreases for the cu rich bath . consequently , highly porous cuscn thin films with totally different crystallographic orientations , namely , the c - axis perpendicular and parallel with the substrate are obtained for the highest end of the rb addition to the scn rich and cu rich baths , respectively . however , it is obvious that such changes are caused by the difference of the chemical stability of the facets of cuscn crystals in different environments , not only by the scn / cu balance but also by the added rb molecules . xrd patterns of cuscn and cuscn / rb hybrid thin films electrodeposited from scn rich ( a ) and cu rich ( b ) baths containing 0 ( a ) , 0.1 ( b ) , 0.2 ( c ) , 0.3 ( d ) , 0.4 ( e ) , 0.5 ( f ) , 0.7 ( g ) , and 1.0 mm ( h ) rb . change of crystallographic orientation of cuscn and cuscn / rb hybrid thin films by changing concentration of rb added to the scn rich and cu rich baths . the amount of rb loaded into cuscn during electrodeposition for 300 s from the scn rich and cu rich baths is plotted as a function of rb concentration ( figure 6 ) . the amount of rb precipitated together with cuscn increases as its concentration in the bath increases , although not proportionally but showing a leveling off trend . the loading efficiency in the scn rich bath is clearly higher than that in the cu rich , especially for the low rb concentration range . some properties of the cuscn / rb hybrid thin films electrodeposited from the scn rich bath are summarized in table 1 . change of the amount of rb loaded into cuscn as a function of rb concentration added to the scn rich and cu rich baths . calculated for = 500 rpm from levich equation by employing the bulk concentration of rb and its diffusion coefficient in water at 25 c ( 3.2 10 cm s ) . calculated by dividing the amount of rb loaded into the film by the amount of rb transported to the electrode surface in 300 s. calculated by using the formulas weight of cuscn ( 121.6 g mol ) , the density of cuscn ( 2.84 g cm ) , the molar weight of rb ( 479.02 g mol ) and the density of rb solid ( 0.79 g cm at 20 c ) . the volume of the void was calculated by subtracting the volumes of cuscn and rb from the total volume of the film determined from the film thickness . increase of the concentration of rb in the bath led to a slight decrease of the consumed charge because of the decrease of the steady - state current and the presence of the incubation time for [ rb ] = 1.0 mm . at the same time , faradaic efficiency for the precipitation of cuscn gradually decreased . the film thickness significantly decreased , as seen in the crosssectional sem pictures in figure 3 . while the amount of electrodeposited cuscn decreased , the amount of precipitated rb increased . the change of the film thickness , however , is mostly caused by the change of the porosity as seen in the volume ratio for cuscn , rb , and void for the hybrid film . as the diffusion coefficient of the rb molecule could be found only as measured in water solution , we took this value to calculate the flux of rb for its given concentration under the rotating condition employed in this study . the sticking efficiency of rb was then estimated by calculating the fraction of rb loaded into the film as compared to its maximum amount transported toward the electrode surface during the electrolysis . the highest efficiency of 17.5% was achieved for [ rb ] = 0.1 mm , and that value decreased as the bulk concentration got higher . it is obvious that the precipitation of rb is not under the control of its transport but rather its stability or rate of attaching to cuscn . when the molar amounts of precipitated cuscn and rb are determined , the fraction of the volume occupied by these components in the hybrid film can be calculated by taking their molar weight and density , either taking into account or not taking into account the total volume of the film . the cuscn : rb ratio gives a rough idea up to how much volume of the precipitated solid can be occupied by rb . an approximately 2:1 ratio seems to be reachable for this system , which is equivalent to that for the zno / eosin y hybrid system reported elsewhere . the cuscn : rb : void ratio correlates to the porosity of the film . as shown in the following section , the loaded rb molecules can be removed from the hybrid films with high rb loading , leaving some empty space within the cuscn grains . the voids in the as - deposited films occur from spaces between the cuscn / rb hybrid grains , while that occupied by rb is converted to the tiny pores formed inside cuscn grains after extraction of rb . when the hybrid films are soaked in dimethylacetamide ( dma ) , the films were bleached as the rb molecules were extracted , as seen from the change of the absorption spectra before and after the treatment ( figure 7 ) . for the films with small rb loading , rb molecules could be only partly removed , whereas those with high rb loading allowed almost complete desorption of rb . the film with the highest rb loading , namely , that deposited from the scn rich bath containing 1.0 mm rb , became nearly colorless because 97% of the loaded dye was removed as estimated from the absorbance change at the maximum of rb absorption . as discussed above , the extraction of rb should result in tiny nanopores enlarging the surface area of the cuscn films . teller analysis of kr sorption measurements ( figure 8) . the surface area of about 6 m g is already reached for the film deposited from the cu rich bath without rb . it only moderately increases by hybridization with rb and its extraction for the cu rich baths . on the other hand , that of the film deposited from the scn rich bath results in only about 4 m g without rb , and it greatly increases up to 12.6 m g when [ rb ] = 1.0 mm . the roughness factor ( rf ) , defined as the ratio of the actual surface area per projected film area , of about 17 is reached as calculated from the amount of electrodeposited cuscn shown in table 1 . considering the small film thickness of 0.8 m , this roughness factor is reasonably high because rf of several hundred can be reached when the thickness is increased to a few tens of micrometers . these thicknesses and rf values are comparable to those of materials typically used for dye - sensitized solar cells . using the reported growth conditions , we were able to extend the growth time and film thickness by roughly a factor of 5 before inducing any cracking in the film . transmission absorption spectra of as - electrodeposited cuscn / rb hybrid thin films ( solid lines ) and those after soaking the films for 1 h in dimethylacetamide ( dma ) ( dashed lines ) . the cuscn / rb thin films were electrodeposited from scn rich ( black lines ) and cu rich ( gray lines ) baths containing 0.1 ( thin lines ) or 1.0 mm ( thick lines ) of rb . change of specific surface area of cuscn / rb hybrid thin films after extraction of rb by dma treatment as determined by kr sorption measurements . as we have succeeded in obtaining highly porous crystalline cuscn thin films , it is of significant interest to check its usefulness as a dye - sensitized photocathode . however , no stably adsorbed sensitizer dye for efficient hole injection to cuscn has been previously discovered . we first checked fluorescence quenching for several fluorescent dye molecules attached to cuscn , such as fluorescein isothiocyanate ( fltc ) , cresyl violet ( cv ) , nile blue ( nb ) , 3,4,9,10-perylene tetracarboxylic acid diimine ( ptcdi ) , quinacridone ( qc ) , and rb . fltc can be coelectrodeposited with cuscn because of the presence of a soft basic thiocyanate moiety that is expected to act as an anchor to the soft acidic cu(i ) sites of cuscn . the electrodeposited cuscn / fltc hybrid thin film in fact exhibited dye - sensitized photocathodic behavior upon visible light illumination . addition of nb and cv to the deposition bath for cuscn also resulted in formation of hybrid thin films because of their cationic character by the presence of ammonium group , the same as that of rb . ptcdi and qc lack such chemical affinity to cuscn , so they were simply vacuum evaporated on top of the porous cuscn . all of these molecules exhibit fluorescence in solution , but nb and cv actually did not show measurable fluorescence in solid state . whereas no quenching was observed for ptcdi and qc , fluorescence of fltc and rb was strongly quenched for their electrodeposited hybrid thin films . in particular , that of rb was almost totally quenched as shown in figure 9 , while imperfect quenching was observed in the case of the cuscn / fltc hybrid ( see figure 2s of supporting information for the spectra ) . because the valence band edge of cuscn is expected to lie around 5.3 ev ( vs vac ) , the highest occupied molecular orbital level of all these dye molecules is deep enough to transfer holes to cuscn from a thermodynamic point of view . it is supposed that the strong chemical affinity of fltc and rb to cuscn is crucial for hole injection to take place actually , unlike ptcdi and qc which are only in physical contact with the surface of cuscn . absorption and fluorescence spectra of electrodeposited cuscn / rb hybrid thin film ( solid lines ) , solid rb thin film prepared by spin coating ( dashed lines ) , and 20 m rb solution in ethanol ( dotted lines ) . the excitation wavelength for the film samples and the solution were 535 and 530 nm , respectively . the fluorescence spectrum of the rb solution is attenuated for better comparison with other spectra because it is far stronger than the other samples . monomeric rb molecules in solution exhibit a sharp absorption peak at 545 nm and a weak shoulder around 505 nm . a strong and sharp fluorescence spectrum occurs like a mirror image of the absorption spectrum , exhibiting a peak at 574 nm and a weak shoulder around 620 nm . the solid thin film of rb aggregate shows a characteristic change of the absorption spectrum with a maximum at 579 nm and another distinctive peak at 541 nm . it has been shown that dimerization of rb molecules in solution leads to an increase of the abosorbance of the short side shoulder because of enhanced intermolecular interaction in parallel arrangement of the chromophore , namely , in h - aggregate form . the great enhancement of the blue - side shoulder band to become a clearly distinguished satellite peak for the solid rb film is obviously caused by the strong electronic interaction in the molecular solid in h - aggregate form . the sharp peak at 613 nm should reflect a monomolecularly confined fluorescence band , which is also red - shifted compared to that of the rb monomer solution . on the other hand , the broad character centered at 687 nm should be related to the transition involving the intermolecular band , which exists only in the aggregates . the absorption spectrum of the electrodeposited cuscn / rb hybrid thin film closely resembles that of the solid rb thin film , exhibiting a character for the h - aggregate . however , both peaks are blue - shifted to 536 and 570 nm for aggregate and monomer bands , respectively , indicating different environments of the rb molecules which are subjected to chemical interaction with cuscn . because characteristic fluorescence was clearly monitored for rb in the solid state , the total quenching of the fluorescence in the hybrid film should be caused by hole transfer from the exited state of rb to the valence band of cuscn . the electrodes were the pure cuscn with a relatively high surface area , that electrodeposited from cu rich bath without rb , and a highly porous cuscn made by electrodeposition from a scn rich bath containing 1.0 mm rb which then was removed by the dma treatment . thus , we prepared a dye - sensitized photocathode with the rb dye shown to undergo photoinduced hole transfer . however , the lack of a suitable redox electrolyte is a significant problem for studying photocathodic sensitization of cuscn . iodide / triiodide redox electrolyte typically used in dsscs reacts with cuscn to convert it to cui that dissolves rather well in many polar organic solvents . adsorption of rb molecules to cuscn was unfortunately not so stable , as recognized by the fact that the electrolyte solution was clearly colored by desorbed rb both for organic and aqueous solutions . for this reason , it was not useful to use co complex redox systems , which are kinetically highly reversible , because fast recombination from the uncovered surface of cuscn or exposed fto actually killed the voltage under illumination . stably adsorbed highly rectifying dyes are needed for effective use of co redox , which are missing at this stage . thus , we have employed an aqueous solution of methylviologen chloride as the electron acceptor . the electrode was reasonably stable in this electrolyte and exhibited a clear photocathodic action under visible light illumination as shown in figure 10 . pure cuscn film sensitized with rb exhibits about 0.2 v photocurrent onset voltage and 22 a cm photocathodic current ; these values increased to ca . 0.3 v and 32 a cm for the porous cuscn electrode prepared by hybrid electrodeposition with rb , extraction of rb , and readsorption of rb . clearly , enlarged surface area made the color of the film more intense than that of the pure cuscn , obviously contributing to the increased current . poorly rectified dark current for pure cuscn ( figure 10a ) can be associated with the the bare fto surface being in contact with the electrolyte , allowing direct charge transfer to methylviologen . because the porous crystalline cuscn ( figure 10b ) covers the fto better than pure cuscn , the dark current is well - rectified and higher voltage is achieved . despite efforts to find a stable dye electrolyte solution combination , the rb dye still desorbs from the surface during measurement , likely contributing to the unusually low photocurrent . i v curves measured at bulk cuscn ( a ) electrodeposited without rb and porous crystalline cuscn ( b ) electrodeposited with 1.0 mm rb and rb subsequently removed by dma treatment , measured in the dark ( hashed lines ) and under illumination ( solid lines ) with visible light ( 100 mw cm ) generated by a 500 w xe lamp equipped with uv and ir cutoff filters . the cuscn films were sensitized by readsorbing rb by soaking in a 0.5 mm rb aqueous solution for 1 h. the electrolyte was a deaerated aqueous solution of 0.1 m methylviologen chloride . we have developed a method for obtaining a highly porous and nicely crystallized p - cuscn thin film by electrodeposition . by selection of the bath composition , it has become possible to load rb molecules in an amount to occupy as much as 30% of the total volume of precipitated solid , from which the loaded rb could be removed by soaking the film in dma . such a process can now be regarded as a counterpart of the entire technology developed for zno / eosin y hybrid electrodeposition for processinghigh - performance porous crystalline zno - based dye - sensitized photoanodes . porous structures are created by the rb molecules within highly crystallized cuscn . not only the porous nanostructure but also the crystallographic orientation of cuscn could be controlled by the choice of the bath composition . the resulting porous crystalline cuscn thin film has been utilized as dye - sensitized photocathode to prove its usefulness as a counterpart of dye - sensitized zno photoanodes . however , limited surface area as well as the lack of good hole - injecting dye stable on the cuscn surface and suitable redox electrolyte remain as challenges for efficiency improvement of the photocathode . because recent studies have discovered a good sensitizer for p - nio in combination with co complex redox systems , there is a good hope of finding one for p - cuscn . the successfully constructed efficient dye - sensitized cuscn photocathode can be combined with existing dye - sensitized photoanodes to achieve high - efficiency tandem dsscs in a very simple structure sandwiching a common redox electrolyte solution by two photoelectrodes .

nanostructured hybrid thin films of cuscn and rhodamine b ( rb ) are electrochemically self - assembled ( esa ) by cathodic electrolysis in an ethanol / water mixture containing cu2 + , scn , and rb . by selecting the solvent , cu2+/scn ratio , and the concentration of rb , we demonstrate several control parameters in the film formation . high loading of rb into the film has been achieved to reach a cuscn : rb volume ratio of approximately 2:1 . the rb solid could almost completely be extracted from the hybrid film by soaking the film in dimethylacetamide ( dma ) , leading to a large increase of the surface area . the crystallographic orientation of the nanostructure with respect to the substrate can be controlled . efficient quenching of fluorescence of rb has been observed for the cuscn / rb hybrid film , implying hole injection from rb excited state to cuscn . photoelectrochemical study on the porous crystalline cuscn obtained after the dma treatment and sensitized with rb revealed sensitized photocathodic action under visible light illumination , indicating the potential usefulness of the porous cuscn electrodes for construction of tandem dye - sensitized solar cells .

pubmed

the nasopharyngeal bursa originates from the area of communication between the notochord and the foregut , ie , the pharyngeal endoderm.1,2 it is believed to be a remnant of embryonic communication between the notochord and the roof of the pharynx , that normally disappears during the second month of intrauterine fetal development . 3 a tornwaldt s cyst develops if the embryonic remnant becomes obstructed ( cystic type ) , and if crusts adhere to the orifice without obstruction , this will form the crust type.4 eagle5 in 1939 indicated that in the crust type the orifice of the bursa is not obstructed despite crust formation . this crust sheds periodically from the nasopharynx , causing an offensive smell and unpleasant taste . the cystic type of nasopharyngeal bursitis is more common and should form part of the differential diagnosis of nasopharyngeal abscesses and cysts . although most patients are symptom - free , some will develop bursitis , a cyst , or an abscess . therefore , the symptoms are of either a space - occupying lesion ( nasal obstruction , eustachian tube obstruction with secretory otitis media , dysphagia , cranial nerve paralysis ) or rhinitis ( choanal discharge , halitosis , pharyngitis , laryngitis , bronchitis , gastritis ) . being located at the posterior wall of the nasopharynx and extending toward the tubercle of the occipital bone , pulsating headache and occipital pain felt at the external occipital tuberculum is often reported . the incidence of nasopharyngeal bursitis is approximately 4% in adults , but it can appear at any age . the peak incidence occurs between 15 and 30 years , with a caucasian predominance and no gender predilection.5,6 nonetheless , most of the published literature comprises selected case reports and describes the cystic type . no single report to the authors knowledge has estimated the true incidence of crust - type nasopharyngeal bursitis or characterized its symptoms . in this clinicoanatomical audit , the aim was to identify the incidence of nasopharyngeal bursa disease , its different types , and its clinical presentation over two years in a single rhinology practice . this was a two - year clinicoanatomical audit of nasopharyngeal bursitis at a rhinology practice in a university unit during 20082009 . all patients diagnosed to have nasopharyngeal bursitis confirmed by endoscopic examination of the nasopharynx and computed tomography scan / magnetic resonance imaging were asked carefully about symptoms of tornwaldt s syndrome . a surgical technique combining an endonasal and transoral approach in the form of disruption of the bursa with electrocauterization was performed to patients presenting with either the cyst type or the crust type . surgical samples of bursitis tissue were fixed in 10% formalin , and paraffin wax blocks were performed . routine hematoxilyn staining of the nasal biopsy was then performed and specimens examined under low and high power fields . representative photos from the slides were taken by a camera attached to software as shown in figure 2 . this was a two - year clinicoanatomical audit of nasopharyngeal bursitis at a rhinology practice in a university unit during 20082009 . all patients diagnosed to have nasopharyngeal bursitis confirmed by endoscopic examination of the nasopharynx and computed tomography scan / magnetic resonance imaging were asked carefully about symptoms of tornwaldt s syndrome . a surgical technique combining an endonasal and transoral approach in the form of disruption of the bursa with electrocauterization was performed to patients presenting with either the cyst type or the crust type . surgical samples of bursitis tissue were fixed in 10% formalin , and paraffin wax blocks were performed . routine hematoxilyn staining of the nasal biopsy was then performed and specimens examined under low and high power fields . representative photos from the slides were taken by a camera attached to software as shown in figure 2 . results of the current audit ( see table ) indicated that six patients ( four males and two females , mean age 54 years ) were diagnosed to have nasopharyngeal bursitis . the incidence was less than 1% of all rhinitis patients seen during the study period . two patients with classical tornwaldt s cyst and one with a fibrosed tornwaldt s cyst were identified , while three patients with crust - type bursitis were diagnosed . the classical cyst type presented with postnasal discharge resulting in hemming , throat irritation , and cough , while the fibrosed type was discovered incidentally in a patient who complained of snoring . on the other hand , the crust type presented with crust expectoration and retching , with or without fetid postnasal discharge , and occasional occipital pain that lasts for a few days , with symptom - free intervals of a few days . for both types of disease , patients who underwent surgery using a combined endoscopic nasal and transoral approach had a complete recovery with no recurrence of symptoms at more than one year postoperatively ( figure 1c ) . interestingly , our audit showed an equal incidence of the cystic and the crust type of the disease . endoscopic examination of the nasopharynx after proper nasal decongestion will clearly show either a cyst or ulcer - like lesion covered with crusts , in the midline of the posterior wall of the nasopharynx ( figures 1a and 1b ) . radiologic investigation ( simple lateral view x - ray , computed tomography scan , and magnetic resonance imaging ) is useful in showing adhesion of the bursa to the cervical vertebrae . although several surgical approaches with good outcomes have been described,69 we believe that the combined endoscopic endonasal and transoral approach with good electrocauterization is a minimally invasive and effective way of treating both types of nasopharyngeal bursitis ( figure 1c ) . nasopharyngeal bursa originate at the interface between the embryologic tissue from which the vertebrae develop . notochord formation is an important change in the embryonic disc that takes place in the third week , and this is used by the embryo as a temporary axial skeleton . during migration of the intraembryonic mesoderm , the node of hansen develops at the cephalic primitive streak , giving rise to the notochord process , in which a small central canal is formed . this canal connects the amniotic cavity and the yolk sac cavity . from this notochordal process , a rod - like solid definitive notochord becomes detached from the endoderm to lie in a position between the ectoderm and endoderm in the midline . this definitive notochord later becomes the permanent vertebral column . in approximately 3%4% of embryos , an invaginated connection remains in the nasopharynx connecting the pharyngeal epithelium with the remnants of the notochord . thus , the lesion is located in the middle of the posterior wall of the nasopharynx and extends to the tubercle of the occipital bone . syndrome10 describes a group of symptoms resulting from inflammation , or cystic or abscess formation . in 1939 , eagle5 indicated that the orifice of the bursa is not obstructed in the crust type , despite the crust formation . this crust will shed from the nasopharynx , causing a bad smell and an unpleasant taste . in this audit , three patients presented with retching , expectoration of crusts , and/or fetid postnasal discharge and occipital pain , with no other described symptoms of nasopharyngeal bursitis . the crust reforms quickly , only to shed again every few days with completely symptom - free intervals in between . although most patients with nasopharyngeal bursa remain symptom - free , trauma to the nasopharynx in the form of nasal packing or adenoidectomy may result in obstruction of the bursa orifice and cyst formation . interestingly , our patients developed their symptoms at an advanced age with no history of an initiating event , such as nasal packing or surgical trauma . also , they remained completely symptom - free for years and presented suddenly with disturbing crusts of the nasopharynx . this indicates that the crust type could be a distinct variant of the disease that may develop at any time , and without recognized predisposing factors . the cystic type of nasopharyngeal bursitis in our study was found to have two subtypes . one type is the classical cyst with postnasal discharge , throat irritation , and cough , while the other fibrosed type presented with irritation of the nasopharyngeal area and snoring . histopathologically , a tornwaldt s cyst can be differentiated from a retention lymphoid cyst in that the former appears as an epithelial cyst lined with columnar epithelium on the surface11 while the latter shows lymphoid tissue only . however , the histologic picture of the crust type is not known . in figure 2 , we show the histologic appearance of the crust type that demonstrates reactive lymphoid mucosa with necrotic tissue . interestingly , reactive lymphoid mucosa was also demonstrated in biopsies from the adjacent tissues , which may explain the cicatricial streaks around the bursa . this interesting histologic finding presents clinically as rapid formation of crusts with shedding and postnasal drip . in conclusion , the crust type is a recognized form of the disease and may produce no symptoms other than retching and irritative expectoration of crusts . awareness of the crust type of nasopharyngeal bursitis would pick up many missed cases and increase appropriate referrals between generalists , pulmonologists , and otolaryngologists . proper endoscopic nasopharyngeal examination with computed tomography and magnetic resonance imaging remains the best method of clinical assessment . nasopharyngeal bursitis should be differentiated from nasopharyngeal carcinoma , which can mimic the ulcerative nature of nasopharyngeal bursitis , but usually does not form overlying crusts . the lack of symptoms , such as epistaxis and metastatic lymphadenopathy , as well as the characteristic midline anatomic site in the nasopharynx , are all in favor of the diagnosis of crust - type nasopharyngeal bursitis . endoscopic interruption of the bursa with electrocauterization at the base is a minimally invasive technique that would suffice , and allows a better view of the operative field .

nasopharyngeal bursitis is a relatively rare syndrome characterized by a collection of symptoms that multidisciplinary specialists should be aware of . here we present an audit of cases presenting to a rhinology clinic over a two - year period , as well as an overview of the relevant embryology and different clinical presentations of nasopharyngeal bursitis . for 20082009 , six patients were diagnosed to have nasopharyngeal bursitis , including four males and two females , of mean age 54 years . two distinct pathologic types were observed , comprising three patients with classical tornwaldt s cyst and three with crust - type bursitis . this audit highlights the importance of recognition of the crust - type of nasopharyngeal bursitis and its anatomic and clinical features . a combined endonasal and transoral endoscopic approach is a minimally invasive procedure and an effective method of treating both types of the disease . our findings are discussed in relation to the embryology of the disorder , with a clinical emphasis on crust- type nasopharyngeal bursitis .

pubmed

the incidence of melanoma skin cancer has been increasing over the past few decades [ 13 ] . estimated 76,250 new cases of invasive melanoma were diagnosed in usa in 2012 , with an estimated number of 9,180 that result in death . australia has one of the highest rates of skin cancer in the world . over 1,890 the most dangerous characteristic of melanoma is that it can spread widely over the body via the lymphatic vessels and blood vessels . thus , early diagnosis of melanoma is a key factor for the prognosis of the disease . however , dermoscopy is a noninvasive diagnostic technique that links clinical dermatology and dermatopathology by enabling the visualization of morphological features which are not discernible by examination with the naked eye . there are different techniques , like solar scan , epiluminescence microscopy ( elm ) [ 10 , 11 ] , cross - polarization epiluminescence ( xlm ) , and side transillumination ( tlm ) [ 12 , 13 ] , that can greatly increase the morphological details that are visualized . dermoscopy enables better diagnosis as compared to unaided eye [ 1416 ] with an improvement in diagnostic sensitivity of 1030% . however , it has also been demonstrated that dermoscopy may actually lower the diagnostic accuracy in the hands of inexperienced dermatologists [ 10 , 1820 ] , since this method requires great deal of experience to differentiate skin lesions . as described in [ 9 , 22 ] only experts have arrived at 90% sensitivity and 59% specificity in skin lesion diagnosis , while for less trained doctors these figures show significant drop till around 62%-63% for general practitioners . the main problem is that the diagnosis is highly dependent on subjective judgement and is scarcely reproducible [ 23 , 24 ] . several scoring systems and algorithms such as the abcd - e rule [ 2527 ] , the seven - point checklist [ 2830 ] , three - point checklist , and the menzies method [ 32 , 33 ] have been proposed to improve the diagnostic performance of less experienced clinicians . although this simplification has enabled the development of these diagnostic algorithms with good accuracy , still they showed problems that have not yet been solved . the most important shortcoming is that the purpose for which they were designed was not achieved , because the within- and between - observer concordance is very low , even for expert observers [ 10 , 25 , 34 , 35 ] . despite extensive research in investigating the varied presentations and physical characteristics of melanoma thus , a growing interest has developed in the last two decades in the automated analysis of digitized images obtained by elm techniques to assist clinicians in differentiating early melanoma from benign skin lesions . application of computational intelligence methods helps physicians as well as dermatologists in faster data process to give better and more reliable diagnoses . studies related to the automated classification of pigmented skin lesion images have appeared in the literature as early as 1987 . after some successful experiments on automatic diagnostic systems for melanoma diagnosis [ 3642 ] , utility of machine vision and computerized analysis is getting more important every year . the importance of the topic is patent if we analyse the enormous quantity of research works related with the melanoma diagnosis . numerous computerized diagnostic systems have been reported in the literature where different border detection , feature extraction , selection , and classification algorithms are used . some researchers [ 37 , 4348 ] reviewed and tried to critically examine image analysis techniques for diagnosis of skin cancer and compared diagnostic accuracy of experts dermoscopists with artificial intelligence and computer aided diagnosis . more research , however , is needed to identify and reduce uncertainties in the automatic decision support systems to improve diagnosis accuracy . a comprehensive up - to - date review of automatic diagnostic model for skin lesions is not available . continuous emergence of new classification algorithms and techniques for dermoscopic image analysis in recent years necessitates such a review . this paper describes a standard automatic decision support system which is based on semantic analysis of melanoma images and further classification of characteristic objects commonly found in pigmented skin lesions . the aim of this review is to summarize and compare advanced dermoscopic algorithms used for the classification of skin lesions and discuss important issues affecting the success of classification . a brief and comprehensive review of feature extraction and selection algorithms that are so far being used for extracting various features of malignant melanoma analysis of various papers is performed with respect to several criteria , such as lesion segmentation , feature extraction , size of data sets , classification techniques , and performance measures used in reporting the diagnosis results . this paper will provide a framework that represents a comprehensive guideline for selecting suitable algorithms needed for different steps of automatic diagnostic procedure for ensuring timely diagnosis of skin cancer . performance evaluation measures and model validation details are presented for analysing various algorithms / models and finally concluding comments are provided . predictive models are used in a variety of medical domains for diagnostic and prognostic tasks . the data can be preprocessed and expressed in a set of rules , such as that it is often the case in knowledge - based expert systems , and consequently can serve as training data for statistical and machine learning models . the general approach of developing a cad system for the diagnosis of skin cancer is to find the location of a lesion and also to determine an estimate of the probability of a disease . the first step in this paper was to establish a standard general scheme of a cad system for skin lesions . the inputs to the computer aided system are digital images obtained by elm , with the possibility to add other acquisition system such as ultrasound or confocal microscopy . in the first phase preprocessing of image is done that allows reducing the ill effects and various artifacts like hair that may be present in the dermoscopic images . once the lesion is localized , different chromatic and morphological features can be quantified and used for classification . differentiation of malignant melanoma images demands very fast image processing and feature extraction and classification algorithms . a detailed research is necessary to make the best choice and to set the benchmarks for diagnostic system development and validation . the following section focuses on the description of the major steps that may be involved in skin cancer diagnosis . numerous imaging modalities are under investigation to determine their usefulness in imaging and ascertaining a correct in vivo diagnosis of melanoma . these include total cutaneous photography , dermoscopy , confocal scanning laser microscopy ( cslm ) , ultrasound , magnetic resonance imaging ( mri ) , optical coherence tomography ( oct ) , and multispectral imaging . we have provided here a bird eye view of the currently available cutaneous imaging devices and new frontiers in noninvasive automated diagnosis of melanoma in table 1 . readers may refer to [ 33 , 4952 ] for analysing performance comparison of some of the existing screening techniques . relative to other specialties , dermatologists have been slow to adopt advanced technologic diagnostic aids . sometimes simple elm does not sufficiently increase the diagnostic accuracy in distinguishing pigmented spitz nevus ( psns ) from melanoma . for obviating the problems of qualitative interpretation , methods based on the mathematical analysis of pigmented skin lesions ( psls ) , such as digital dermoscopy analysis ( dda ) and d - elm , efficient image processing techniques must therefore be developed to help physicians in making a diagnosis . the introduction of digital elm and sophisticated image processing software has opened up a new horizon in the evaluation of cutaneous benign and malignant pigmented skin lesions ( psls ) as it enables the observation , storage , and objective evaluation of many parameters . in this paper we have focussed on automatic diagnostic system based on digital dermoscopy images normally collected from different dermoscopy atlases [ 55 , 56 ] or from dermatologists since it is the most widely used . however , we anticipate that multimodal systems that combine different imaging technologies will further improve the ability to detect melanoma at an earlier stage and reduce the trauma of dermatologic diagnosis . the main processing step towards a complete analysis of pigmented skin lesion is to differentiate the lesion from the healthy skin . detection of the lesion is a difficult problem in dermatoscopic images as the transition between the lesion and the surrounding skin is smooth and even for trained dermatologist ; it is a challenge to distinguish accurately . it has been observed that dermoscopy images often contain artifacts such as uneven illumination , dermoscopic gel , black frames , ink markings , rulers , air bubbles , and intrinsic cutaneous features that can affect border detection such as blood vessels , hairs , and skin lines and texture . these artifacts and extraneous elements complicate the border detection procedure , which results in a loss of accuracy as well as an increase in computational time . thus , it requires some preprocessing steps to facilitate the segmentation process by the removal of unwanted objects or artifacts and colour space transformation . everything that might corrupt the image and consequently affect the results of image processing must be localized and then removed , masked , or replaced . many approaches can be used that include image resizing , masking , cropping , hair removal ( or attenuation ) , and conversion from rgb color to intensity grey image . it is meant to facilitate image segmentation by filtering the image and enhancing its important features . the most straightforward way to remove these artifacts is to smooth the image using a general purpose filter such peer group filter ( pgf ) , mean filters , median filter [ 5860 ] , gaussian filters [ 61 , 62 ] , or anisotropic diffusion filters ( adf ) . a major issue with these aforementioned filters is that these filters are originally formulated for scalar images . for vector images one can apply a scalar filter to each channel independently and then combine the results , a strategy referred to as marginal filtering . despite being fast another noteworthy thing is setting mask size proportional to the image size to manage a tradeoff between smoothing of image and blurring of edges . inspite of taking care of all the forementioned things , it is still not guaranteed to get an image free of all artifacts . an alternative strategy for artifact removal is to use specialized methods for each artifact type . many methods have been suggested ; very few [ 6466 ] discussed different aspects of artifacts together , but none of them have discussed all cases of artifacts . for this rationale , we have presented an overview of effective preprocessing methods , namely , color space transformation , color quantization , contrast enhancement , and artifact removal , which are being used for reducing all the possible ill effects present in the dermoscopic images . dermoscopy images are commonly acquired using a digital camera with a dermatoscope attachment . due to the computational simplicity and convenience of scalar ( single channel ) processing , the resulting rgb ( red - green - blue ) color image is often converted to a scalar image using different methods like retaining only the blue channel as lesions are often more prominent in this channel or applying the luminance transformation or karhunen - love ( kl ) transformation and retaining the channel with the highest variance . skin lesions come in a variety of colors but absolute colors are not very useful in segmenting images . normally the analysis is based on changes in color within the lesion or with the surrounding skin particularly color changes belonging to the lesion boundary . therefore , it is quite common to transform the images that are in rgb color coordinates into other color spaces like ciel*a*b * , ciel*u*v * , kl , and hsi ( hue - saturation - intensity ) . typical 24-bit color images have thousands of colors , which are difficult to handle directly . for this reason color quantization is commonly used as a preprocessing step for color image segmentation . the process of color quantization consists of two - phases palette design ( i.e. , selection of a small set of colors that represents the original image colors ) and pixel mapping ( i.e. , assignment of one of the palette colors to each input pixel ) . . showed that , for skin lesion , the color quantization method should reduce the number of colors in image to 20 for getting precise quantization . one of the factors that complicate the detection of borders in dermoscopy images is insufficient contrast . the contrast of image is enhanced to ensure that edges of the lesion are eminence . gmez et al . proposed a contrast enhancement method based on independent histogram pursuit ( ihp ) . an easy , yet powerful way to enhance the image contrast another very popular technique is histogram equalization , which alters pixel values to achieve a uniform distribution . homomorphic filtering , fft , and high pass filter can be used to compensate for uneven illumination or specular reflection variations in order to obtain the high contrast lesion images . for the removal of black frames produced in the digitization process , celebi et al . [ 59 , 70 ] proposed an iterative algorithm based on the lightness component of the hsl ( hue - saturation - lightness ) color space . in order to remove air bubbles and dermoscopic gels , adaptive and recursive weighted median filter developed by dehghani tafti and mirsadeghi can be utilized . a method that can remove bubbles with bright edges was introduced in where the authors utilized a morphological top - hat operator followed by a radial search procedure . line detection procedure based on the 2d derivatives of gaussian ( dog ) and exemplar - based object removal algorithm can be used for removing dark lines like ruler marking . in most cases one of the most undesirable elements that are most commonly present in dermatoscopic images is hair . erosion / dilation with straight line segments can efficiently eliminate ( or at least weaken the effect of ) hairs [ 77 , 78 ] . [ 79 , 80 ] suggested a scheme based on a morphological closing operator , while in they applied to the three components of the l*u*v * uniform color space . however , it is being observed that most of these techniques often leave behind undesirable blurring ; disturb the texture of the tumor ; and result in color bleeding . due to these problems , it is very difficult to use the color diffuse image for further skin tumor differentiation . in contrast , a new artifact removal algorithm that focuses on accurate detection of curvilinear artifacts and pays special attention to lesion structure during the removal stage has been introduced by zhou et al . . this approach effectively removes artifacts such as ruler markings and hair , but it has high computational requirements . to address all these issues abbas et al . abbas et al . presented a comparative study about hair removal methods which indicate that hair - repairing algorithm based on the fast marching method achieves an accurate result . all the above mentioned strategies are meant to facilitate the segmentation and feature extraction stages which consequently lead to better diagnostic results . segmentation refers to the partitioning of an image into disjoint regions that are homogeneous with respect to a chosen property such as luminance , color , and texture . the goal of segmentation is to simplify and/or change the representation of an image into something that is more meaningful and easier to analyse . some researchers argued that manual border detection is better than computer - detected borders in order to separate the problems of feature extraction from the problems of automated lesion border detection . however , for the development of automated diagnostic system for skin lesion detection , it is very important to develop automatic segmentation algorithms . as segmentation is a crucial early step in the analysis of lesion images , it has become one of the important areas of research and many algorithms and segmentation techniques are available in the literature . we have briefly provided an overview of various segmentation algorithms being used for dermoscopic image analysis as tabulated in table 2 . several comparative studies [ 59 , 61 , 66 , 88 , 89 ] are also present in the literature which provides performance analysis of several segmentation algorithms . there are several issues that should be kept in mind for selecting a suitable algorithm , for example , scalar versus vector processing , automatic versus semiautomatic , and the number of parameters whose values need to be determined a priori . interested readers may check relevant references to identify a suitable approach for a specific study . it is important to identify the most effective features to extract from melanoma , melanoma in situ and clark nevus lesions , and to find the most effective pattern - classification criteria and algorithms for differentiating those lesions . thus , the next stage of the image analysis process is to extract the important features of the image . the purpose of feature extraction is to reduce the original data set by measuring certain properties , or features , that differentiate one input pattern from another . the feature extraction is performed by measurements on the pixels that represent a segmented object allowing various features to be computed . unfortunately , the feature extraction step is often subject to error . in most of the publications dealing with this topic , many features are extracted to feed a sophisticated classifier , but there is very little discussion about the real meaning of those features and about objective ways to measure them . thus , we investigate this topic in detail to come up with a guideline for future research . different feature extraction methods found in the literature include statistical and model - based and filtering - based methods , among which multichannel filtering is the most efficient and accurate one . various researchers used principal component analysis ( pca ) of a binary mask of the lesion , wavelet packet transform ( wpt ) [ 9094 ] , grey level cooccurrence matrix ( glcm ) [ 61 , 95 ] , fourier power spectrum , gaussian derivative kernels , and decision boundary feature extraction [ 98100 ] in order to reduce data redundancy . some of the typically used filter banks are laws masks , the dyadic gabor filter bank , and wavelet transform . a particular problem in the related literature is that a significant number of studies do not report the details of their feature extraction procedure ; see table 6 . the abcd - e system [ 25 , 26 , 102 ] , 7-point checklist [ 29 , 103 ] , 3-point checklist , pattern analysis , and menzies method offer alternative approaches in deciding the differentiating features that need to be extracted . according to the conclusion made by johr , the automatic extraction of characteristics that take into account the rule abcd [ 25 , 102 , 106 ] is computationally less expensive than the ones that take into account 7-point checklist [ 29 , 103 ] or the menzies method [ 32 , 107 ] . furthermore , the reliability in the clinical diagnosis is very high for abcd - e rule . so , most of the automated decision support systems also use abcd rule as the base of their feature extraction step . however , abcd is more prone to over classification of atypical melanocytic nevi as melanomas . . showed , in a comparative study , that menzies method achieved the highest sensitivity , 84.6% , for the diagnosis of melanoma , followed by the 7-point checklist ( 81.4% ) , the abcd rule ( 77.5% ) , pattern analysis ( 68.4% ) , and assessment of a macroscopic image ( 60.9% ) . pattern analysis and assessment of the macroscopic image showed the highest specificity , 85.3% and 85.4% , respectively . so many researchers [ 109114 ] are trying to develop efficient automatic diagnostic systems based on 7-point criteria and pattern analysis . numerous methods for extracting features from clinical skin lesion images have been proposed in the literature as figure 2 illustrated the distribution of features used in dermoscopic studies . several studies have also proven the efficiency of border shape descriptors for the detection of malignant melanoma on both clinical- and computer - based evaluation methods [ 115 , 116 ] . very simple parameters , such as area and perimeter , are extracted in [ 117119 ] . measurements of shape features are also used like fragmentation index [ 120122 ] , thinness ratio / circularity factor [ 61 , 123125 ] asymmetry index [ 77 , 116 , 122 ] , aspect ratio [ 118 , 126 ] , compactness [ 118 , 126 ] , symmetry axis , bulkiness score , irregularity index [ 129 , 130 ] , fractality of borders , convex hull ratio , and skin line pattern . some groups use the sharpness of the transition from the lesion interior to the skin [ 61 , 123 , 125 ] as descriptors of the structure and irregularity of the border . lacunarity is another measure that can be used to characterize a property of fractals and quantifies aspects of patterns that exhibit changes in structure . color features are mainly statistical parameters calculated from different colour channels , like average value and standard deviation of the rgb [ 120124 ] or hsv colour channels . other color features used in different studies include colour asymmetry , centroidal distance , and luv histogram distance . cotton and claridge found that all normal skin colours lie on a two - dimensional surface patch within a three- dimensional ( 3d ) colour space ( cie - lms ) . some researchers [ 61 , 117 , 118 , 134 , 135 ] used glcm - based texture features [ 136138 ] like dissimilarity , contrast , energy , maximum probability , correlation , entropy , and so forth . parameters for the description of dermatoscopic structures and elm criteria are difficult to find in the literature . major issues are concerned with the difficulty in relating such information as lesion shape and color to medical structures ( tissues , vessels , etc . ) which experts are more familiar with . some of the dermoscopic feature extraction studies include atypical pigment networks [ 72 , 110 , 139 ] , globules / dots / blotches [ 72 , 140143 ] , streaks , granularity , and blue - white veil [ 87 , 146 ] . it is noteworthy that diagnostic systems based on extraction of critical high level features show an increase in the diagnostic accuracy of computerized dermoscopy image analysis systems . thus , in addition to general features like area , border , shape , and color , these high level features should also be integrated in the automated diagnostic system to gain greater clinical acceptance . some researchers used some unique features for classification , but we know from skin cancer research that a unique feature is not sufficient to diagnose precisely skin cancer and that the combination of different criteria is the key to the early detection of malignant melanoma and other types of skin cancer . the evolution of competing dermoscopic algorithms with variable definitions of specific attributes complicates dermoscopic diagnosis . it is necessary to identify features that are the most reproducible and diagnostically significant and formulate them into a single algorithm . for clinical purposes , similarly , features selection is a critical step for successfully distinguishing between malignant melanoma , benign , and dysplastic nevi . many potential features may be used , but it is important to select a reasonable reduced number of useful features while eliminating redundant , irrelevant , or noisy features . however , it is important to make sure that there may not be loss of significant information . from the classification perspective , there are numerous potential benefits associated with feature selection : ( i ) reduced feature extraction time and storage requirements , ( ii ) reduced classifier complexity for better generalization behaviour , ( iii ) increased prediction accuracy , ( iv ) reduced training and testing times , and ( v ) enhanced data understanding and visualization . there are many methods available for feature selection which include principle component analysis and search strategies like sequential forward selection ( sfs ) , sequential backward selection ( sbs ) , plus - l - take - away - r ( pta ( l , r ) ) , floating search methods [ 54 , 151 ] , sequential forward floating selection ( sffs ) , sequential backward floating selection ( sbfs ) ) and fisher score ranking . all these algorithms use stepwise inclusions and exclusions of features into / from the subset of consideration , but they differ in their strategy of applying them . although the floating methods are considered to be more intelligent , they are still suboptimal and even more there is no warranty that they yield better results . in addition to these , some of the filter - based methods include relieff , mutual information - based feature selection ( mifs ) , and correlation - based feature selection ( cfs ) . filter methods are usually very fast and allow one to compare several alternative methods within an optimization framework . it is possible , and also desirable , to use clinical criteria or statistical methods to reduce the number of candidate variables , thus reducing the risk of an overoptimistic model . a particular problem in the related literature is that there is very little number of studies that report the details of their feature selection procedure . normally we do not find details of feature selection procedures that are used for choosing the appropriate features for skin cancer diagnosis . handels et al . described feature selection as an optimization problem and compared several approaches including heuristic strategies , greedy and genetic algorithms . zagrouba and barhoumi proposed an accelerated system for melanoma diagnosis based on subset feature selection . the number of features retained by the feature selection algorithm ( k ) is an important parameter . sometimes a small number of features are not likely to discriminate between the classes well . on the other hand , a large number of features might lead to overfitting . green et al . showed by calculating correlation coefficients that the size of the lesion is the most important feature in their system . they achieve a tremendous reduction to five features starting with 87 features calculated from surface profiles of skin lesions . used sbfs and sffs and showed that the best selection performances were with subset size of between 10 to 15 and performance degrades with subsets size of more than 20 features . on the other hand by inspecting individual sensitivities on the malignant class of several subset sizes , it turns out that an acceptable performance is only achieved with subsets of more than 20 features . while celebi et al . showed by using cfs feature selection algorithm that auc peaks can be obtained with the use of 18 features and inclusion of features beyond this value does not add much to the classifier performance . presented a study particularly based on feature selection for melanoma recognition and showed a strong increase in performance for small subsets followed by a slight increase up to medium sized subsets . larger subsets cause a drop in the recognition rate . ruiz et al . also confirmed this thing in the evaluation done using sbfs and sffs and showed that minimum error rate was observed using subset of 6 features and a significant increase in classification error rate is observed by using a subset of more than 20 features . by inspecting the overall achieved performances one even could imagine that using 5 to 20 features is enough to get acceptable classification results . the aim of feature selection is to find the optimum number of features to obtain the best achievable performance ( i.e. , recognition rate ) in classification . therefore , the feature selection algorithms should be evaluated to get performance estimation on some standard classifier by applying tenfold cross - validation ( xval ) , that is , repeating feature selection ten times with slightly different data for all algorithms . classification phase of the diagnostic system is the one in charge of making the inferences about the extracted information in the previous phases in order to be able to produce a diagnostic about the input image . there are two different approaches for the classification of dermoscopic images : the first considers only a dichotomous distinction between the two classes ( melanoma and benign ) and assigns class labels 0 or 1 to data item . the second attempts to model p(y | x ) ; this yields not only a class label for a data item , but also a probability of class membership . logistic regression , artificial neural networks , k - nearest neighbours , and decision trees are all members of the second approach , although they vary considerably in building an approximation to p(y | x ) from data . we do not intend in this paper to delve deeply into the technical aspects of all the classification algorithms . however , to make the reader analyse the performance of algorithms that are mostly used for dermoscopic image analysis , we believe that it is helpful to air them briefly . readers who wish to have a detailed description of a specific classification approach should refer to cited references . the k - nearest neighbour classifier [ 161 , 162 ] is a nonparametric method of pattern recognition . for a lesion belonging to the test set ( query vector ) , it is found that the k vectors are the closest to the query vector in the training set . the unclassified sample is then assigned to the class represented by the majority of the k closest neighbours . the most critical requirement of the k - nearest neighbour classifier is to have a training set including enough examples of each class of pigmented lesions to adequately represent the full range of measurements that can be expected from each class . optimizing the procedures of feature selection and weight definition could additionally improve the performance of the k - nearest neighbour classifier . in medicine , most applications use nearest - neighbour algorithms as benchmarks for other machine learning techniques [ 156 , 164 ] . classification based on the k - nearest neighbour algorithm differs from the other methods considered here , as this algorithm uses the data directly for classification , without building a model first [ 162 , 165 ] . the only adjustable parameter in the model is k , the number of nearest neighbours to include in the estimate of class membership , and the value of p(y | x ) is calculated simply as the ratio of members of class y among the k - nearest neighbours of x. by varying k , the model can be made more or less flexible ( small or large values of k , resp . ) . k - nn algorithm permits retrieval and visualization of the most similar cases to those at hand . this aspect partly resembles the medical reasoning and allows a dermatologist to directly compare unknown lesions with other known skin lesions . this case - based explanation can provide an advantage in areas where black - box models are inadequate . k - nn can also be used for the evaluation of feature subset selection process because it allows incorporating / eliminating characteristics easily and it has low computational cost . the major drawback of k - nearest neighbour lies in the calculation of the case neighbourhood . thus , it needs to define a metric that measures the distance between data items . in most application areas , it is not clear how to , other than by trial and error , define a metric in such a way that the relative ( but unknown ! ) importance of data components is reflected in the metric . it is popular for its simplicity in constructing , efficient use in decision making , and simple representation , which is easily understood by humans . this algorithm repeatedly splits the data set according to a criterion that maximizes the separation of the data , resulting in a tree - like structure [ 167171 ] . it does this by identifying a variable and a threshold in the domain of this variable that can be used to divide the data set into two groups . the best choice of variable and threshold is the one that minimizes the disparity measures in the resulting groups . the most common criterion employed is information gain ; this means that at each split , the decrease in entropy due to this split is maximized . the estimate of p(y | x ) is the ratio of y class elements over all elements of the leaf node that contains data item x. various modifications of decision trees like adwat and lmt are also used for dermoscopic image classification . advantages and disadvantages of decision trees in medicine have been widely investigated [ 172 , 173 ] . the advantage of decision trees over many of the other methods is that they are not black - box models but can easily be expressed as rules . decision tree classifiers have been preferred to other solutions ( also including ann and svm ) because they are often fast to train and apply and generate easy - to - understand rules . a major disadvantage of decision trees is given by the greedy construction process . in this process at each step , the combination of single best variable and optimal split - point is selected . however , on the other hand if we use multistep look ahead , it considers combinations of variables which may obtain different ( and better ) results . given a large training set , decision tree classifiers , in general , generate complex decision rules that perform well on the training data but do not generalize well to unseen data . in such cases , a further drawback lies in the fact that continuous variables are implicitly discretised by the splitting process , losing information along the way . logistic regression is an algorithm that constructs a separating hyperplane between two data sets , using the logistic function to express distance from the hyperplane as a probability of class membership . although the model is linear in parameters and can thus only calculate linear decision boundaries , it is nevertheless a widely used predictive model in medical applications [ 155 , 175 , 176 ] . the main advantage that this method has over other algorithms is its ease of use ( it is implemented in numerous software packages ) , allowing the interpretation of results as probabilities and variable - selection capability . . showed in a comparative study that logistic regression performs on about the same level as artificial neural networks and support vector machines , which are both capable of implementing nonlinear separating surfaces . artificial neural network [ 165 , 177180 ] is one of the great vital parts of soft computing . the ann consists of several small processing units ( the artificial neurons ) that are highly interconnected . the supervise ann is an iterative process which requires many presentations of the training set ; the system is said to learn from examples . it has conspicuous capacity to obtain idea from complex data and is used to take out patterns and determine trends that are too difficult to be noticed by humans or any other computer skills . a lot of research is being carried out nowadays on dermoscopic image analysis using anns . the general working mechanism for artificial neural network many of the early implementations required a significant amount of parameter tuning to achieve satisfactory results , a process that needed too much time and expertise for a nonexpert . over the past few years , statistically motivated bayesian methods and implementations of faster learning algorithms have allowed nonexperts use to sophisticated methods that require little to no parameter tuning . various neural networks - based clustering techniques and algorithms are being used in this regard which include back propagation network ( bpn ) , radial basis function network ( rbf ) and extreme learning machine ( elm ) . support vector machines ( svms ) are a machine learning paradigm based on statistical learning theory [ 184 , 185 ] . performances on par with or exceeding that of other machine learning algorithms have been reported in the medical literature . algorithmically , support vector machines build optimal separating boundaries between data sets by solving a constrained quadratic optimization problem . while the basic training algorithm can only construct linear separators , different kernel functions ( i.e. , linear , polynomial , radial basis function , and sigmoid ) can be used to include varying degrees of nonlinearity and flexibility in the model . svms have several advantages over the more classical classifiers such as decision trees and neural networks . therefore , there is no risk of getting stuck at local minima as in the case of back propagation neural networks . svms are based on the structural risk minimization ( srm ) principle which minimizes the upper bound on the generalization error . therefore , svms are less prone to overfitting when compared to algorithms such as back propagation neural networks that implement the erm empirical risk minimization principle . another advantage of svms is that they provide a unified framework in which different learning machine architectures ( e.g. , rbf networks and feed forward neural networks ) can be generated through an appropriate choice of kernel . the disadvantage of support vector machines is that the classification result is purely dichotomous , and no probability of class membership is given . the only difference is that the hidden elements can be independent from the training data and target functions . because of this independence of hidden elements , this feed forward network provides better generalization performance and it can learn much faster as compared to the other conventional algorithms . the important features of extreme learning machine are that even simple math is enough for it . the learning speed is extremely fast . unlike the traditional classic gradient - based learning algorithms which often face several issues like local minima , improper learning rate , and overfitting . this learning algorithm looks much simpler than many other learning algorithms like neural networks and support vector machines . there is very less work being done on the classification of dermoscopic images using extreme learning machine . research work done on extreme learning machine shows that extreme learning machine needs much less training time as compared to popular bp and svm . the prediction accuracy of elm is usually slightly better than bp and close to svm in many applications . compared with bp and svm , extreme learning machine can be implemented easily since there is no parameter to be tuned except an insensitive parameter l. it should be noted that many nonlinear activation functions can be used in extreme learning machine . extreme learning machine needs more hidden nodes than bp but much less nodes than svm . this implies that extreme learning machine and bp have much shorter response time to unknown data than svm . the papers propose that , for skin lesion classification , three different classification tasks should be used as benchmarks : the dichotomous problem for distinguishing common nevi from dysplastic nevi and melanoma , the dichotomous problem for distinguishing melanoma from common nevi and dysplastic nevi , and the trichotomous problem for correctly distinguishing all the three classes . the two criteria to assess the quality of a classification model are discrimination and calibration . discrimination is a measure of how well the two classes in the data set are separated and calibration is a measure of how close the predictions of a given model are to the real underlying probability based on expert knowledge . some of the common measures of analysing discriminatory power of different methods are reported in this paper as can be noticed in table 3 . accuracy can be used as a single parameter , but if there is imbalance between the classes ( melanoma , benign ) , then accuracy is not a suitable approach of evaluation . a better performance measure in unbalanced domains is the receiver operating characteristic ( roc ) curve . auc is a statistically consistent and a more discriminatory measure than accuracy [ 191 , 192 ] . the log diagnostic odds ratio is also sometimes used in meta - analyses of diagnostic test accuracy studies due to its simplicity ( being approximately normally distributed ) . dclass is a measure to compare different classifiers presented by sboner et al . that enable giving a simple estimation of how useful one classifier is with respect to another . by using this parameter instead of accuracy , out the comparison between classifiers can be carried in an accurate but intuitive way , avoiding the unbalanced class problem . to provide an unbiased estimate of a model 's discrimination and calibration there are some important considerations like the effect of class imbalance , train / test ratio , and cross - validation . several studies have demonstrated that the accuracy degradation on unbalanced data sets is more severe when the classes overlap significantly [ 190 , 194 , 195 ] which is the case in skin lesion classification . most classifiers focus on learning the large classes which leads to poor classification accuracy for the small classes such as classifying the minority ( melanoma ) samples as majority ( benign ) which implies serious consequences . it has been observed that as the training - set size increases , the results improve . the effect of train / test ratios on classification accuracy is studied in and the best classification results were reached with 70/30 train to test ratio . there are two approaches for selecting training and test data : either to separate test and training feature vectors or pick training feature vectors as a subset of the test vectors . a classification result may be overly optimistic if performance can not be measured on a data set not used for model building . in the ideal case , testing on a separate data set will provide an unbiased estimate of generalization error . if the original data set is too small for this approach , the recommended strategy is to use cross - validation or bootstrapping to make the best possible use of the limited amount of data . one way is to divide the whole data into n pieces , n 1 pieces used for training , and the last piece as the test set . this process of n - fold cross - validation builds n models ; the numbers reported are the averages over all n test sets . the extreme case of using only one data item for testing is known as leave - one - out cross - validation . bootstrapping is rarely used in the literature for skin lesion case , but it has shown to be superior to cross - validation on many other data sets . the increasing number of electronic data bases containing dermoscopic images has led to an increasing interest in their utilization for building classification models that can learn from examples . the need to use data and learning techniques in order to make correct diagnosis requires proper choice of the learning algorithms and of their statistical validation . the problem is difficult given the relative paucity of lesion data and consequently the low quality of training data available and the imbalance between the classes . a variety of statistical and machine learning approaches are used for the classification of dermoscopic images . as illustrated in table 4 , while figure 5 presents the percentage of classification methods as used by existing diagnostic systems in literature . the question of which classification approach is suitable for a specific study is not easy to answer . different classification results may be obtained depending on the classifier(s ) chosen , differences in sample sizes , proportion of melanomas in the sample , and the number of features used for discrimination as can be notice in table 5 . many factors , such as different sources of obtaining dermoscopic images , availability of classification software , time consumption , computational resources , and the number of melanoma and benign images available for training must be taken into account when selecting a classification method for use . very few researchers provided comparisons of different classification algorithms using the same set of images [ 46 , 94 , 126 , 166 , 196 ] . the review of all these comparative studies reveals that mlp gives better performance than bayesian and knn classifiers , while svm with rbf kernel normally outperforms mlp , decision trees , and other statistical methods . the results of an experimental assessment of the different designs can be the basis for choosing one of the classifiers as a final solution to the problem . it had been observed in such design studies that although one of the designs would yield the best performance , the sets of patterns misclassified by the different classifiers would not necessarily overlap . , all the designs , or their subset , are used for decision making by combining their individual opinions to derive a consensus decision . some classifier combination schemes have been devised [ 126 , 193 , 200 ] for dermoscopic images and it has been experimentally demonstrated that some of them consistently outperform a single best classifier . however , there is presently inadequate understanding why some combination schemes are better than others and in what circumstances . such models do not always work well in practice , so it is widely recommended that they need to be validated [ 201 , 202 ] . to be useful , a prognostic index should be clinically credible and accurate and have generality ( i.e. , be validated elsewhere ) , and the study should be described in adequate detail . to gauge the current state of reporting results in the literature , we sampled many papers on dermoscopic images data sets analysis . the paper is proposing some criteria as quality assessment criteria which can be noticed in table 6 . it includes lack of calibration in image acquisition , unspecified method for extracting and selecting variables in the model , and risk of overfitting through too few events per variable . many researchers did not specify the test / train or used uneven number of melanoma and benign images for training which may lead to biased classification . some articles do not report comparisons and cross - validation ; instead they just reported the performance of a single method . it is imperative that these details should be presented in papers as otherwise the validity of the claims in the papers can not be assessed by the reader . when assessing the quality of the results obtained using any diagnostic models , the work should consider the quality of the data set employed in model building , the care with which adjustable model parameters were chosen , and the evaluation criteria used to report the results of the modelling process . this is important in distinguishing between overly optimistic claims ( such as when performance is reported on the training set ) and needlessly pessimistic ones ( when model parameters are chosen in a suboptimal manner ) . apart from all this , in order to judge the performance of an automatic diagnostic model it is important to mention who is going to use that model . if automated diagnostic systems will be used by general practitioners or in pharmacies and shopping centres , these systems should be used with very high sensitivity and reasonably good specificity . that is , it should recognize the greatest number of melanomas in early stage , without misclassifying too many nevi so that unnecessary excision of benign lesions could be avoided . if the target is the expert user , studies should be designed with the aim to help clinicians in distinguishing between benign lesions , dysplastic nevi , and malignant tumors of the skin . an increase in specificity might be the goal for an automated system directed to expert users together with sensitivity at least equal to that achieved by the expert . overall , our objective is to get a classifier with the sensibility and the specificity balanced . it should be noted that the ability to diagnose correctly melanoma is by far the most important property that an automated system must have . the consequence of failure to diagnose correctly a malignant tumor may lead to the eventual death of the patient . on the other hand , if we get a classifier with a high sensibility but a low specificity , it is not going to be useful as a screening method to avoid biopsies ( an invasive technique ) . and , off course , we want a classifier with a high sensibility to avoid false negatives .

image - based computer aided diagnosis systems have significant potential for screening and early detection of malignant melanoma . we review the state of the art in these systems and examine current practices , problems , and prospects of image acquisition , pre - processing , segmentation , feature extraction and selection , and classification of dermoscopic images . this paper reports statistics and results from the most important implementations reported to date . we compared the performance of several classifiers specifically developed for skin lesion diagnosis and discussed the corresponding findings . whenever available , indication of various conditions that affect the technique 's performance is reported . we suggest a framework for comparative assessment of skin cancer diagnostic models and review the results based on these models . the deficiencies in some of the existing studies are highlighted and suggestions for future research are provided .

pubmed

type 2 diabetes ( t2d ) is a chronic disease characterized by high blood glucose which is induced by both insulin resistance and relative insulin deficiency , and the complications of t2d , such as cardiovascular disease , renal failure and blindness , always bring heavy burden to society . the latest studies from the international diabetes federation ( idf ) reported that currently there were 366 million people with diabetes in the world by 2030 this number is expected to rise to 552 million with diabetes if no action is taken . some 90% of patients with diabetes have t2d , which is always as a result of a combination of lifestyle and genetic factors . peroxisome proliferator - activated receptor coactivator 1 ( pgc-1 ) gene is originally identified as a coactivator of ppar. it is a multifunctional regulatory factor involved in mitochondrial biosynthesis , -oxidation of fatty acids , beta - cell insulin secretion , hepatic gluconeogenesis , glucose transport in muscle [ 46 ] . our and other genetic association studies showed that the associations of pgc-1 loci with t2d were found in different populations [ 710 ] . recently , it was reported that estrogen - related receptor- ( esrra ) protein binding pgc-1 protein could regulate the expression of mitofusin-2 ( mfn2 ) gene by stimulating the activity of the mfn2 promoter . esrra gene is an orphan nuclear receptor ( nr ) belonging to the nr superfamily , group iii , and it is a vital regulator involved in a wide variety of cell functions such as mitochondrial biogenesis , mitochondrial oxidative metabolism , carbohydrate metabolism , adaptive energy metabolism , and lipid metabolism [ 1214 ] . therefore , we hypothesize that esrra gene may be a candidate gene involved in the pathogenesis of t2d . mfn2 gene maps on chromosome 1p36.22 . mfn2 serve as a mitochondrial fusion protein regulating their morphology and distribution [ 15 , 16 ] . tissues with high energetic requirement , such as skeletal muscle and heart , show high expression of mfn2 gene . some evidences demonstrated that it also played an important role in the oxidative metabolism and er - mitochondria juxtaposition , and repression of mfn2 gene reduced glucose oxidation , mitochondrial membrane potential , cell respiration , and mitochondrial proton leak . in addition , the mfn2-dependent mechanism of mitochondrial control is disturbed in skeletal muscle in animal or human obesity and in patients with t2d [ 17 , 19 ] , but no study has confirmed that mfn2 gene was associated with t2d . in the present study , we evaluated the associations between the mfn2 and esrra genetic polymorphisms and t2d , and then combining our published data of pgc-1 , we estimated the interactions between genetic variants in the mfn2 , esrra , and pgc-1 on the t2d risk in han chinese . the total of stage 1 populations comprised 1204 unrelated subjects , including 555 patients with t2d and 649 nondiabetic control subjects , ascertained from han chinese . all subjects were randomly enrolled from three top - grade hospitals in beijing in 2010 . total case subjects were diagnosed according to the world health organization criteria ( who 1998 ) . as total control subjects , the age was between 35 and 70 years old , and they had normal fasting plasma glucose levels ( 5.0 0.5 mmol / l ) without diabetic record or t2d family history . the clinical characteristics of stage 2 populations are also summarized in table 1 ; it included 546 unrelated patients with t2d and 419 nodiabetic control subjects and of han chinese origin , as described previously . international hapmap project snp database ( http://www.hapmap.org/ ) was implemented to search for snps in mfn2 and esrra gene . using tagging with an r > 0.8 and minor allele frequency ( maf ) of > 5% and sequencing data in 20 nondiabetic control subjects , we selected 5 tag snps ( rs873458 , rs2878677 , rs2236058 , rs3766742 , rs3766741 ) in mfn2 gene and 3 tag snps ( rs731703 , rs650008 , rs11600990 ) in esrra gene ; they were all located in intron region and could capture 100% of common variations across mfn2 and esrra gene region ( containing 21 hapmap snps ) . 7 of 8 snps were successfully genotyped using the taqman method on a bio - rad iq5 system . primers and probes of rs873458c_8861262_10 , rs3766741c_25606040_10 and rs650008 ahwr23 m were purchased from applied biosystems , the others from shanghai genecore biotechnologies ( primers showed in table 2 ) . only one snp ( rs3766742 ) in the mfn2 was genotyped with pcr - restriction fragment - length polymorphism ( pcr - rflp ) method , using the following primers ( 5-agcaggacatgataggttag-3and 5-cacagcttgtcacagtttag-3 ) and pcr tm was 57c , pcr product length is about 900 bp , and restriction enzyme ( hha i ) provided by mbi fermentas . a 10 l aliquot of pcr product was digested overnight at 37 in a 20 l reaction containing 5 units of hha i. after overnight digestion , the products were separated on a 3% agarose gel stained with ethidium bromide . tt genotypes were represented by a dna band with a size of 900 bp , tc genotypes were represented by dna bands with sizes of 900 , 700 , and 200 bp , and cc genotypes were represented by dna bands with sizes of 700 and 200 bp . 20 samples randomly selected from the whole sample bank were sequenced for 8 tag snps and the results showed excellent correspondence between sequencing and taqman genotyping or pcr - rflp technique . genotype distributions for all studied snps were tested for hardy - weinberg equilibrium ( hwe ) by chi - square tests and no significant deviation was found in control subjects . allele frequencies and genotype distributions , linkage disequilibrium ( ld ) , and haplotypes were tested using the online software shesis ( http://analysis.bio-x.cn/myanalysis.php/ ) . logistic regression analysis with additive model was used to adjust for sex , age , and body mass index ( bmi ) , and the analysis was performed by spss ( version 16.0 ) . tests for association between genotypes and quantitative traits were performed in control subjects using kruskal - wallis analysis of ranks for traits with nonnormal distribution or , alternatively , anova for normally distribution in spss . we examined the analyses of gene - gene interactions on the risk of t2d using multifactor dimensionality reduction ( mdr ) version 2.0 beta 6 ( http://www.epistasis.org ) . in stage 1 populations , we found that the a allele and c allele frequencies of rs873458 and rs2878677 of mfn2 gene in the diabetic group were lower than in the control group ( p = 0.005 , or = 0.79 , 95% ci= 0.670.93 ; p = 0.01 , or = 0.81 , 95% ci = 0.680.95 ) ( table 3 ) ; it showed a allele and c allele of rs873458 and rs2878677 of mfn2 gene confered protection agasist t2d . after adjusting for age , sex , and body mass index by using analysis of logistic regression with additive model , the results showed that the genotype distributions of rs873458 and rs2878677 of mfn2 gene also were significantly different in case - control subjects ( pc = 0.01 , or = 0.80 , 95% ci = 0.670.95 ; pc = 0.02 , or = 0.83 , 95% ci = 0.710.97 ) ( table 3 ) . replicating the two associated snps in stage 2 populations , data suggested that the c allele of rs2878677 of mfn2 gene in the diabetic group was lower than in the control group ( p = 0.01 , or = 0.79 , 95% ci = 0.650.94 ) ( table 3 ) . and it revealed that there was a borderline association between genotype distributions of rs873458 of mfn2 gene and type 2 diabetes ( adjusted pc= 0.08 , or = 0.84 , 95% ci = 0.691.02 ) ( table 3 ) . additionally , analyses in the combined populations showed that the allele frequencies and genotype distributions of rs873458 and rs2878677 of mfn2 gene had more significant effects on type 2 diabetes susceptibility ( p = 0.002 , or = 0.83 , 95% ci = 0.730.93 ; p = 0.0001 , or = 0.79 , 95% ci = 0.690.89 and adjusted pc = 0.002 , or = 0.82 , 95% ci = 0.720.93 ; pc = 0.0004 , or = 0.81 , 95% ci = 0.720.91 ) ( table 3 ) . no significant association of other snps in mfn2 or esrra gene with type 2 diabetes was found ( see supplementary table 1 in supplementary material available online at doi:10.1155/2012/205752 ) . analyses of association between haplotypes and phenotypes were performed in nondiabetic control subjects , for most patients with t2d included in the present study had medical treatments , which may affect the real parameters . we found that the genotype distributions of snps in mfn2 and esrra gene in control subjects displayed no significant association with quantitative traits of type 2 diabetes including body mass index , diastolic blood pressure , systolic blood pressure , fasting plasma glucose , triglycerides , and glycated haemoglobin a1c ( data not shown ) . we further examined the degree of linkage disequilibrium of snps in mfn2 and esrra gene ( supplementary tables 2 and 3 ) . to test whether haplotypes represent the causal variants better than single snps , we estimated the frequencies of haplotypes between the case - control subjects in stage 1 populations ( all those frequencies < 0.03 were ignored ) . it was showed that a - c - g - t - c and g - t - c - t - c in mfn2 gene were significantly associated with t2d ( p = 0.007 , or = 0.79 , 95% ci = 0.660.94 ; p = 0.009 , or = 1.26 , 95% ci = 1.061.49 ) ( table 4 ) , while the frequencies of haplotypes in esrra gene did not significantly differ in case - control subjects ( data not shown ) . the model was considered to be statistically significant with a testing accuracy > 50% and p < 0.05 via permutation test of 1000 iterations . the rs2878677/rs3766741/rs731703/rs11600990 model ( p < 0.0001 ) in mfn2-esrra from stage 1 populations , the rs2878677/rs3774923/rs7656250 model ( p < 0.0001 ) and the rs2878677/rs3774923/rs7656250/rs13131226 model ( p < 0.0001 ) in mfn2-pgc-1 gene from stage 2 populations ( shown in table 5 ) , data of pgc-1 gene from our previously study , suggest that there are interactions between genetic variants in the mfn2 , esrra , and pgc-1 gene . in the present study , we examined eight common variants in mfn2 and esrra genes and confirmed that g allele of rs873458 and t allele of rs2878677 in mfn2 gene are associated with higher t2d risk compared with a and c allele , respectively . consistently , in haplotypes analyses of mfn2 gene , results show that individuals carrying g - t - c - t - c present increased t2d risk compared with those carrying a - c - g - t - c . because rs873458 and rs2878677 map within intron 2 and 3 , it is difficult for us to shed light on their downstream consequences , given our rudimentary knowledge of the mechanics of gene regulation . in multifactorial disease , most susceptibility variants , mapping outside the coding regions of genes , are assumed to influence transcript regulation rather than gene function . locations of rs873458 and rs2878677 are surrounded by the regions coding the gtpase domain of mfn2 which drive gtp hydrolysis to provide energy for the mitochondrial fusion activity induced by mfn2 [ 16 , 22 ] . in addition , mfn2 gene is the first structural tether identified gene in the molecular basis of er - mitochondria juxtaposition , it has a role in the intercommunication during ca signaling . a study revealed that ablation or silencing of mfn2 gene in mouse embryonic fibroblasts or hela cell created confusion in er morphology and increased distance between the er and mitochondria , hence retarding the mitochondrial ca uptake , which may lead to insulin secretion disorder and insulin resistance . besides in addition , the mfn2-dependent mechanism of mitochondrial control is disturbed in skeletal muscle in animal or human obesity , and in patients with t2d [ 17 , 19 ] . it is possible that the associated snps may be in strong linkage disequilibrium with unidentified causal variant(s ) that may regulate the expression or function of mfn2 gene . although many genomewide association studies ( gwass ) of t2d are now emerging , mfn2 gene is not involved . indeed , the effect sizes of the known , common variants influencing the risk of type 2 diabetes are modest , and the proportion of overall predisposition explained is approximately 5 to 10% for t2d . it has been found that t2d loci identified by linkage analyses and gwas lack the correlation of t2d . differences in genetic background , risk - factor profile , environment , and study design may lead to ethnic differences in susceptibility loci . different allele frequencies of rs873458 and rs2878677 among the hapmap populations indicate that differences in genetic architecture may play a role ( supplementary table 4 ) . esrra is an orphan nuclear receptor , it could regulate a number of downstream genes to control energy balance in animals . esrra gene ko mice showed altered expression of several target genes implicated in the regulation of adipogenesis and energy metabolism . however , in the present study , no association between the phenotypes of t2d , such as bmi , was found . our data also showed that there were three common variants ( rs731703 , rs650008 , rs11600990 ) in esrra in han chinese populations , while only one common variant rs11600990 was found in danish populations . three tag snps could capture 100% of common variations ( maf > 5% ) across esrra gene region , but we can not exclude rare causal genetic polymorphisms in esrra gene , for the rare variations may be responsible for disease [ 2931 ] . pgc-1 is an important transcriptional coactivator involved in the regulation of genes related to energy metabolism . mice lacking pgc-1 developed fasting hepatic steatosis which may lead to lower rates of fatty acid oxidation that might play a causative role to develop insulin resistance [ 33 , 34 ] . in addition , pgc-1 maintained higher number of active mitochondria and oxphos proteins that were reduced in t2d subjects . as it relates to the human genetics , several snps of pgc-1 gene in certain populations the analysis of association between pgc-1 gene and t2d has been published in our previous paper . in addition , it was found that the interactions between genetic variants of the mfn2-esrra and mfn2-pgc-1 in the pathogenesis of type 2 diabetes . moreover , a positive result of association analyses regarding single snp rs2878677 in mfn2 gene indicates mfn2 gene may be the main factor during the interaction and has an important role in the pathogenesis of t2d . it is consistent with the study that it proved the interactions between mfn2 , esrra , and pgc-1 proteins , which revealed the stimulatory effect of pgc-1 on the activity of mfn2 gene needed to integrate the esrra , esrra , and pgc-1 caused a synergic effect on activity of the mfn2 gene expression , and the stimulatory effect of pgc-1 gene on mitochondrial membrane potential can be weakened by mfn2 loss of function . therefore , further studies are needed to explore the functions and interactions of the mfn2 , esrra , and pgc-1 genes . in most patients , t2d is due to alterations of many genes , each of which has a partial and additive effect . consequently , we hypothesize that these three genes may serve as partial or additive factors leading to t2d , while the intrinsic mechanism is unknown . functional studies , multiplex - gene analyses , and computational biology are needed to further characterization of their interactions in the pathogenesis of t2d . however , some limitations should be noted in the present study . first , the effect of gene - environment interactions was not evaluated ; environmental risk factors may influence the effect estimates . second , given the study samples were relatively small , we need to replicate our finding in additional samples in future studies . , the present analysis shows that mfn2 gene has significant associations with t2d in han chinese . but more studies with different ethnic populations are needed to verify this finding and the intrinsic mechanism is expected to be uncovered .

mfn2 and esrra are candidate genes involved in the pathogenesis of t2d . five tag - snps in mfn2 gene and three in esrra gene were selected and genotyped with taqman or pcr - rflp method in stage 1 populations ( 555 patients with t2d and 649 control subjects ) and stage 2 populations ( 546 patients with t2d versus 419 control subjects ) in han chinese . and combining our published data , we estimated the interactions between genetic variants in the mfn2 , esrra , and pgc-1 genes on the t2d risk using mdr . rs873458 ( g > a ) and rs2878677 ( c > t ) in mfn2 gene were significantly associated with t2d ( p = 0.005 and 0.01 ) in stage 1 populations , and the association of other snps with t2d was not found . in stage 2 populations , we further confirmed the association between rs2878677 and t2d ( p = 0.01 ) . combining the two stage populations , the data supported more significant effect of rs873458 and rs2878677 on t2d risk ( p = 0.003 and 0.0001 ) . a - c - g - t - c and g - t - c - t - c in mfn2 had significant association with t2d ( p = 0.007 and 0.009 ) . the present study also provided the evidence that mfn2 had interactions with pgc-1 ( p < 0.0001 ) or esrra ( p < 0.0001 ) . this study suggested a role of mfn2 polymorphism in the risk of t2d ; however , further studies are needed .

pubmed

interindividual variations of ex vivo platelet aggregation during clopidogrel therapy have been correlated to reduced exposure of active clopidogrel thiol metabolite and differences in p2y12 receptor occupancy.1 high on - treatment platelet reactivity during therapy with clopidogrel and low platelet inhibition are associated with risk of ischemic events after coronary stenting in particular , stent thrombosis.15 as compared with extensive metabolizers , carriers of cytochrome p450 2c19 ( cyp2c19 ) nonfunctional alleles have been shown to have reduced platelet inhibition by clopidogrel and are at increased risk of adverse events and stent thrombosis after coronary.59 recent advances in the understanding of clopidogrel metabolism suggest that the inactive prodrug undergoes two steps of bioactivation . first , clopidogrel undergoes oxidation to 2-oxo - clopidogrel by hepatic cyp450 enzymes ( 1a2 , 2b6 , 2c9 , 2c19 , and 3a4/5 isoenzymes ) . then , in the second step of the biotransformation , thiol metabolite is generated.10 a recent study employing sophisticated metabolomic methods suggested a hydrolytic process involving paraoxonase-1 ( pon1 ) as the rate - limiting enzyme.10 a common polymorphism of pon1 ( q192r ) is associated with reduced paraoxonase activity in carriers of the qq ( aa ) haplotype . in two cohorts of patients with coronary stenting , and using a case study design , the investigators found the pon1 ( qq192 ) genotype was associated with increased risk of stent thrombosis.10 a group of patients with a history of prior stent thrombosis but who were not receiving current therapy with clopidogrel were administered a clopidogrel 600 mg loading dose . within this group , reduced plasma activity of pon1 and pon1 ( qq192 ) genotype were associated with reduced active thiol metabolite exposure and reduced platelet inhibition by clopidogrel.10 only one genome - wide association study ( gwas ) has been performed examining the association of common genetic polymorphisms on platelet inhibition.11 this gwas examined platelet aggregation data from 429 generally healthy white subjects of amish descent after administration of a clopidogrel 300 mg loading dose , followed by maintenance clopidogrel dosing of 75 mg daily for 6 days . platelet aggregation was assessed before administration of clopidogrel and again after 6 days of maintenance dosing . the results from the gwas only found single nucleotide polymorphisms ( snps ) on chromosome 10q24 in linkage equilibrium with the cyp2c19 * 2 loss - of - function variant to be significantly associated with clopidogrel platelet inhibition.11 the gwas did not find evidence of a significant association of snps located on or near the pon1 gene on chromosome 7 with variation in platelet inhibition by clopidogrel.11 the aim of the current study was to assess the impact of pon1 ( q192r ) and cyp2c19 * 2 polymorphisms on clopidogrel platelet inhibition , as measured by light transmittance aggregometry and verifynow ( vn ) p2y12 assay , in a north american study population of mixed racial background . the indiana university school of medicine s institutional review board for research approved the study protocols . subjects were eligible to be enrolled if they had established coronary disease and were on dual antiplatelet therapy with clopidogrel and aspirin 81325 mg daily for secondary prevention . subjects were included in this study either if they had been taking clopidogrel 75 mg for at least 14 days prior to enrollment or if they had received a clopidogrel 600 mg loading dose during a percutaneous coronary intervention ( pci ) . subjects were excluded if they had a history of drug or alcohol abuse , bleeding disorder , myelodysplastic or myeloproliferative disorders , chronic liver disease , or current warfarin use . subjects were also excluded if they were pregnant , if the platelet count was less than 150,000/mm , and if there was planned glycoprotein iib / iiia antagonist use during pci . after 14 days of taking clopidogrel 75 mg daily , peripheral venous blood samples were obtained from subjects prior to the next dose of clopidogrel and aspirin to determine on - treatment platelet aggregation . for patients who received clopidogrel 600 mg at the time of a pci , a baseline blood sample was obtained from the arterial access sheath prior to administration of the clopidogrel loading dose and administration of heparin or bivalirudin . peripheral venous blood samples were drawn at 4 and 1624 hours after administration of the clopidogrel loading dose . for subjects who received a clopidogrel 600 mg loading dose , the 16- to 24-hour sample was used to determine the final on - treatment platelet aggregation used for the primary analysis of platelet aggregation among genotypes . all blood samples were directly transferred into vacutainer tubes containing 3.2% sodium citrate and were analyzed within 2 hours . ex vivo platelet function was assessed by light transmittance aggregometry ( lta ) at 37c with an optical lumi- aggregometer ( model 700 with aggro / link 8 software ; chrono - log corporation , havertown , pa ) . platelet - rich and platelet - poor plasma were obtained by differential centrifugation , as previously described.12,13 platelet aggregation in platelet - rich plasma was induced with adenosine diphosphate ( adp ) at 5 , 10 , and 20 mol / l . a vn - p2y12 point - of - care assay ( accumetrics , inc , san diego , ca ) was used to assess platelet inhibition in whole blood in subjects during maintenance clopidogrel dosing , as previously described.14 genomic dna was isolated from whole blood with the aid of the qiaamp dna blood midi kit ( qiagen , germantown , md ) . subjects were genotyped for cyp2c19 * 2 ( 681g > a ; rs4244285 ) , cyp2c19 * 3 ( 636g > a ; rs4986893 ) , and pon1 ( q192r ; 575 a > g ; rs662 ) snps using a bio - rad laboratories real - time icycler thermal cycler ( bio - rad laboratories , inc , hercules , ca ) . sequence - specific primers were used to amplify the alleles of interest , along with two allele - specific taqman probes ( applied biosystems , foster city , ca ) . allelic discrimination software ( optical system , v 3.1 ; bio - rad laboratories ) was used to determine individual genotypes . all statistical tests were two - sided , and values are represented as the mean plus or minus the standard deviation , unless otherwise specified . unpaired two - sided student s t - test was used to compare normally distributed continuous data between two groups and for genotype group comparisons using one - way analysis of variance . non - normally distributed continuous data were compared across two groups with the two - sided unpaired wilcoxon test and genotype group comparisons with the kruskal genotypes for cyp2c19 and pon1 were identified and included in univariate and multivariate linear regression , along with clinical variables that are known to affect platelet reactivity and response to clopidogrel and variables with p < 0.1 in univariate analysis ( table 2 ) . the indiana university school of medicine s institutional review board for research approved the study protocols . subjects were eligible to be enrolled if they had established coronary disease and were on dual antiplatelet therapy with clopidogrel and aspirin 81325 mg daily for secondary prevention . subjects were included in this study either if they had been taking clopidogrel 75 mg for at least 14 days prior to enrollment or if they had received a clopidogrel 600 mg loading dose during a percutaneous coronary intervention ( pci ) . subjects were excluded if they had a history of drug or alcohol abuse , bleeding disorder , myelodysplastic or myeloproliferative disorders , chronic liver disease , or current warfarin use . subjects were also excluded if they were pregnant , if the platelet count was less than 150,000/mm , and if there was planned glycoprotein iib / iiia antagonist use during pci . after 14 days of taking clopidogrel 75 mg daily , peripheral venous blood samples were obtained from subjects prior to the next dose of clopidogrel and aspirin to determine on - treatment platelet aggregation . for patients who received clopidogrel 600 mg at the time of a pci , a baseline blood sample was obtained from the arterial access sheath prior to administration of the clopidogrel loading dose and administration of heparin or bivalirudin . peripheral venous blood samples were drawn at 4 and 1624 hours after administration of the clopidogrel loading dose . for subjects who received a clopidogrel 600 mg loading dose , the 16- to 24-hour sample was used to determine the final on - treatment platelet aggregation used for the primary analysis of platelet aggregation among genotypes . all blood samples were directly transferred into vacutainer tubes containing 3.2% sodium citrate and were analyzed within 2 hours . ex vivo platelet function was assessed by light transmittance aggregometry ( lta ) at 37c with an optical lumi- aggregometer ( model 700 with aggro / link 8 software ; chrono - log corporation , havertown , pa ) . platelet - rich and platelet - poor plasma were obtained by differential centrifugation , as previously described.12,13 platelet aggregation in platelet - rich plasma was induced with adenosine diphosphate ( adp ) at 5 , 10 , and 20 mol / l . a vn - p2y12 point - of - care assay ( accumetrics , inc , san diego , ca ) was used to assess platelet inhibition in whole blood in subjects during maintenance clopidogrel dosing , as previously described.14 genomic dna was isolated from whole blood with the aid of the qiaamp dna blood midi kit ( qiagen , germantown , md ) . subjects were genotyped for cyp2c19 * 2 ( 681g > a ; rs4244285 ) , cyp2c19 * 3 ( 636g > a ; rs4986893 ) , and pon1 ( q192r ; 575 a > g ; rs662 ) snps using a bio - rad laboratories real - time icycler thermal cycler ( bio - rad laboratories , inc , hercules , ca ) . sequence - specific primers were used to amplify the alleles of interest , along with two allele - specific taqman probes ( applied biosystems , foster city , ca ) . allelic discrimination software ( optical system , v 3.1 ; bio - rad laboratories ) was used to determine individual genotypes . all statistical tests were two - sided , and values are represented as the mean plus or minus the standard deviation , unless otherwise specified . unpaired two - sided student s t - test was used to compare normally distributed continuous data between two groups and for genotype group comparisons using one - way analysis of variance . non - normally distributed continuous data were compared across two groups with the two - sided unpaired wilcoxon test and genotype group comparisons with the kruskal genotypes for cyp2c19 and pon1 were identified and included in univariate and multivariate linear regression , along with clinical variables that are known to affect platelet reactivity and response to clopidogrel and variables with p < 0.1 in univariate analysis ( table 2 ) . baseline characteristics of the study subjects are described according to cyp2c19 * 2 and pon1 q192r genotypes in table 1 . overall clinical variables were well balanced among genotype groups . a total of 96 subjects on maintenance clopidogrel therapy for over 14 days and 55 subjects who received a clopidogrel 600 mg loading dose at the time of pci were enrolled . the prevalence of african - americans was significantly different across pon1 q192r genotypes , with a higher prevalence of african - americans in carriers of pon1 qq192 ( table 1 ) . these findings are consistent with the increased prevalence of qq192 genotypes previously described in the african population as compared with populations of european descent.15 among the 151 patients included in this study , 63 ( 42% ) were carriers of the qq192 genotype , 67 ( 44% ) were heterozygous allele carriers ( qr192 ) , and 21 patients ( 14% ) were homozygous rr192 genotype carriers . for the cyp2c19 * 2 allele , 107 ( 71% ) were wild - type homozygous for the * 2 allelic variant ( * 1/*1 ) , 39 ( 26% ) were heterozygous * 2 allele carriers ( * 1/*2 ) , and five patients ( 3% ) were homozygous * 2 allele carriers ( * 2/*2 ) . for both genotype distributions , no significant deviations from the hardy weinberg equilibrium were observed ( p = 0.64 for pon1 q192r ; p = 0.54 for cyp2c19 * 2 genotypes ) . there were no cyp2c19 * 3 alleles detected in the study population , which is consistent with the very rare frequency ( < 1% ) of this loss - of - function allele in caucasians and african- americans . platelet aggregation induced by adp demonstrated wide interindividual variability during therapy with clopidogrel , as previously documented ( table 2 , figures 14).15 pon1 qq192 was not associated with increased platelet aggregation induced by adp at various concentrations during clopidogrel therapy before and after adjustment for 2c19 * 2 , diabetes mellitus , obesity , smoking , and proton pump inhibitor therapy ( table 2 ) . pon1 qq192 was also not associated with increased vn - p2y12 reactivity ; in fact , there was a trend towards lower platelet reactivity in pon1 qq192 individuals . no significant difference in the percentage of platelet inhibition measured by vn was documented among individuals of varying pon1 genotype ( table 2 , figure 2 ) . carriers of 2c19 * 2 alleles had higher platelet aggregation and higher vn - p2y12 reactivity with significantly lower vn platelet inhibition than noncarriers ( table 2 , figure 1 ) . among the subgroup of patients who received a clopidogrel 600 mg loading dose prior to undergoing pci , platelet aggregation measured before and after administration of clopidogrel was not significantly different between subjects of pon1 qq192 and qr192/rr192 genotypes ( figure 3 ) . there was no significant difference in platelet inhibition among homozygous carriers of reduced - function pon1 ( qq192 ) and qr192 and rr192 genotypes at 4 and 1624 hours after administration of a clopidogrel 600 mg loading dose ( figure 3 ) . in the same subgroup of patients who had received a clopidogrel 600 mg loading dose and who had a baseline platelet aggregation measurement , 2c19 * 2 carriers status was associated with significantly lower percentage of platelet inhibition measured by adp 10 mol / l , without reaching statistical significance for other concentrations of adp ( figure 4 ) . in the univariate linear regression analysis , only 2c19 * 2 carrier status ( adp 10 mol / l ; p = 0.034 ) , current smoking ( adp 10 mol / l ; p = 0.025 ) , and weight ( adp 10 mol / l ; p = 0.018 ) were significantly associated with adp - induced platelet aggregation . pon1 q192r homozygous reduced - function genotype ( qq192 ) ( adp 10 mol / l ; p = 0.7 ) , diabetes ( adp 10 mol / l ; p = 0.22 ) , and proton pump inhibitor use ( adp 10 mol / l ; p = 0.65 ) were not significantly associated with adp - induced platelet aggregation ( table 2 ) . covariates with p < 0.1 were included in a multivariate regression analysis , as well as variables such as diabetes and proton pump inhibitor use that have previously been established as affecting clopidogrel platelet inhibition . in a multivariate linear regression analysis that included diabetes , smoking , obesity , and proton pump inhibitor use as variables in addition to pon1 q192r and cyp2c19 * 2 genotypes , cyp2c19 * 2 carrier status ( adp 10 mol / l ; p = 0.036 ) , but not pon1 q192r ( adp 10 mol / l ; p = 0.89 ) , were associated with significant differences in adp - induced platelet aggregation ( table 2 ) . clopidogrel is transformed into 2-oxo - clopidogrel by cyp450 enzymes and , in a second step , into the active thiol metabolite . 10 a recent metabolomic study suggested that pon1 , a serum esterase , may represent the rate - limiting enzyme by hydrolysis of 2-oxo - clopidogrel into thiol clopidogrel . common snps of pon1 have been suggested to affect enzymatic activity of the enzyme . in bouman et als10 study , pon1 activity was significantly reduced in subjects homozygous for the wild - type allele ( pon1 qq192 ) compared with carriers of the mutant allele . in addition , in a group of patients with stent thrombosis and matched controls without stent thrombosis , pon1 qq192 was associated with decreased platelet inhibition by clopidogrel and decreased plasma exposure to active thiol metabolite after a clopidogrel 600 mg loading dose . also , pon1 qq192 genotype was associated with an odds ratio of 3.3 for the occurrence of stent thrombosis as compared with qr192 or rr192 genotypes.10 the only gwas on clopidogrel response had previously not found an association with snps located on or near the pon1 gene , but the platelet assays in that study were performed after 7 days of therapy with clopidogrel.11 the patients included in the gwas were generally healthy white subjects , who had received a clopidogrel 300 mg loading dose followed by 75 mg daily for 6 days prior to analysis of platelet aggregation.11 similar to findings from other investigators since the publication of the study by bouman et al,10 the current study could not document an association of pon1 ( q192r ) genotype with platelet inhibition by clopidogrel in patients of mixed racial background with established coronary artery disease during treatment with clopidogrel.1621 neither platelet inhibition measured by vn - p2y12 assay nor on - treatment platelet aggregation using various doses of adp showed significant differences among genotypes of pon1 q192r . among the group of patients who received a clopidogrel 600 mg loading dose immediately before pci , platelet studies performed before and after administration of 600 mg of clopidogrel showed no significant differences in platelet aggregation among genotypes of pon1 q192r ( figure 3 ) . the results of the current study are consistent with findings of shuldiner et al,11 who did not demonstrate an association of snps in or near the pon1 gene with clopidogrel nonresponse during clopidogrel maintenance dosing , and they confirm the association of 2c19 * 2 polymorphism with reduced platelet inhibition by clopidogrel.22,23 in the current study , cyp2c19 * 2 carrier status was associated with reduced platelet inhibition , as measured by vn - p2y12 assay and lta 10 mol / l , and increased on - treatment platelet reactivity , as measured by both lta and vn - p2y12 assay ( table 2 , figures 1 and 4 ) . cyp2c19 * 2 polymorphism has been demonstrated to be a strong determinant of reduced active clopidogrel metabolite formation , increased on - treatment adp - induced platelet reactivity , and clinical ischemic events after coronary stenting.59,11,22,23 in a meta - analysis investigating the effect of reduced - function 2c19 alleles on recurrent ischemic events in patients receiving clopidogrel after coronary stenting , the presence of one reduced - function allele was associated with a hazard ratio of 2.67 and the presence of two reduced - function alleles was associated with a hazard ratio of 3.97 for the occurrence of stent thrombosis . these findings support the clinical importance of the reduced - function cyp2c19 * 2 polymorphism and clopidogrel nonresponse after coronary stenting.9 although not supported by the findings of this study , that pon1 q192r polymorphisms could affect the generation of active thiol metabolite with administration of 600 mg or greater loading doses ( as suggested by the findings of bouman et al)10 and that it could be associated with small differences in platelet inhibition can not be excluded.10 however , in contrast to bouman et als10 study , several investigators were unable to confirm the association between pon1 q192r genotypes and stent thrombosis in other independent large cohorts of patients undergoing pci who were treated with clopidogrel.1621 limitations of the study include a relatively small sample size and a lack of pharmacokinetic analysis to detect differences in active metabolite concentrations . although considered the gold standard for measuring platelet aggregation and clopidogrel platelet inhibition , lta is subject to operator variability , and on - treatment platelet aggregation may not be a direct reflection of p2y12 receptor inhibition by active clopidogrel metabolites . this study confirms the previously reported impact of reduced - function cyp2c19 * 2 polymorphism on platelet inhibition by clopidogrel . polymorphisms of pon1 q192r appear not to be predictive of clopidogrel response when measured by lta and vn - p2y12 assay in patients with coronary artery disease .

backgroundthe metabolic activation of clopidogrel is a two - step process . it has been suggested that paraoxonase-1 ( pon1 ) is a rate - limiting enzyme in the conversion of 2-oxo- clopidogrel to an active thiol metabolite . conflicting results have been reported in regard to ( 1 ) the association of a common polymorphism of pon1 ( q192r ) with reduced rates of coronary stent thrombosis in patients taking clopidogrel and ( 2 ) its effects on platelet inhibition in patient populations of european descent.methodsblood samples from 151 subjects of mixed racial background with established coronary artery disease and who received clopidogrel were analyzed . platelet aggregation was determined with light transmittance aggregometry and verifynow p2y12 assay . genotyping for cytochrome p450 2c19 ( cyp2c19)*2 and * 3 and pon1 ( q192r ) polymorphisms was performed.resultscarriers of cyp2c19 * 2 alleles exhibited lower levels of platelet inhibition and higher on - treatment platelet aggregation than noncarriers . there was no significant difference in platelet aggregation among pon1 q192r genotypes . homozygous carriers of the wild - type variant of pon1 ( qq192 ) had similar on - treatment platelet reactivity to carriers of increased - function variant alleles during maintenance clopidogrel dosing , as well as after administration of a clopidogrel 600 mg loading dose.conclusioncyp2c19*2 allele is associated with impaired platelet inhibition by clopidogrel and high on - treatment platelet aggregation . pon1 ( q192r ) polymorphism does not appear to be a significant determinant of clopidogrel response .

pubmed

toxoplasmosis is caused by the obligate intracellular protozoan parasite , toxoplasma gondii , for which preferred primary host is a cat and human may become infected as an intermediate host.1 most people with toxoplasmosis are asymptomatic . however , if a pregnant woman is infected , the parasite can enter the fetal circulation through placenta and may cause congenital toxoplasmosis in the fetus . congenital toxoplasmosis may present with a broad spectrum of clinical manifestations , ranging from mild chorioretinitis to severe central nervous system ( cns ) involvement , including hydrocephalus , mental retardation , seizures and intracerebral calcifications.2,3 seizure is one of the main presenting symptoms in patients with cns infection of congenital acquired toxoplasmosis and many patients with congenital toxoplasmosis took anti - epileptic drugs ( aed ) constantly . however , there have been few reports of detailed description of seizure semiology and besides few reported cases of a patient with congenital toxoplasmosis who was suffering from temporal lobe epilepsy with hippocampal sclerosis ( hs ) , which is one of the most common epileptic syndromes and is considered medically intractable but surgically remediable in any causes.4,5 hereby we described a patient with congenital toxoplasmosis who showed intractable mesial temporal lobe epilepsy . a 34-year - old left handed arabian female was presented with our hospital with medically intractable seizure . she had hydrocephalus and right ventriculoperitoneal ( vp ) shunting was performed at 1 month old . she also complained of decreased visual acuity in both eyes . on past history , she had no history of febrile seizure , metabolic disease or head trauma and her family also had no history of seizure . in a neurological examination , contracture and her mental status was evaluated using mini - mental state examination with the help of a translator and her score was 22 , suggesting memory impairment and inattention . in ophthalmologic evaluation for visual impairment , macular inferior chorioretinal scar in right eye and large gray - whitish retinal necrotic lesion on posterior pole in left eye was found compatible with chorioretinitis which was characteristic finding usually seen in the patients with congenital toxoplasmosis ( fig . test using enzyme linked fluorescent assay , toxoplasma - specific igg test was positive and igm test was negative , suggesting chronic phase of t. gondii infection.6 the patient and her mother reported that her seizures were started with psychic aura such as anxiety initially and then secondarily generalized seizure with right eyeball deviation and head version . in the beginning , her seizures occurred one to three times a month . however the frequency of her seizure were gradually increased over time , and though the dose of aed was increased , the frequency of seizure was not reduced . finally , she had the polytherapy of aeds including levetiracetam , lamotrigine and topiramate , but seizures were still poorly controlled . for further evaluation and management of her seizure , she was referred to our epilepsy clinic . she underwent 3.0 t high resolution brain magnetic resonance imaging ( mri ) . on brain mri , multifocal encephalomalacic change related to vp shunt , left hs and left amygdala hypertrophy , and multifocal parenchymal calcification including left perihippocampal region were observed . the parenchymal calcification was confirmed again by brain computed tomography ( ct ) , which was compatible with congenital toxoplasmosis . video / electroencephalogram ( eeg ) monitoring was performed for 6 days . during video - eeg monitoring , 5 complex partial seizures ( cps ) were observed , and 2 of those 5 cps were led to generalized tonic clonic ( gtc ) seizure . all cpss were started with psychic aura such as anxiety , and dialeptic seizure was followed . in secondary gtc seizures , tonic posture of right arm and leg and head version to right side were accompanied . the rhythmic delta activity was observed initially on left temporal area , and then led to rhythmic theta waves in the same area , spreading into left hemisphere . intermittent slowing in left temporal area was also seen in interictal eeg monitoring and no epileptiform discharges were seen . she underwent ictal and interictal 99m - technetium ( tc ) hexamethylpropyleneamineoxime single photon emission computed tomography ( hmpao - spect ) . ictal hyperperfusion was seen in the bilateral frontotemporal areas , more pronounced on the left side . interictal spect , taken after at least 24 hour seizure freedom , showed hypoperfusion in the left frontotemporal area . substracted ictal spect coregistered to mri ( siscom ) shows the ictal hyperperfusion zone in left mesial temporal areas . brain f - fluorodeoxyglucose positron - emission tomography ( fdg - pet ) demonstrated focal hypermetabolism in left medial temporal cortex compatible with postictal status and hypometabolism in the left fronto - parieto - temporal cortex . wada test was performed to determinate the lateralization of hemispheric speech and memory dominance and it revealed right hemisphere language and memory dominance , predictive of good outcome after left anteromesial temporal resection . considering that her seizure was medically intractable and epileptogenic focus was determined to be in left temporal lobe by presurgical evaluation , we decided to perform epilepsy surgery with left anteromesial temporal resection including calcified lesion to achieve seizure control . this is a case report of medically intractable mesial temporal lobe epilepsy in a patient with congenital toxoplasmosis , diagnosed by toxoplasma - specific serologic test with typical symptoms and signs including chorioretinitis , growth retardation , cerebral palsy and hydrocephalus . in patients with congenital toxoplasmosis , it is well known that infection may invade tissues in central nervous system and can cause diffuse and multifocal parenchymal necrosis forming calcifications , which are main characteristics of this disease . in addition , the distribution and severity of calcifications vary according to the date of maternal infection and also correlate with neurologic signs and symptoms.7,8 in our case , multifocal small parenchymal calcifications including a calcified nodule in left perihippocampal area was found in brain imaging findings , and hs and amygdala hypertrophy on left temporal lobe were also observed . our patient presented predominantly dialeptic seizure accompanied by psychic aura during ictal events , which was a commonly observed finding in tle . tle was confirmed with ictal and interictal eeg findings , and neuroimaging including spect and siscom . hs is the most common finding in mesial temporal lobe epilepsy , closely associated with medically intractable epilepsy . though the cause of hs remains unknown , many hypotheses including febrile seizures , genetic susceptibility or acquired damage such as inflammation , are considered . also , it is well known that recurrent seizures may cause hippocampal neuronal damage in animals similar to hippocampal sclerosis , which may be considered as a mechanism in patients with dual pathology , coexistence of hs with extrahippocampal lesion.5,9 our patient had no history of febrile seizure and no family history of epilepsy , but on brain imaging study , hs and amygdala hypertrophy were demonstrated concomitant with a calcified nodule in perihippocampal region by congenital toxoplasmosis . in this case , the pathogenesis of hs , which may be the cause of the tle , can be considered by several different mechanisms . one of possible mechanism of hs is that seizure due to a calcified nodule constitutes an initial precipitating illness , leading to the development of hs . the kindling phenomenon of epilepsy is known as a process in which repeated low - level stimuli result in seizure development until a plateau is reached . this phenomenon is one of well - known model of epileptogenesis and also has been closely related to mesial temporal lobe epilepsy in respect of inducing progressive neuronal loss in hippocampal formation by repetitive seizure.10,11 this hypothesis has been suggested in several case reports of other disease such as neurocysticercosis . another possible mechanism is that hippocampus may be impacted by the inflammatory reaction and resulting gliosis around the calcified nodule . there was a case report of a patient with neurocysticercosis and intractable mesial temporal lobe epilepsy.12 in that case , calcified mass near the hippocampus and abundant corpora amylacea were found , suggesting that degenerating cysticercus induced inflammatory injury to hippocampus . also , the possibility that a calcified nodule by congenital toxoplasmosis and hs may coexist purely by chance can not be excluded . tle accompanied with hs is usually medically intractable , and hippocampal damage is known to progress in patient with medically intractable tle . so it needs to be very careful when managing with seizure in patients with congenital toxoplasmosis , accompanied with calcification in the perihippocampal area . comprehensive evaluation including video - eeg monitoring , spect , fdg - pet and mri might be needed for defining epilepsy and localizing epileptogenic foci accurately , and be necessary to consider the surgical treatment for managing seizure if possible . in korea , there have been many surveys on seroprevalence of toxoplasmosis , which are in the range from 1.9 to 7.7% in the 198090s.1315 these result was significantly lower than those of the other endemic countries of 3070%.16 however , with increase of the number of domestic cats and change of eating habits to meat preference , seroprevalence of toxoplasmosis in korea has increased to 1325%.17,18 furthermore , considering high prevalence of t. gondii in the stray cats in korea,19,20 these data imply that the number of patients with congenital toxoplasmosis like our case may increase , thus careful prenatal and neonatal monitoring for congenital toxoplasmosis is necessary and essential in korea . in our opinion , the association of congenital toxoplasmosis with mesial temporal lobe epilepsy has been poorly reported so far , and also patients with congenital toxoplasmosis showing seizure who underwent video eeg monitoring and appropriate neuroimaging study were reported rarely . we report a case with medically intractable mesial temporal lobe epilepsy with congenital toxoplasmosis , in which presented both hs and a calcified nodule in perihippocampal area . there may be several hypotheses for the observed hs in patients with congenital toxoplasmosis , and limits to define a cause - effect relationship between the two conditions among several mechanisms . when managing seizure disorder in patients with congenital toxoplasmosis , neurologists should be aware of this association , and the surgical treatment should be considered for managing seizure when patients with congenital toxoplasmosis presented with medically intractable tle and a calcified nodule in perihippocampal area .

toxoplasmosis is a rare disease caused by intracellular protozoan parasite , toxoplasma gondii . though most patients with toxoplasmosis are asymptomatic , congenital toxoplasmosis in the fetus can cause ocular involvement such as chorioretinitis and central nervous system disease including intracerebral calcification , nystagmus , hydrocephalus and microcephaly . also , these brain lesions can cause seizure secondarily . our patient was diagnosed with congenital toxoplasmosis , based on toxoplasma - specific serologic test with typical clinical symptoms , including chorioretinitis , nystagmus , hydrocephalus and cerebral palsy . her brain imaging findings revealed not only the multifocal encephalomalacia , but also multifocal cerebral calcification including intracerebral calcification in left perihippocampal region . her epileptogenic zone was defined as mesial temporal lobe including hippocampus on left side by seizure semiology , electroencephalogram and neuroimaging including single photon emission computed tomography and 18f - fluorodeoxyglucose positron - emission tomography . her seizures were refractory to multiple anti - epileptic drugs . we report a patient with congenital toxoplasmosis who showed intractable mesial temporal lobe epilepsy .

pubmed

approximately 20 million people ( 10 - 15% of the adult population ) have gallstones and 1 million new patients are diagnosed with cholelithiasis every year . as a result , the treatment of cholelithiasis has become the most costly digestive disease , with an estimated annual cost of more than $ 5 billion . in september , 1992 , the national institute of health convened a consensus development conference to discuss the treatment of cholelithiasis and laparoscopic cholecystectomy . after considerable discussion and review of the current literature , the panel concluded that laparoscopic cholecystectomy offered substantial advantages over the traditional open cholecystectomy with no apparent increase in morbidity or mortality . because of the reduction in pain and disability , the conference concluded that treatment costs would be equal to or slightly less than the cost of open cholecystectomy and that there would be substantial savings to patients and society because of the shortened period of disability . the disability and loss of income following standard open cholecystectomy further increase cost , but these items are generally not measured by traditional appraisal methods . this convalescent period and , consequently , costs have been greatly reduced by laparoscopic cholecystectomy . this example of cost analysis should be requested by business and possibly patients , as they are impacted the most . indeed , charges ( not costs ) for laparoscopic cholecystectomy often surpass those of open cholecystectomy . this is surprising because charges should reflect the costs resulting from increased length - of - stay ( los ) , nursing and hospital support required by open cholecystectomy . the charges for equipment and other technical requirements for laparoscopic procedures probably explain the excess charges noted in laparoscopic cholecystectomies . surgeons at maricopa medical center ( mmc ) identified excessive los and charges by comparing the cholecystectomy drg ( diagnostic related group ) 195 with 16 other hospitals in the region . the remainder were either old patients with complications or young patients with advanced disease and complicated courses . little could be done to reduce charges except to either lessen the conversion rates or costs for laparoscopic cholecystectomy . in order to get a precise estimate of laparoscopic charges , match pair comparisons from the same data base of charges between open cholecystectomy and converted laparoscopic cholecystectomy were accomplished . since the comparisons matched age , sex and similar diagnosis ( ex . : acute vs. chronic cholecystitis , pancreatitis , etc . ) , many confounding variables could be eliminated . this suggested that the only reasons for the increased charges were the charges for laparoscopy . other reasons for conversion , such as intraoperative complications , were eliminated because none of the matched pairs had serious complications or concomitant disease . therefore , this comparison provided a relatively straightforward technique for statistical comparison to determine the charges for laparoscopy . a retrospective review of 70 cholecystectomy patients was conducted from a four - year data base of 633 patients who had undergone cholecystectomy . thirty - five patients with converted laparoscopic cholecystectomies and thirty - five patients with open cholecystectomies were selected from the data by matching for age , sex and similar diagnosis . a paired sample t - test was used for statistical analysis with significance at the p=0.05 level . decision tree and sensitivity analyses were then used to choose the most cost - effective operation and then determine what the limitation of charges would be needed to make laparoscopic cholecystectomy cost - effective . the average age was 37 years ( range = 15 - 70 years ) ( standard deviation [ sd ] = 15.9 ) . the average charge for converted laparoscopic cholecystectomies was $ 19,922 ( sd = $ 7,134 ) , and that of open cholecystectomies , $ 13,042 ( sd = $ 9,715 ) ( p=<0.01 ) . the amount attributed to laparoscopy was $ 6,880 ( $ 19,922 - $ 13,042 ) . decision tree analysis showed that , regardless of the rate of conversion , open cholecystectomy was more cost - effective . however , if charges could be reduced to certain levels , depending on the conversion rate , laparoscopic cholecystectomy would become cost - effective . steiner reviewed surgical rates and operative mortality for open and laparoscopic cholecystectomy in maryland and found the overall mortality rate had been reduced by 33% . however , the total number of cholecystectomy - related deaths had not fallen because of a 28% increase in the rate of cholecystectomies . now that the technique has proven to be beneficial , the current challenge to all health providers is to improve these results by further reducing mortality and morbidity , while improving cost - efficiency . to achieve this , databases for cholecystectomies must be studied to determine which aspects of the preoperative evaluation , surgery and postoperative treatment could be modified to achieve these goals . this paper attempts to address these goals with studying the charges for open , laparoscopic and converted laparoscopic cholecystectomy by evaluating matched pairs of patients in a large data set . matched - pair comparisons of open and converted laparoscopic cholecystectomy provide a simple method of determining the true charges for laparoscopic cholecystectomy ( including increased operative time , anesthesia time , cholangiograms , instruments , etc . ) . with similar length - of - stay ( los ) , and averaging the charges over a large number of matched pairs , the main independent variable remaining is that of the charge for the laparoscopy . in this comparative series , reduction of this charge by applying reusable instruments and having surgeons with advanced laparoscopic skills operate on patients that have a higher probability of conversion would make laparoscopic cholecystectomy more cost - effective . consider that the average charge for laparoscopic cholecystectomy in the 633 patients equaled $ 16,011 and the average charge for open cholecystectomy was $ 13,042 . a decision - tree analysis of these values shows that managed - care contractors would prefer open cholecystectomy for all patients with cholecystitis at mmc ( figure 1 ) . however , most patients would not enroll in a health plan or go to a hospital which did not provide laparoscopic cholecystectomy , so an evaluation of what could be done to reduce the charges for laparoscopic cholecystectomy must be done to make laparoscopic cholecystectomy attractive to the buyer . the most important question is , " how much of a reduction in charge ( cost ) would be necessary to make laparoscopic cholecystectomy cost - efficient ? " this depends not only on a reduction in charges but on the percentage of conversions ( because of the high cost of conversions ) . sensitivity analysis of these data shows that if the conversion rate equals 5% , a charge of < $ 12,679.90 would be needed to make laparoscopic cholecystectomy cost - effective ( figure 2 ) . therefore , a $ 3,331 ( $ 16,011 - $ 12,680 ) reduction in the charge of laparoscopic cholecystectomy would be needed before all patients could be considered for laparoscopic cholecystectomy . therefore , a $ 4,689 ( $ 16,011 - $ 11,322 ) reduction would be needed to consider all patients for laparoscopic cholecystectomy . therefore , the greater the amount of reduction needed , the less likely efficiency can be achieved by reducing laparoscopic cholecystectomy charges . vanek and borguet reviewed the cost of laparoscopic versus open cholecystectomy in a community hospital.5 they demonstrated that conversion from laparoscopic to open cholecystectomy resulted in a 57% increase in total charges over laparoscopic cholecystectomy . they suggested that this is due to the increase in hospital los of 4.7 days . they further demonstrated a charge for open cholecystectomy of $ 8,160 versus $ 10,210 ( p<0.001 ) for laparoscopic cholecystectomy . furthermore , the mean total hospital charge for converted laparoscopic cholecystectomies was $ 16,070 . given the literature average of return to work of 8.4 days for laparoscopic cholecystectomy and 27.5 days for open cholecystectomy , a savings of $ 2,483 accrues with the selection of laparoscopic cholecystectomy . comparative analysis is difficult because many of these studies include all cholecystectomy categories while other studies were limited to elective laparoscopic cholecystectomy patients . furthermore , these studies reflect marked geographical and socioeconomic factors as well as severity of disease mixes . it therefore becomes advisable that health care payers evaluate charge data for each of the regional hospitals before contracting with them for medical services . since laparoscopic cholecystectomy is demanded by most patients , knowledge based on laparoscopic charges and conversion rates give the hospital or health care provider information to use in evaluating surgical costs . obviously , the lower the charge , all else being equal , the more likely the contract . health care providers , including hmos , are looking for the most cost - effective provider and institutions which wish to compete must evaluate their procedures to inform health care providers that they are cost - efficient . matched - pair analysis , decision - tree and sensitivity analyses , similar to those described above , facilitate appraisal by each institution as to what can be done to improve care , reduce charges and become cost - efficient in performing laparoscopic cholecystectomy . since there is reduced los and short - term dis - ability , hospital charges should not be in excess of that for open cholecystectomy . only evaluation and modification of the charges which contribute to the excess will lead to validated cost efficiencies and subsequent recruitment by providers for hospital service .

background and objectives : maricopa medical center ( mmc ) was found to have higher charges and length - of - stays than 16 other regional hospitals in an analysis of drg categories for gallbladder disease . these comparative figures identified mmc as being inefficient and demanded review to determine the reasons for the inefficiencies.methods:in an attempt to determine the reason for inefficiency of charges and length - of - stay for the laparoscopic portion of laparoscopic cholecystectomy , matched pairs of open cholecystectomy and converted laparoscopic cholecystectomy from a data base of 633 patients with cholecystectomies were reviewed . thirty - five matches for age , sex and similar diagnosis were successful.results:matched pair evaluation disclosed a $ 6,880 difference in charges , which was attributed solely to the charge for laparoscopy . subsequent chart analysis showed a high charge for instrumentation , prolonged anesthesia and operative times and longer preoperative delays before surgery . moreover , no matter what the conversion rate is , open cholecystectomy was more cost effective . however , if there is a conversion rate of 5% , total hospital charges for laparoscopic cholecystectomy would have to be reduced to $ 12,679 ( a reduction of $ 3,332 from $ 16,011 ) to make laparoscopic cholecystectomy cost-effective.conclusions:cost-effective decision tree analysis of matched pair comparisons and sensitivity analysis proves to be an effective technique in evaluating the cost - effectiveness of laparoscopic cholecystectomy in a hospital population .

pubmed

type 1 diabetes is characterized by progressive autoimmune destruction of pancreatic cells that leads to symptoms of insulinopenia when -cell mass is deficient . glucose homeostasis is restored with administration of exogenous insulin and some patients experience a reduction of their daily insulin requirements ( dir ) with maintenance of optimal glycemia and hba1c ( a1c ) levels . due to its impact on disease control , this phenomenon is called partial remission ( pr ) or honeymoon period and is variable both in intensity ( with sometimes interruption of insulin ) and duration . definition of pr was recently revised based on dir and a1c levels ( i.e. , insulin - dose - adjusted a1c , idaa1c ) , after evidence of strong correlation between idaa1c and stimulated c - peptide levels 6 and 12 months after diagnosis . underlying mechanisms of pr remain largely unknown , but restoration of residual -cell mass activity in an otherwise insulin - sensitive environment is believed to account for the condition . pr has driven much interest as a crucial target period for interventions aiming at preserving the residual -cell mass , but immunomodulation trials have not proven to be efficient so far except for secondary study endpoints . besides potential therapeutic implications , the significance of pr is unclear , in terms of both disease progression and complication risks . reduction of diabetes - related complications ( retinopathy , nephropathy ) has been described in patients with residual c - peptide secretion , but association factors are unknown and regular c - peptide measurements are included neither in current pr definition nor in clinical routine guidelines . moreover , pr impacts preliminary experiences of patients with type 1 diabetes , which change abruptly ( with rise of both dir and a1c ) as pr ends . the objectives of this retrospective study are to evaluate the characteristics and potential determinants of pr according to the new definition and to evaluate the presence of correlations between pr and long - term type 1 diabetes evolution ( metabolic control and complications ) and occurrence of severe hypoglycemia . in our study , we included a total of 242 children aged 0.9 to 16.4 years , followed up in our pediatric diabetes clinic from diagnosis ( established from 1994 to 2008 ) to adulthood ( 1820 years of age ) . type 1 diabetes was diagnosed according to international society for pediatric and adolescent diabetes guidelines and based on symptoms of insulinopenia , elevated blood glucose ( bg ) and a1c , positive anti - islet antibodies ( gad65 , ia2 , and insulin ) , and lack of family history of genetic diabetes . biometrics ( age , height z - score , and bmi z - score ) and biological features ( bg , a1c ) were collected at diagnosis and at each consultation ( postdiagnosis consultations occurred at 15 days and 1 month and then every 3 months ; only fully adherent patients were recorded ) . at diagnosis , measures included screening of dka ( defined as ph < 7.3 and/or bicarbonate < 16 mm ) and postprandial c - peptide levels ( autodelfia c - peptide , perkinelmer life and analytical sciences ) , which were assayed every year . bmi was calculated by using the formula ( bmi = weight [ kg]/height [ m ] ) . pr was defined as idaa1c 9 , according to definition by mortensen et al . : a1c ( % ) + [ 4 insulin dose ( u / kg / day ) ] . insulin doses were adjusted for pre- and postprandial glycemic targets according to ispad guidelines , when available , or to our institution 's guidelines . screening for complications was performed yearly , starting from 2 years after diagnosis until adulthood , and included determination of microalbuminuria ( urine spot ) , ophthalmoscopy , peripheral and autonomic neuropathy by physical examination , and blood lipids . a patient was considered having complications when a single anomaly or more anomalies were diagnosed . severe hypoglycemia was defined as loss of consciousness , coma with or without convulsions , or alteration of consciousness impeding the capacity for oral sugar ingestion ( need of a tier for i m glucagon administration ) . occurrence of severe hypoglycemia was monitored at each consultation ( as per our institution 's guidelines ) . for data analysis , 3 age subgroups were constituted ( 04 years , 59 years , and > 10 years ) in order to include most pubertal patients into one subgroup ( > 10 years ) . categorical variables were analyzed using chi - square test and continuous variables were analyzed using chi - square trend test , unpaired t - test , or mann - whitney u test , according to the statistical distribution . data were submitted to d'agostino and pearson omnibus normality test and levene 's test for equality of variances . correlation analysis was used to evaluate relationship between variables . to assess the relative contribution of each variable ( initial c - peptide , initial a1c , sex , and dka at diagnosis ) to chances of remission , logistic regression models were built . in our clinical series , pr occurred in 56.2% of patients with type 1 diabetes ( table 1 ) , without any case of complete remission . the proportion of girls ( 47.5% ) and boys ( 52.5% ) was comparable among remitters and nonremitters . mean age at diagnosis was 8.8 3.8 years with no difference between subgroups ( pr versus no pr , girls , boys , and age subgroups ) . height and bmi z - scores , measured at first follow - up consultation to avoid influence of insulinopenia on weight , were also similar between subgroups and to the distribution of healthy belgian children from the reference database . at diagnosis , most patients had no dka ( 74.4% versus 25.6% with dka , p < 0.0001 ) and the absence of dka was significantly higher in pr ( 82.3% ) than in no pr ( 63.5% ) patients ( p = 0.0047 ) ( table 2 ) . this effect was more pronounced in boys , who had a higher pr frequency ( 69.4% versus 30.6% no pr , p = 0.0023 ) when no dka occurred at diagnosis . furthermore , multivariate logistic regression analysis showed that chances of pr were higher when there was no dka at diagnosis ( or = 0.43 , p = 0.018 ) ( cf . below and in table 3 ) . mean pr duration was 279.6 days ( range 151722 ) or 9.2 months ( range 0.556.6 ) with no differences between gender and age subgroups ( figure 1(a ) ) . in 71.6% of patients , pr duration was shorter than 1 year and pr mostly occurred during the first 6 months ( figure 1(b ) ) . however , 7.5% of patients experienced pr during more than 2 years ( mean 992 days ; 95% ci [ 797 , 1187 ] ) . at diagnosis , patients had an overall a1c at 10.4 2.8% ( figure 1(c ) ) , which then dropped after 2 , 3 , and 5 years . patients entering pr had a lower baseline a1c ( 10.1 2.7% ) than patients with no subsequent pr ( 10.8 2.8% ) ( p = 0.049 ) ( figure 1(d ) ) . this effect was evidenced in girls entering pr who had a lower a1c at diagnosis ( 10.0 2.9% ) than girls without pr ( 11.2 3.1% ) ( p = 0.028 ) . however , boys had similar a1c levels whether they entered pr or not . in multivariate logistic regression models , lower a1c at diagnosis was associated with higher chances of pr ( or = 0.87 , p = 0.03 ) ( table 3 ) , but levels of a1c at diagnosis did not correlate with pr duration when analyzed for all patients ( cf . yet when age subgroup 04 years was considered , a1c levels at diagnosis negatively correlated with longer pr durations ( p = 0.01 , r = 0.15 ) . initial a1c did not impact long - term a1c levels since no correlation could be observed between a1c at diagnosis and a1c at 2 years after diagnosis ( a1c+2y ) or at 3 years after diagnosis ( a1c+3y ) , either in pr or in no pr subgroups ( see figures s1(a)-s1(b ) in supplementary material available online at http://dx.doi.org/10.1155/2014/851378 ) . a1c+2y was significantly lower in the pr ( 7.5% ; 95% ci [ 7.3 , 7.7 ] ) than in the no pr population ( 8.2% ; 95% ci [ 7.9 , 8.5 ] ) ( p < 0.0001 ) ( figure 1(e ) ) , which is partly explained by the fact that pr duration is longer than 2 years in 7.5% of the pr patients . this observation was corroborated by the fact that a1c at 3 and 5 years after diagnosis ( resp . , a1c+3y and a1c+5y ) did not differ between pr and non - pr groups . however , a1c+3y was lower ( 6.7% ; 95% ci [ 4.7 , 8.8 ] ) in patients having a remission lasting between 510 and 570 days as compared to nonremitters ( 8.2% ; 95% ci [ 7.8 , 8.5 ] ) ( p = 0.045 ) , suggesting a prolonged effect on a1c after the pr period in this subgroup . furthermore , a1c levels at 3 , 6 , 9 , and 12 months after diagnosis were strongly associated with pr duration when subgrouped ( figure 2(a ) ) . indeed , low ( 6% ) a1cs at 3 to 12 months were associated with longer pr ( mean 320 to 515 days ) , whereas pr durations dropped to mean 169 to 222 days for a1cs > 6 to 7% ( p < 0.01 ) and to mean 62 to 82 days for a1cs > 7 to 8% ( p < 0.01 ) . with high a1cs ( > 8 to 9% ) , pr durations were only negligible ( 1446 days , p < 0.05 ) and not different from patients with poorly controlled diabetes ( a1c > 9% ) . hence , a1c levels at 3 months allowed us to estimate the proportion of patients that will experience pr for more than 1 year at 34% ( n = 15/44 ) when a1c levels were 6% , 16% ( n = 16/102 ) for a1cs > 6 - 7% , and 5% ( n = 2/36 ) for a1cs > 7 - 8% . similarly , at 6-month follow - up , these proportions changed to 46% ( n = 23/50 ) for a1cs 6% , 8% ( n = 6/74 ) for a1cs > 6 - 7% , and 4% ( n = 2/50 ) for a1cs > 7 - 8% . no differences could be observed in the titers of anti - gad65 and anti - ia2 antibodies at diagnosis among the different analyzed populations ( girls , boys , and age subgroups ) ( table s1 ) . however , pr duration was significantly shorter ( 85.8 days ; 95% ci [ 33.9 , 137.6 ] ) in patients having 2 positive anti - gad65 and anti - ia2 antibodies at diagnosis compared with patients with only 1 positive antibody ( 198.0 days ; 95% ci [ 70.9 , 325.0 ] ) ( p = 0.04 ) . additionally , anti - ia2 antibody titers positively correlated with a1c+2y levels ( p = 0.006 , r = 0.16 ) , but this correlation was not found with a1c+3y and a1c+5y levels or with the other antibodies , partly due to smaller sample sizes . c - peptide secretion was significantly different in pr versus no pr patients , from diagnosis until 3 years after diagnosis ( figure 2(b ) ) . however , this difference could not be observed after 4 and 5 years after diagnosis , perhaps in part because of a bias of patient selection for c - peptide screening . higher baseline c - peptide concentrations were observed both in girls and in boys entering pr compared to patients without pr and this effect was more pronounced in boys ( figure 2(c ) ) . an age effect on c - peptide levels was observed not only at diagnosis where higher c - peptide levels were found in patients aged > 10 years , as compared with other age groups ( 04 years , 59 years ) ( p < 0.0001 ) ( figure 2(c ) ) , but also later on since age at diagnosis positively correlated with c - peptide levels at 2 years after diagnosis ( p < 0.0001 , r = 0.21 ) . in multivariate logistic regression , other variables than a1c levels and dka at diagnosis ( i.e. , age , sex , height and bmi z - scores , c - peptide levels , and anti - gad65 and ia2 titers ) were not significantly associated with a higher chance of pr ( table 3 ) . when pr was considered on a continuous scale ( pr duration ) , none of the estimates was significant in linear regression , even if consistent with logistic regression estimates . pr duration or a1c+2y levels did not correlate with occurrence of severe hypoglycemia as these parameters were not different in patients without documented episodes of severe hypoglycemia during the follow - up period as compared to patients having at least one episode ( n = 97 ; 39.7% ) or more than 2 ( n = 32 ; 13.2% ) episodes of severe hypoglycemia . furthermore , only few patients ( n = 11 ) developed diabetes - related complications during follow - up until adulthood ; thus , no differences with complication - free patients could be observed in terms of pr duration and a1c+2y . in our study , overall occurrence of pr was 56.2% and mean pr duration was 9.2 months , which was comparable to other studies using idaa1c or dir and a1c levels as pr definition ( table s2 ) . table s2 shows studies that included a1c and dir in pr definition since pr defined with dir only or a1c only overestimates pr rates . while some data showed that young children ( < 5 years ) have lower rates of pr , in our study , young children ( 04 years ) had similar pr rates ( 47.7% ) than older children ( 60.2% and 56.3% , resp . similar to other studies [ 10 , 11 ] , even with different pr definitions [ 7 , 12 ] , we did not find gender differences . however , dost et al . reported longer pr in boys below 10 years of age , but in the same study pr durations were shorter for patients < 10 years when the global cohort was analyzed . furthermore , we did not observe any complete remission as per idaa1c definition or defined as a complete interruption of insulin administration , contrary to data from other groups [ 11 , 14 ] showing complete remission in up to 4% of patients for a mean period of 3 months . our finding that dka is associated with a lower frequency of pr is in concordance with the literature . however , this effect was more pronounced in boys than in girls in our series . furthermore , we did not find a higher frequency of dka in younger children ( 04 years ) as described elsewhere [ 9 , 10 , 15 , 16 ] . whether dka is a variable independent of pr is unclear , since dka might reflect rapid -cell destruction and thus preclude ongoing pr development , by contrast to patients without dka whose -cell mass is partially preserved . in our study , however , only girls had lower a1c levels at diagnosis when entering pr , and only young patients ( 04 years ) had their a1c levels at diagnosis correlating negatively with pr duration . we did not find similar gender- or age - related correlations in the literature , whether as it was not measured or not found . interestingly , our findings show that early a1c levels might be helpful to predict pr durations since , as soon as 3 months after diagnosis , lower a1cs were significantly associated with a higher incidence of longer prs , this association being present at 6- , 9- , and 12-month follow - up . likewise to a1c , patients entering pr had higher c - peptide levels at diagnosis , and this effect was more pronounced in older ( > 10 years ) patients . also , older children at diagnosis had more chance to have higher c - peptide levels after 2 years . however , initial c - peptide levels were not significantly associated with higher chances of pr . in our study , we randomly assayed postprandial c - peptide levels , making definitive analysis of -cell reserve difficult . proper analysis of stimulated c - peptide with mixed meal tolerance testing was not available in our retrospective study . however , since c - peptide secretion is not affected by exogenous insulin and higher c - peptide levels may be reached by random rather than stimulated sampling , we believe that our data provide a valuable basis for correlation analyses . furthermore , the age effect in c - peptide levels observed in our series might in part be explained by differences in treatment modalities . indeed , in our center during the study period , adolescent patients were systematically proposed multiple daily injections ( mdi ) whereas younger children were offered bis in die insulin regimens . whereas similar glycemic and a1c targets were applied in both groups , mdi - treated patients might have benefited from intensive insulin therapy which was shown to prolong c - peptide levels in the dcct trial . nevertheless , as for dka , a1c and c - peptide levels at diagnosis may perhaps not be considered independent of pr , since they reflect , directly or not , residual -cell mass . while young children were shown to have either higher or lower rates of anti - islet antibodies at diagnosis of type 1 diabetes , we found no difference in anti - gad65 or anti - ia2 titers in any of the analyzed subgroups . only in patients without pr and aged 04 years a trend towards higher anti - gad65 titers was observed , while this was not significant ( p = 0.06 ) . our finding that pr duration was shorter in patients with 2 positive autoantibodies , as compared to patients with a single autoantibody , corroborated a recent study showing higher probability for developing type 1 diabetes in children having 2 anti - islet antibodies ( 69.7% ) versus one autoantibody ( 14.5% ) . in this prospective cohort , progression towards type 1 diabetes after seroconversion was also accelerated in the group having 2 autoantibodies whereas no data were provided regarding evolution of type 1 diabetes after diagnosis . finally , we did not observe long - term effects of pr on diabetes complications or incidence of severe hypoglycemia , partly due to low numbers of events . yet a short - term effect of pr on metabolic control was suggested in patients having pr durations between 510 and 570 days , since they had lower a1c levels after 3 years . however , no difference in a1c levels was detected after 5 years , showing the rapid loss of this effect . in conclusion , our comprehensive analysis of clinical and biological features of newly diagnosed belgian type 1 diabetes patients shows that , at diagnosis , parameters directly or indirectly associated with -cell mass reserve ( a1c and dka ) correlate with pr and that longer pr duration is associated with a lower a1c even 18 months after pr ends . further long - term research is needed to determine if this effect is associated with a reduced rate of complications and severe hypoglycemia .

to evaluate the characteristics and determinants of partial remission ( pr ) in belgian children with type 1 diabetes ( t1d ) , we analyzed records of 242 children from our center . clinical and biological features were collected at diagnosis and during follow - up . pr was defined using the insulin - dose - adjusted a1c definition . pr occurred in 56.2% of patients and lasted 9.2 months ( 0.5 to 56.6 ) . 25.6% of patients entered t1d with dka , which correlated with lower pr incidence ( 17.6% versus 82.3% when no dka ) . in our population , lower a1c levels at diagnosis were associated with higher pr incidence and in young children ( 04 years ) initial a1c levels negatively correlated with longer pr . early a1c levels were predictive of pr duration since 34% of patients had long prs ( > 1 year ) when a1c levels were 6% after 3 months whereas incidence of long pr decreased with higher a1cs . c - peptide levels were higher in patients entering pr and remained higher until 3 years after diagnosis . initial antibody titers did not influence pr except for anti - ia2 titers that correlated with a1c levels after 2 years . presence of 2 versus 1 anti - islet antibodies correlated with shorter pr . pr duration did not influence occurrence of severe hypoglycemia or diabetes - related complications but was associated with lower a1c levels after 18 months . we show that , at diagnosis of t1d , parameters associated with -cell mass reserve ( a1c , c - peptide , and dka ) correlate with the occurrence of pr , which affects post - pr a1c levels . further research is needed to determine the long - term significance of pr .

pubmed

oral health is important to the quality of life of individuals of all age groups . oral lesions can lead to interference of daily activities due to discomfort or pain that interferes with mastication , swallowing , and speech , producing additional symptoms such as halitosis , xerostomia , or oral dysesthesia , which hampers an individual s daily social activities . as has been established by the who ( 1984 ) , a population aging more than 60 years old should be considered , to be an elderly population . the last national census of population conducted in india in 2011 showed that 8.6% of the population was 60 years or more . these figures indicate that geriatric health care should be given due importance , since there has been little research done in the field of gerodontology . the prevalence of oral lesions in the major adult has been reported by few researchers in colombia , mexico , chile , spain , brazil , israel , argentina , usa , and a few asian countries like cambodia and china . these studies were mainly based on clinical evaluation of the lesions . the prevalence based on the biopsies of the observed lesions the oral lesions and treatment needs of the elderly patients differ according to every nation , geographic region within a country , and even in different communities . therefore , identification of the prevalence of oral lesions in this section of the population is due to socioeconomic differences , medical and dental history , treatment expenditure , and any handicap . no previous studies have been done till date in the indian subcontinent estimating the prevalence of oral mucosal lesions in the elderly population . epidemiologic studies provide information that is important to understand the prevalence , incidence , and severity of oral disease in a specific population , but the results of such studies have rarely been published worldwide . earlier epidemiologic studies of the oral health status of the general population in india provided very little information about oral mucosal lesions particularly as seen in the elderly . the present study was carried out to find the prevalence of oral mucosal lesions in 5,100 geriatric patients in india . this study consisted of 5,100 patients , 3,100 ( 60.8% ) men and 2,000 ( 39.2% ) women with age range , 60 to 98 years , attending the department of oral medicine and radiology , jodhpur dental college general hospital , from september 2008 to october 2012 . a written informed consent was obtained from the patients prior to the inclusion for the study . all elderly patients who were not willing or could not be allowed the clinical oral examination were excluded . all the examinations were carried out by one researcher . before recording the clinical parameters , the examiner and another researcher calibrated the clinical examination . both a dental and a general medical history of the patients were obtained , and a questionnaire was completed by each patient , after which a clinical examination was performed . some of the mucosal changes were diagnosed solely by clinical examination ( e.g. , linea alba , fissured tongue ) . when clinical features were not diagnostic , a biopsy was undertaken . the clinical diagnosis was established according to the correlation in the etiological factor associated to the lesion by systematic examination of oral mucosa and classifying those alterations according to the epidemiology guide for the diagnosis of oral mucosal diseases ( who ) . the elements to evaluate during the questionnaire included : general status of the patient , systemic diseases , medications used , age , gender , alcohol and tobacco consumption , habits ( trauma ) , and prosthetic or other appliances use . during the clinical examination the following elements were analyzed : features of the lesion , anatomical location , extension , etiological factors or related factors , dental status , alcohol , tobacco , trauma , use of prosthesis and whether or not these were well adapted . all non - pathological conditions or developmental such as leukoedema , lingual varices , fordyce granules , benign migratory glossitis , and fissured tongue were excluded from the present study . there were 3,100 ( 60.8% ) men and 2,000 ( 39.2% ) women with the age range 60 to 98 years . of these 5,100 patients , 3,264 patients ( 64% ) presented with various oral lesions . males were more affected ( 66% ) than females ( 34% ) , and this difference was not statistically significant ( p > .05 ) . in the majority of the cases , only one lesion was found ( 42% ) ; however , some patients exhibited more than one oral lesion simultaneously . the majority of the oral lesions were observed in patients between 65 to 70 years of age . the hard palate had most lesions ( 23.1% ) , followed by the tongue ( 16.4% ) and gingiva ( 14.7% ) , whereas soft palate ( 3.6% ) was the least affected site . the lesions identified during the study were classified according to clinical , histopathological , and microbiological diagnosis and were distributed in 27 different clinical entities ( table 2 ) . nicotinic stomatitis ( 43% ) due to tobacco use and denture stomatitis ( 34% ) due to long use of prostheses were the most commonly diagnosed lesions . osmf ( 30% ) was also a commonly seen premalignant lesion due to high percentage of people with the habit of betel nut chewing . according to different studies , it is a common finding to observe oral pluripathology in the elderly , due to the aging process , systemic complexity involvement in these patients , metabolic changes , nutritional factors , prosthetic use , medications , psychobiological habits , and alcohol or tobacco habits . therefore , several conditions are to be encountered in this particular age group which include neoplasms , infections , immunological , hematological , and systemic disorders , leading to oral pain and discomfort in the elderly patient . among the various causes leading to changes in the oral mucosa due to aging are bacterial , fungal , viral , parasitic infections and infections caused by other agents , physical and thermal alterations , immune system changes , systemic diseases , tumors , trauma , and other factors . the prevalence of oral mucosal lesions has been found to be higher in older patients than in the younger population . the prevalence of mucosal changes or lesions was 64% in the present study , which was higher than that of the findings of mujica et al . and espinoza et al . who noted the prevalence of mucosal changes to be 57% and 53% , respectively however , cebeci et al . observed the prevalence of oral changes to be 15.5% in the elderly patients . most population - based studies correlate oral mucosal disease with oral cancer and precancerous conditions , but few authors have recorded overall oral mucosal lesions or mucosal changes . the most common mucosal lesion found in the present study was nicotinic stomatitis ( 43% ) , whereas mujica et al . and cebeci et al . showed that the most common lesion was denture stomatitis ( 18% ) and anatomical changes ( 7% ) , respectively . in the present study , denture stomatitis was the second common lesion and was observed in 34% of patients , which was more when compared to the other studies . in the present study , osmf was recorded in 30% of population , but this condition was not at all seen in the studies by mujica et al . who reported leukoplakia to be the most common premalignant lesion ( 13% ) . leukoplakia was seen in 22% of the patients , in the present study which , however , was more when compared to results of the above study and the findings of cebeci et al . who noted leukoplakia in 0.4% . in the current study , lichen planus was detected in 18% of patients , whereas in the mujica and cebeci studies , lichen planus was noted in 3% and 0.8% . respectively . in the present investigation , 110 patients ( 2% ) were diagnosed as having malignancies , all of whom were diagnosed as having squamous cell carcinoma . the oral cavity is one of the most suitable locations for the development of cancerous diseases . most oral cancers , especially squamous cell carcinomas involving the mucosal tissue , are usually evident . however , all such potentially malignant lesions should be confirmed by microscopic analysis . in the present study , tobacco and its related products the odds of having a lesion increased with age , even after adjusting the tobacco use , the presence of faulty dentures , race / ethnicity and sex . the use of prostheses or their condition in the elderly helps to determine the number of oral lesions present in these individuals . in the present study , 42% of patients exhibited one lesion , similar to the findings of mujica et al . espinoza et al . stated that the use of poorly adapted prostheses should be considered as a relevant factor , explaining the fact that in some aged populations , where the use of prosthesis is low or limited , the prevalence of oral pathologies is relatively low . this fact could be evidenced in reports from hoad - reddick and corbet et al . who noted a prevalence of 41% and 31% , respectively , due to poorly adapted prosthesis . in the present study , however , of the lesions related to prosthesis use , denture stomatitis , was the most common one , representing 1,734 cases ( 34% ) as reported in the literature . the length of denture use and diabetes mellitus can be considered as significant risk factors for denture stomatitis and denture hyperplasia . some important associations between denture - related lesions , systemic diseases , and medication use which require further investigation has also been observed . certain lesions are more commonly seen on lower lip , while few are seen most commonly on upper lip . different interactions between genetic and environmental factors in the oral mucosa lead to the formation of different lesions . hard palate ( 23.1% ) was the most commonly affected site in the present study , whereas the soft palate was the least involved ( 3.6% ) . knowledge of site predilection for different diseases will be useful in acknowledging the factors responsible for the same . according to gender distribution , the majority of the studies have not demonstrated differences in the prevalence of the lesions . in contrast to these findings , a slight male preponderance is noted in the present study . in conclusion , the present study has provided information about the epidemiologic aspects of oral mucosal lesions in the geriatric population that may prove valuable in the planning of future oral health studies of this group of the population in india . the increased number of aged individuals implies an important demographic change all over the globe . therefore , it is important to plan accordingly in the social- , economic- , and health - related areas . further , community programs should be enhanced to educate the elderly population along with the younger generation to get the elderly screened for any oral - mucosal lesions by availing adequate clinical and pathological laboratory facilities .

backgroundoral health is important to individuals of all age groups . previous epidemiologic studies of the oral health status of the general population in india provided very little information about oral mucosal lesions in the elderly . hence , the purpose of the present study was to determine the prevalence of the oral lesions in a geriatric indian population.methods5,100 patients were clinically evaluated , with age ranging from 60 to 98 years . there were 3,100 males and 2,000 females , with a mean age of 69 6.3 yrs . the statistical analysis was done using the spss software , where p < .05 was considered to be significant.results64% of the patients presented with one or more oral lesions , associated to tobacco , betel nut consumption , and lesions secondary to trauma and prosthesis . males were more affected than females and this difference was clinically not significant ( p > .05 ) . the lesions were more frequently observed between 65 to 70 yrs . the most common alterations observed were smoker s palate ( 43% ) , denture stomatitis ( 34% ) , oral submucous fibrosis ( 30% ) , frictional keratosis ( 23% ) , leukoplakia ( 22% ) , and pyogenic granuloma ( 22% ) . hard palate was the most commonly affected site ( 23.1%).conclusionsthe findings of the present study provide important information when clinically evaluating oral cavity in elderly . close follow - up and systematic evaluation is required in the elderly population to plan future treatment needs .

pubmed

chondroblastoma is a rare osseous tumor which accounts for less than 1% of all primary bone tumors , and affects the epiphyseal regions of the tubular bones , especially the ends of tibia , femur , and humerus . chondroblastoma localized in the head and neck region is uncommon but characteristically occurs in the temporal bone or the temporomandibular joint ( tmj ) and results in various symptoms such as occlusal abnormalities , trismus , clicking of the tmj , hearing impairments , dizziness , and otorrhea . malignant changes in chondroblastoma , although rare , have been observed after irradiation [ 3 , 4 ] . in order to avoid the inherent risk of malignant progression , radiation therapy should not be considered . however , chondroblastoma in the temporal bone has unusual histological features that make diagnosis difficult , and incomplete resection carries a high risk of recurrence [ 5 , 6 ] . thus , it would seem advisable , provided the diagnosis is accurate , to attempt a complete surgical removal for the curative treatment of chondroblastoma . following 5 years of unsuccessful treatment , the patient was referred to our department presenting with chronic hearing impairments and subsequently progressive trismus . a tumor mass was present in the middle cranial fossa , including the tmj and cochlea , in spite of earlier treatment regimens . by reporting this initially misdiagnosed case an accurate diagnosis was delayed because the original diagnosis relied on imaging analysis with neither careful magnetic resonance imaging ( mri)/computed tomography ( ct ) scanning analysis nor histopathologic examination . this less than thorough approach led not only to a delay of an accurate diagnosis and to unnecessary treatments , but also to a delay of suitable treatments for this rare chondroblastoma with malignant potential . this study presents methods which are helpful in diagnosing chondroblastoma , as well as suitable surgical procedures for maintaining postoperative functionality . a 41-year - old male with tmj disorder , initially diagnosed with otitis media , presented with a history of chronic ( lasting more than 1 year ) hearing loss on the right side . the patient underwent several attempts at antibiotic therapy and treatment for eustachian tube blockage , but his symptoms were unresponsive to both systemic antibiotic agents and tubing therapy . his initial diagnosis of tmj disorder was based on physical assessment of malocclusion and complaints of subsequently developed trismus . after these 2 years , endoscopic surgery , followed by several attempts to correct the malocclusion for the tmj disorder , was implemented but resulted in no significant improvement in clinical manifestations . the patient was further assessed and a ct scan and pathologic examination showed the presence of a giant cell tumor in the temporal bone . the patient harbored the primary disease and consulted us for persistent restriction of the mouth opening as well as hearing loss and dizziness , which he had noticed 5 years earlier . re - examination of the ct scan revealed that the tumor mass had progressively enlarged and had widely destroyed the middle cranial fossa in the temporal lobe ( fig . 1 ) . mri displayed the presence of a poorly defined mass , with predominantly low signal and relatively high signal intensities , on t1- and t2-weighted sequences , respectively , involving the right middle cranial fossa and infratemporal fossa floor . the tumor mass invaded the right temporal lobe and destroyed the subzygomatic fossa ( fig . hearing loss in the right ear , trismus , and paralysis in v2 had not been reported by the patient before visiting our department . no paralysis was observed in v1 and v3 , nor in the facial , glossopharyngeal , or hypoglossal nerves . re - examination of combined mri / ct scanning indicated rapid enlargement and widespread invasion of the mass into the cranial bone , with enlarged lymph nodes in the cervical region , providing evidence for chondrosarcoma or chondroblastoma in the temporal bone ( data not shown ) . preoperative histopathologic studies of the biopsy specimens for s-100 protein ( rabbit polyclonal antibody ; dako , denmark ) were performed on formalin - fixed , paraffin - embedded tissues , using an avidin - biotin - complex method as instructed by the manufacturer . histopathologic findings showed extensive proliferation of multinucleated giant cells with circular nuclei showing a sheet - like structure , formation of a small quantity of acidophilic substrate in the tumor mass , large cystic areas , and the presence of hemorrhage and aneurismal bone cysts indicating secondary aneurism - like bone cyst formation ( fig . preoperative re - examinations of combined mri / ct scanning and pathologic findings revealed the features of chondroblastoma . a generous c - shaped postauricular incision was made through the skin and subcutaneous tissue , which was elevated to expose the underlying fascia , periosteum , and muscle . the incision was extended down to the cervical region to allow exposure of the sternocleidomastoid ( scm ) muscle , the carotid artery , and the internal jugular vein . neck dissection was performed ranging in scope from removal of level ii and iii nodes to possible radical neck dissection , and total parotidectomy was performed while leaving facial nerves intact ( fig . a radical mastoidectomy was performed to achieve complete exposure of the mastoid and tympanic segments of the facial nerve . subtotal resection of the temporal bone and skull base dissection with resection of tmj using a zygomatic - extended middle fossa approach was carried out . the temporal lobe dura was resected about 30 cm together with part of the temporal lobe . the dura was reconstructed using fascia lata and covered with the pericranial flap and the temporalis muscle , and reconstruction using the scm and the platysma muscle was performed to prevent infection and dysfunction ( fig . reconstruction of the facial tissue allowed the patient to open his mouth to the extent of 40 mm with no pain ( fig . this study underlines an attempt to use conventional surgical procedures , with some modifications , for the treatment of chondroblastoma in order to prevent postoperative tmj dysfunction , including malocclusion , trismus , and dysphagia , while preserving the masseter , the coronoid process , and the anterior part of the temporal muscle . reconstruction for facial tissues was carried out by inserting the scm muscle flap into the infratemporal fossa . the scm flap was then fixed to the resected end of the zygoma inside the mandible . four and a half years after surgery , enhanced ct imaging showed no residual tumor . the patient had no clinical evidence of tumor recurrence during this period , nor of dizziness , with improvement of the occlusal abnormalities and trismus , indicating no postoperative dysfunction . the patient did not undergo postoperative irradiation since this is contraindicated with a diagnosis of chondroblastoma . the present study underlines the importance of an accurate diagnosis especially for a mass in the temporal bone . tumors such as chondroblastoma , which may originate as benign , may become malignant under inappropriate treatment . in fact , a combined radiotherapeutic - surgical approach may provoke neoplastic transformation in the residual tumor tissues of chondroblastoma [ 4 , 5 , 7 ] . following an inaccurate diagnosis , the misdiagnosis was attributable to the difficulty of a differential diagnosis since the clinical manifestations can be indistinguishable from giant cell tumor , chondrosarcoma , aneurismal bone cyst , enchondroma , or chondromyxoid fibroma . immediately before being referred to our department , the patient was misdiagnosed with giant cell tumor in the temporal region on the basis of ct scan / mri plus pathology . re - examination of ct / mri indicated rapid enlargement and widely - spread invasion of a mass into the cranial bone , along with enlarged lymph nodes in the cervical region , showing a possibility of chondrosarcoma in the temporal bone . however , the widely- and rapidly - enlarged mass was found to have infiltrated the temporal lobes , and to have destroyed the middle cranial fossa . these findings provided evidence of malignant chondroblastoma in the temporal bone . the subsequent immunohistochemical examination of the lesion showed reactivity of the neoplastic protein mass for s-100 protein . the presence of s-100 protein in tumor cells differentiates chondroblastoma from giant cell tumor , chondrosarcoma , and aneurismal bone cysts , favoring the diagnosis of chondroblastoma [ 8 , 9 , 10 ] . the presence of associated aneurismal bone cysts is , while not absolute , a supplemental criteria for the diagnosis of chondroblastoma . these findings strongly suggest potentially malignant chondroblastoma . treatment of a chondroblastoma in the temporal bone requires total en bloc excision , curettage , and irradiation . postoperative irradiation has been performed with the intention to reduce the risk of postoperative recurrence . however , it is not universally recommended due to malignant changes in chondroblastoma observed after irradiation . , a total en bloc excision should be the first choice as a therapeutic approach for chondroblastoma in the temporal bone . in the case presented here , the tumor was located in a site that included important neurovascular components , which generally makes surgical resection impractical . thus , accurate , early - stage diagnosis is a vital component to successful treatment . for patients with later - stage chondroblastoma , our modified surgical procedure is a valuable alternative .

we present a case involving a late diagnosis of chondroblastoma of the temporal skull base involving the temporomandibular joint ( tmj ) . following an initial misdiagnosis and unsuccessful treatment over a period of 5 years , the patient was referred to our department for further evaluation and possible surgical intervention for occlusal abnormalities , trismus , clicking of the tmj , and hearing impairment . based on preoperative immunochemical studies showing positive reaction of multinucleated giant cells for s-100 protein , the final diagnosis was chondroblastoma . the surgical approach postauricular incision and total parotidectomy , with complete removal of the temporal bone , including the tmj via the extended middle fossa was successful in preserving facial nerves and diminishing clinical manifestations . this study highlights a misdiagnosed case in an effort to underline the importance of medical examinations and accurate differential diagnosis in cases involving any tumor mass in the temporal bone .

pubmed

the benefits of regular structured exercise are well documented and include improvements in cardiovascular and metabolic function , which reduce the risk of chronic health conditions such as type 2 diabetes and cardiovascular disease . recent evidence also demonstrates that exercise alone ( in the absence of concomitant dietary restriction ) reduces visceral adipose tissue ( vat ) and intrahepatic lipid ( ihl ) [ 35 ] , both of which are independently associated with cardiovascular and metabolic risk . while there is strong evidence for the benefits of regular structured exercise ( i.e. , exercise which is planned and involves repetitive movement ) , it is currently unclear whether increased levels of habitual activity ( activity that is unstructured and accumulated during daily activities outside of structured exercise ) and reduced sedentary behavior ( activity with low metabolic rate ( < 1.6 mets ) and in a reclining or seated position ) have similar health benefits in the reduction of these adipose tissue depots . understanding such associations has implications for policy as , because of low participation rates in structured exercise , there is an increasing emphasis in public health messages on the importance of habitual physical activity levels and reduced sedentary time . cross - sectional analyses indicate an inverse relationship between central adiposity as inferred by waist circumference , dual energy x - ray absorptiometry , or computed tomography ( ct ) and self - reported physical activity levels . a similar inverse relationship has been observed for nonalcoholic fatty liver disease ( nafld ) [ 1214 ] . prospective studies have further supported this association [ 1517 ] . in the finnish twin cohort ( twinactive ) study , physically inactive cotwins were found to have liver fat scores ( assessed via difference in mean signal intensity from magnetic resonance imaging ) 170% greater than that of their active cotwin , and habitual physical activity , performed outside of work hours , was the strongest correlate of central abdominal fat after controlling for both environmental and genetic factors . however , self - reported behavior is subject to reporter bias and tends to overestimate activity levels , especially in overweight and obese populations . emerging evidence using objectively measured physical activity via accelerometry supports these observations [ 2124 ] ; however research using accurate assessments of vat and ihl is limited . if the above epidemiological data [ 1114 ] can be confirmed by high quality objective assessments , encouraging habitual physical activity and reducing sedentary behavior will have important clinical ramifications . hence , in this study , we aimed to evaluate whether levels of accurately quantified sedentary behavior and habitual physical activity could predict levels of vat and ihl in adults with overweight / obesity . based on current guidelines for the management of obesity which recommend high volumes of moderate intensity activity , we hypothesised that there would be a negative relationship between time spent in moderate intensity physical activity and levels of vat and ihl and a positive relationship between time spent in sedentary behavior and vat and ihl content . furthermore , we hypothesised that levels of sedentary behavior and moderate physical activity would explain a significant proportion of variance in vat and ihl when modelled with known obesity - related health risk factors ( including age , sex , body mass index ( bmi ) , waist circumference , fasting glucose , fasting insulin , high density lipoprotein cholesterol , triglycerides , and resting blood pressure ) . participants were recruited via noticeboards , electronic bulletins , and clinical databases between june 2011 and january 2015 from a community - based sample of adult men and women ( aged 1860 years ) with overweight or obesity ( bmi 25.0 kgm ) . volunteers were excluded if they reported being physically active ( exercising > 3 days per week or meeting current guidelines for physical activity ) or were cigarette smokers . participants provided clearance for participation from a medical practitioner prior to involvement and were excluded if there was evidence of an unstable cardiac condition , type 2 diabetes , hypertension , or liver disease other than nonalcoholic fatty liver disease ( nafld ) or secondary causes of steatohepatitis . other exclusion criteria included taking lipid - lowering or insulin sensitizing medication , reporting taking n-3 polyunsaturated fatty acids within the last 6 months , or a change in medication within the last 12 months , regular alcohol consumption > 20 g / day , or a significant change in body weight ( > 5% weight change ) in the previous three months . the analysis included 82 volunteers from larger intervention trials ( anzctrn12610000351011 ; actrn12614000723684 ) , the results of which have been published elsewhere . the study conformed to the ethical guidelines of the 1975 declaration of helsinki and was approved by the university of sydney human research ethics committee and the sydney local health district ethics review committee . eligible participants were screened via telephone interview and those meeting criteria provided informed consent and were invited to attend baseline assessments which included measures of anthropometry , a fasting blood test , magnetic resonance imaging ( mri ) and spectroscopy ( h - mrs ) , and accelerometry . stature was recorded by stadiometer ( seca model 220 telescopic height rod , hamburg , germany ) . weight was measured ( tanita bc-418 body composition analyzer ; tanita corporation , tokyo , japan ) to the nearest 0.1 kg and bmi ( kgm ) was calculated . waist circumference was measured in triplicate at the horizontal plane , midway between the inferior margin of the ribs and the superior boarder of the iliac crest at end - expiration . systolic ( sbp ) and diastolic ( dbp ) blood pressure were measured manually on each arm , after 1015 min of quiet sitting . venous blood was collected from the antecubital vein after an overnight fast ( > 10 hours ) . on the same day serum glucose , insulin , total cholesterol ( tc ) , triglycerides ( tg ) , high density lipoprotein cholesterol ( hdl ) , low density lipoprotein cholesterol ( ldl ) , alanine aminotransferase ( alt ) , aspartate aminotransferase ( ast ) , and high sensitivity c - reactive protein ( hs - crp ) were analysed by a commercially accredited laboratory . visceral adipose tissue was assessed via mri and intrahepatic lipid via h - mrs using a 1.5 tesla achieva whole - body system ( philips medical systems ; best , netherlands ) as previously outlined [ 5 , 26 ] . the cross - sectional areas for abdominal vat were analysed using automated software ( hippo fat ) with manual editing as necessary . total vat volumes were calculated by summation of slice areas with adjustment for slice thickness . liver spectra data were analysed by magnetic resonance user interface software ( jmrui version 4.0 , eu project ) . current mean time spent in physical activity , sedentary behavior , daily steps , and energy expenditure were assessed by triaxial accelerometer ( sensewear , bodymedia inc . data were collected from all participants for four days ( 3 weekdays and 1 weekend , which was the minimum inclusion criteria ) for 24 hours per day , except during water - based activities including showering , bathing , or swimming . physical activity levels and sedentary behavior were quantified ( bodymedia software , v8.0 ) with sedentary behavior defined as < 1.6 mets , light activity as 3 < 6 mets , vigorous activity as 6 < 9 mets , and high activity as 9 mets . data were included if the monitor was worn for > 85% of a 24-hour day . all descriptive data are reported as means standard error from the mean ( se ) . differences between groups were compared by student 's t - test with homogeneity of variances assessed via levene 's test for equality of variance or for categorical data . simple anthropometric and demographic variables were entered at block 1 for both vat and ihl . known cardiometabolic risk variables were entered into the regression at block 2 , based on the variables that were significantly associated with vat and ihl , respectively , in our sample ( table 2 ) . these included tg , glucose , insulin , sbp , dbp , alt and bmi for vat and glucose , insulin , alt , hdl , and bmi for ihl . while tg was not significantly associated with vat in our analysis , it was included given its previously established association with excess vat . finally , mean time spent in sedentary behavior , moderate intensity activity , and vigorous intensity activity were entered into block 3 of the regression analysis . data were analysed using statistical package for the social sciences ( spss version 22.0 ; imb corp . , armonk , ny , usa ) ; p values were based on two - sided tests and considered statistically significant at p < 0.05 . mean age was 40.1 1.9 years , bmi 30.9 0.5 kgm , waist circumference 100.0 1.1 cm , and liver fat 5.3 0.5% . thirty - five percent of the participants were classified as having nafld based on ihl > 5.5% . participants with nafld had a significantly higher bmi , waist circumference , sbp , vat , fasting glucose , and fasting insulin ( p < 0.05 ) . there were no differences in objectively measured levels of physical activity , sedentary behavior , total daily energy expenditure , or the number of steps taken per day between participants with and without nafld ( table 1 ) . time spent in high intensity activity ( > 9 mets ) was negligible ( mean 37 27 seconds ) with only 3/82 participants ( 4% ) engaging in high intensity activity for more than an average of one minute in total . when high intensity activity was included in the analysis , the results were not altered ( data not shown ) . while there are no established critical thresholds for vat volume with respect to morbidity and mortality risk , based on gender specific waist circumferences for caucasian populations , 51% of men and bivariate correlations between vat and ihl and biochemical , anthropometric , and accelerometry assessed activity variables are summarised in table 2 . a higher volume of vat was associated with higher ihl , bmi , waist circumference , sbp , dbp , fasting glucose , fasting insulin , and alt ( table 2 ) . there was a weak association between higher levels of vat and higher amounts of moderate intensity physical activity ( r = 0.294 , p = 0.007 ) . higher levels of ihl were associated with higher bmi , waist circumference , vat , fasting glucose , and fasting insulin and lower levels of hdl , but there was no association between ihl and total energy expended , time spent in sedentary behavior , and time spent in any level of activity ( table 2 ) . demographic and anthropometric variables ( waist circumference , sex , and age ) accounted for 54% and 30% of the variance in vat and ihl , respectively ( p < 0.001 ; tables 3 and 4 ) . the addition of tg , glucose , insulin , sbp , dbp , and bmi in block 2 accounted for a further 9% of variance in vat ( p = 0.032 ) . the addition of glucose , hdl , alt , insulin , and bmi increased the prediction of ihl to 40% ; however this was not significant ( p = 0.06 ) . the addition of time spent in sedentary behavior , moderate and vigorous intensity activity ( block 3 ) , increased the prediction of vat to 65% ( p = 0.14 ) and did not change the prediction of ihl ( p = 0.96 ) . waist circumference , triglyceride , and fasting glucose contributed significantly to the prediction of high levels of vat while waist circumference and fasting insulin contributed significantly to the prediction of high levels of ihl . we aimed to evaluate whether levels of accurately quantified sedentary behavior and habitual physical activity could predict levels of visceral adiposity and liver fat . surprisingly , while traditional risk factors for cardiovascular disease ( e.g. , sex , age , and waist circumference ) were associated with vat and ihl , no relationship was observed between objectively measured physical inactivity and raised liver fat . further , we unexpectedly observed a weak positive association between vat volume and time spent in moderate activity ( i.e. , increased vat was associated with higher time spent in moderate intensity physical activity ) and also total energy expenditure . the inclusion of objectively measured levels of moderate activity and sedentary behavior did not improve the potency of traditional risk factors ( waist circumference , triglycerides , fasting glucose , or fasting insulin ) for predicting the liver fat concentration or level of visceral adiposity . as a cross - sectional analysis , this study demonstrates that current habitual activity levels are not related to vat or liver fat ; however it does not account for lifetime levels of habitual activity . further , while objective assessment of activity via accelerometry is considered more accurate than self - report methods , participants may still have altered their usual behavior during monitoring . while a significant reduction in liver fat has been observed with just 4 weeks of structured aerobic exercise , it is uncertain how long current habitual activity patterns may take to affect vat and liver fat . on the basis of epidemiological data at the population level , physical inactivity is associated with the development of nafld [ 1214 ] and visceral adiposity , and reducing sedentary behavior ( including sitting time ) should be generally advocated in all populations and is now recognized in public health messages . however , our findings suggest that objectively measured physical activity is not greatly predictive of risk at an individual level and the associations between habitual physical activity levels , sedentary behavior , and metabolically detrimental fat depots such as vat and ihl remain uncertain . while our findings appear counterintuitive to epidemiological data , unlike these studies , we accurately quantified both liver fat and vat , as well as 24-hour habitual physical activity behavior , suggesting that the relationship may not be as straightforward . there is now strong and consistent evidence that regular structured physical activity ( exercise training ) reduces vat and ihl [ 35 ] . this reflects current physical activity guidelines which advocate structured exercise for health benefits . with the less potent stimulus of general habitual activity , the volume of activity required for benefit is much greater ( e.g. , the accumulation of > 470 min of physical activity for weight loss ) . while increasing incidental activity is important for increasing total daily energy expenditure , there is an established dose potency of structured exercise for a range of cardiovascular and metabolic health risk variables . additionally , there are well - established inverse relationships between cardiorespiratory fitness and nafld , histological severity of liver disease , and vat . indeed high levels of cardiorespiratory fitness is a characteristic of the phenomenon termed metabolically healthy obesity and low cardiorespiratory fitness is an independent predictor of all - cause and cardiovascular disease mortality . in consideration of this , the emphasis for behavior change in this population should arguably be on adopting and adhering to structured exercise programs , cognizant of individual barriers to participation in exercise and exercise preferences , which may require facilitation with behavioral management strategies . obesity in developed nations is linked with an obesogenic environment which encompasses a calorie - dense diet ( overnutrition ) and a physical activity hostile ( or physical inactivity - promoting ) environment . however , while both physical inactivity and excess adiposity are independently associated with a greater risk of mortality , it is contentious whether physical inactivity is a cause or an effect of obesity [ 39 , 40 ] . therefore , while it is established that low amounts of physical activity and high amounts of sedentary behavior predict mortality , the role of these behaviors in the development of abdominal obesity and liver fat is unclear and perhaps minimal . recent research has suggested that prolonged sitting ( 8 hours / day ) is associated with an increased risk of all - cause mortality [ 41 , 42 ] and type 2 diabetes . however , in support of our findings , the association between sedentary behavior and markers of cardiometabolic risk disappear when adjusted or modified for total physical activity levels . hence only individuals with low activity and high levels of sedentary behavior such as sitting may be at increased health risk . while there is a heightened understanding of the detrimental effects of increased sedentary behavior and sitting time [ 42 , 45 ] and interrupting sitting time may confer some health benefit , this study suggests that a direct link between unstructured , objectively measured levels of physical activity and sedentariness and levels of metabolically risky fat depots is insignificant . in concordance with these findings , despite significant associations with moderate - to - vigorous physical activity , no associations were observed between objectively quantified sedentary time and hepatic steatosis ( estimated via ct ) in adults with nafld or with vat volume in men or women . a large analysis in 3056 volunteers observed that individuals with nafld ( defined by the fatty liver index ) spent less time in light , moderate , or vigorous activity than non - nafld counterparts and that the lowest quartile of physical activity was found in individuals with nafld and comorbid type 2 diabetes . it has also been observed that individuals meeting 150 minutes / week of moderate - to - vigorous physical activity as assessed via accelerometry had the lowest odds of hepatic steatosis ( assessed via ct ) independent of bmi ( or 0.77 ) . however , in these investigations , it is unclear whether the low levels of physical activity caused the elevated liver fat content or whether nafld itself caused patients to have low participation in physical activity . a limitation of this cross - sectional analysis is that the non - nafld cohort had low mean liver fat levels which reduced the range in the data . an important consideration is that we recruited adults with overweight / obesity who had no known comorbid cardiovascular or metabolic disease , which probably contributed to this limited spread . further research examining the interaction between sedentary behavior , physical activity levels , and metabolically detrimental fat depots , employing gold - standard methods , across a more diverse population , is therefore warranted . another limitation is that the aforementioned associations between physical activity , fitness , and nafld may not translate to disease severity . a retrospective analysis of 813 adults with biopsy - proven nafld demonstrating that only those who reported meeting the minimal guidelines for vigorous intensity activity ( 75 min / week ) and not moderate physical activity guidelines ( 150 to 300 min / week ) reduced the odds of developing nash ( or 0.65 ) with further reduced odds ( or 0.56 ) when the extensive guidelines for vigorous intensity activity ( 150 min / week ) were met . furthermore , the odds of advanced fibrosis were only reduced in patients meeting the extensive guidelines for vigorous activity . these data suggest that vigorous activity per se may be necessary for improvements in the progressive forms of nafld although longitudinal data from clinical trials are lacking and limited by the invasive nature of liver biopsy to inform disease severity . in conclusion , there were no associations between sedentary behavior and levels of ihl or vat and low levels of objectively measured physical activity did not predict high levels of vat or ihl . these findings suggest that while traditional risk factors such as waist circumference , triglycerides , fasting glucose , or fasting insulin may predict the incidence of nafld or high levels of vat , objectively quantified physical activity levels and sedentary behavior did not predict those at risk .

objective . epidemiologic studies suggest an inverse relationship between nonalcoholic fatty liver disease ( nafld ) , visceral adipose tissue ( vat ) , and self - reported physical activity levels . however , subjective measurements can be inaccurate and prone to reporter bias . we investigated whether objectively quantified physical activity levels predicted liver fat and vat in overweight / obese adults . methods . habitual physical activity was measured by triaxial accelerometry for four days ( n = 82 ) . time spent in sedentary behavior ( met < 1.6 ) and light ( met 1.6 < 3 ) , moderate ( met 3 < 6 ) , and vigorous ( met 6 < 9 ) physical activity was quantified . magnetic resonance imaging and spectroscopy were used to quantify visceral and liver fat . bivariate correlations and hierarchical multiple regression analyses were performed . results . there were no associations between physical activity or sedentary behavior and liver lipid . sedentary behavior and moderate and vigorous physical activity accounted for just 3% of variance for vat ( p = 0.14 ) and 0.003% for liver fat ( p = 0.96 ) . higher levels of vat were associated with time spent in moderate activity ( r = 0.294 , p = 0.007 ) , but there was no association with sedentary behavior . known risk factors for obesity - related nafld accounted for 62% and 40% of variance in vat and liver fat , respectively ( p < 0.01 ) . conclusion . objectively measured levels of habitual physical activity and sedentary behavior did not influence vat or liver fat .

pubmed

this is a retrospective , observational , consecutive case series conducted in a tertiary eye care hospital in north india . all patients with history of fireworks - related injuries , who attended the emergency eye care services and outpatient services during the 5 days of the diwali festival 2 days before diwali , day of diwali , and 2 days after diwali for 5 consecutive years from 2011 to 2015 , were included in this study . all relevant data related to age , sex , laterality , type of firework , whether user or bystander , baseline visual acuity ( va ) , details of injuries , diagnosis , whether requiring surgical or conservative management , and final best - corrected va ( bcva ) at the last follow - up were recorded . patients with lid tears , corneal tears , scleral tears , and intraocular foreign body ( iofb ) were admitted and were given immediate medical and surgical attention , while those with nonpenetrating injuries were treated on an outpatient basis . the immediate management of ocular burns comprised thorough eye wash with normal saline , removal of dust , soot particles and debris , and finally ruling out open - globe injury . depending on the severity of ocular injury , patients were started on copious lubricants , topical antibiotics , and steroid combination eye drops and cycloplegics . a total of 53 eyes of 45 patients were seen during the 5 consecutive days of diwali festival from 2011 to 2015 . the right eye was affected in 30 eyes , while in 23 , the left eye was affected . the characteristics of injury at presentation are summarized in table 1 . among the patients who could recall the type of the firecracker causing injury , the severity of ocular damage was more among the patients who ignited the fireworks themselves than the bystanders [ table 1 ] . five eyes ( 9.43% ) had an open - globe injury while 48 ( 90.56% ) had closed - globe injury . six patients had undergone primary treatment elsewhere and were referred to our hospital for further management . one patient with corneal epithelial defect required amniotic membrane transplant and glue with bandage contact lens . eighteen ( 33.96% ) eyes underwent surgical management at our institute , 34 ( 64.15% ) eyes were managed conservatively , and only 1 ( 1.88% ) eye was advised primary enucleation . age distribution of patients presenting with fireworks - related ocular injuries characteristics of ocular injury out of the 18 surgically managed eyes , three eyes underwent corneal tear repair , one eye underwent scleral tear repair , cataract extraction with posterior chamber intraocular lens implantation , and lensectomy in one eye each . pars plana vitrectomy for the management of vitreous hemorrhage , retinal detachment , and iofb removal was done in 5 , 2 , and 1 eyes , respectively . twenty - two eyes had bcva of 6/6 at final follow - up , 11 eyes had between 6/60 and 6/9 , < 6/60 to perception of light ( pl+ ) in 7 eyes , and no pl in 1 eye [ table 2 ] . five eyes eventually developed phthisis bulbi , of which three eyes underwent secondary enucleation with ball implant and ocular prosthesis . va could not be assessed in 12 injured eyes , of which three patients ( 6 eyes ) were of the pediatric age group . three patients ( 6 eyes ) were lost to follow - up . visual outcome following ocular firecracker injury diwali or deepawali is described as a festival to celebrate lord rama 's return from exile . this 5-day festival is also called the festival of lights , where people light their homes with this gesture brings out a feeling of joy and happiness and an expression of gratitude to the lord . the firecracker industry gives livelihood to over 0.3 million people directly and indirect employment to about 1 million people . sound and dust pollution and firework - related burn injuries are the most common problems related to the use of firecrackers . horrifying industrial accidents arising from the manufacture of fireworks have also been reported . our study was a hospital - based , single - center , retrospective case series of firecracker injuries documented over a 5-day long period during diwali over the past 5 years . our hospital is a tertiary eye care hospital located in the central district of the state of new delhi . we cater our services to a large population of northern and central india including the adjoining states of haryana , rajasthan , uttar pradesh , and punjab . in our study , the age range was 353 years with mean age of 20.55 years . among the 45 patients , the majority ( 39 ) were males , consistent with findings from previous studies , probably reflecting the males to be more adventurous and aggressive . a similar study was conducted in a multispecialty hospital located in the vellore district , tamil nadu , by john et al . , which included patients < 18 years during the 3 days of diwali over the past 5 years . eighty - four children presented with firework - related ocular injuries during the study period , of which 44% required hospitalization . the prevalence of unilateral blindness in children due to fireworks was 8% . in our study , 53 eyes of 45 patients presented with firework - related eye injuries . we included patients of all age groups and all cases who presented to our hospital during the 5 days of diwali in the past 5 years . the possible reasons for the lesser incidence of firework - related injuries in our study were as follows : ( 1 ) our hospital is a tertiary eye care center where only cases related to eye pathologies are referred . ( 2 ) our hospital is surrounded by muslim - dominated population who do not celebrate the hindu festival of diwali . ( 3 ) the urban location of our hospital may be one of the reasons for the fewer cases being reported . there are multiple tertiary referral centers in the city that are capable of managing ocular trauma - related cases . ( 4 ) more firecracker manufacturing industries are located in south india ; increased risk of fireworks - related trauma . ( 5 ) better awareness regarding safety and legislation related to fireworks among the urban population . in our study , only one eye ( 1.88% ) with ocular injuries did not have even pl while seven eyes ( 13.2% ) had vision worse than 3/60 . five eyes had phthisis bulbi three eyes following irreparable retinal detachment , one eye following severe ocular hypotony , and one eye following a failed retinal detachment surgery . , three children ( 5.1% ) with ocular injuries did not have even pl while 10 ( 11.9% ) children had vision worse than 3/60 in the injured eye . this clearly demonstrates the extent of ocular morbidity and visual loss due to firecracker injuries . in our study , serious ocular injuries occur due to the sudden appearance of unpredictable firecrackers before a person can deploy any defense mechanism . the most common types of firecracker causing injury were bombs as reported by other indian studies . high - velocity fireworks such as rockets and bombs are more frequently associated with facial lacerations , iofb , endophthalmitis , and subsequently , poorer visual outcome . a study from china documenting fireworks - related injuries during the spring festival have reported a higher preponderance of open - globe injuries . in our study , corneal abrasions , corneal foreign body , and hyphema were the most common findings involving the anterior segment , while the most common posterior segment finding was vitreous hemorrhage . unfortunately , fireworks - related ocular trauma can be vision - threatening and can cause permanent facial disfigurement . the central pollution control board estimates that 95% of firecrackers violate noise and pollution norms . in india , the manufacture , sale , and use of firecrackers generating noise levels exceeding 125 db or the supreme court has banned setting off firecrackers between 10 p.m. and 6 a.m. during festival seasons . however , in india , laws regarding displays and celebrations involving firecrackers and those regulating bursting of firecrackers by individual members of the public are virtually absent . deregulating the sale of fireworks can cause increased burden of firework related morbidity as seen in studies from northern ireland and south africa . chan et al . reported tripling of ocular injuries after the removal of a ban on fireworks in 1996 in ireland . kuhn et al . showed a reduced incidence of firework related injuries , following the strict enforcement of fireworks ban at private places . the limitation of our study was the retrospective method of capturing data and inclusion of cases that got injured only during the 5 days around diwali . this would have resulted in missing out additional cases of firework - related eye trauma and underestimating the actual magnitude of this vision - threatening problem . a significant percentage of patients end up losing their vision due to firework - related eye injuries . immediate measures such as prohibiting the manufacture and sale of dangerous fireworks such as rockets and those which produce loud noise are required in the reducing significant ocular morbidity and noise pollution . public awareness about the possible dangers , prophylactic measures , and expert primary ophthalmic care of the injured are the most realistic options available at present . firework displays at public places by professionals should be encouraged during diwali and other major celebrations . firework - related eye injuries are an important cause of preventable blindness , especially in the younger economically productive population . ocular trauma from firecrackers when serious can lead to blindness , facial burns , and permanent disfigurement . in a country like india , festivals have their own social , cultural , and economic significance . with the concept of cracker - free festival not working , a combined approach of creating public awareness and strong legislation is required to regulate the manufacture , sale , and use of consumer fireworks to reduce this preventable cause of ocular morbidity .

purpose : to evaluate the epidemiological characteristics and outcomes of ocular injuries resulting from the use of firecrackers during the diwali festival in all age groups.materials and methods : a single - center , retrospective , hospital - based case series presenting with ocular trauma consequent to fireworks usage in a tertiary eye care center in north india during the 5 days of diwali festival from 2011 to 2015 was conducted.results:a total of 53 eyes of 45 patients were included in the study , out of which the vast majority ( 39/87% ) were males . the mean age was 20.55 years . almost an equal number of bystanders ( 25/55.5% ) were affected as compared to people handling the fireworks ( 20/44.44% ) . five ( 9.43% ) eyes had open - globe injury , whereas 48 ( 90.56% ) eyes had closed - globe injury . eighteen ( 33.96% ) eyes underwent surgical intervention . thirty - three ( 62.26% ) eyes had final vision > 20/200 with eight ( 15.09% ) eyes being vision < 3/60 in the affected eye.conclusion:firework-related ocular trauma can lead to serious visual impairment . mandatory legislative laws pertaining to the manufacture , sale , and use of fireworks and creating public awareness can reduce the incidence of this preventable cause of blindness in the society . initiating new policies for retailers involved in sale of these firecrackers can also bring in decrease of such morbidities .

pubmed

subclinical hypothyroidism ( sch ) , which is defined as an isolated elevation of thyroid - stimulating hormone ( tsh ) levels with a normal free thyroxine ( ft4 ) level , is a relatively common disorder . most experts agree that sch represents early , mild thyroid failure and this issue is becoming increasingly recognized because the performance of thyroid function tests have substantially increased in primary care settings . despite the intuitive appeal of the relationship between elevated tsh levels or sch and adverse cardiovascular ( cv ) outcomes , it remains unclear whether variations in thyroid hormone levels within the normal range are also associated with mortality endpoints . the precise definition and clinical significance of sch are also confounded by controversies regarding the correct upper limit of the reference range for serum tsh . in fact , the optimal tsh cutoff values and clinical significance of these subclinical abnormalities are even more controversial . typically , the treatment of sch is considered only for patients who are pregnant , infertile , exhibit associated symptoms , or have a high risk of progression to overt hypothyroidism . although there is a high prevalence of sch , the evidence supporting screening for this disorder and the benefits and risks of its treatment remain controversial . therefore , the primary goals of the present narrative review were to assess current evidence regarding the clinical aspects of sch and cardiovascular disease ( cvd ) and to evaluate the mechanisms that are likely to underlie this relationship . to accomplish these goals , an updated search of pubmed was conducted for the most recent articles investigating the risks and treatment of sch that were published prior to 2015 . sch has various causes of which the most common ( 60% to 80% ) is chronic autoimmune thyroiditis associated with the presence of anti - thyroid peroxidase antibodies , which is a marker of chronic lymphocytic ( hashimoto 's ) thyroiditis . hashimoto 's thyroiditis is more common in girls and women , but the overall incidence increases with age in both sexes . patients who have been treated for overt thyroid failure often exhibit sch due to inadequate thyroid hormone supplementation , poor adherence to the treatment regimen , drug interactions , or the inadequate monitoring of treatment . the prevalence of sch varies according to population , age , sex , race , region , and method of tsh measurement . thus , it is necessary that the normal reference range for tsh levels be standardized and that appropriate quality control procedures be established by each laboratory investigating this phenomenon . in general , sch is defined as a tsh level greater than 4.0 to 6.0 miu / l . the prevalence of sch is relatively high and ranges from 4% to 20% in the adult population with women , elderly people , and iodine - sufficient populations being affected more often . for example , in a recent study using korean population - based cohorts , the prevalence of sch was reported to be 11.3% . the adverse clinical effects associated with overt thyroid disorders are well known and , given the multiple actions of thyroid hormones on the heart , vessels , bones , and brain , long - term adverse outcomes may manifest as subclinical levels of dysfunction . table 1 provides an overview of the findings from studies investigating the associations of sch with various clinical conditions . for example , the relationship between pathological thyroid dysfunction and body weight is well established . cross - sectional studies using population cohorts have demonstrated that variations in thyroid function within the population reference range have a substantial impact on weight and body mass index ( bmi ) . higher levels of tsh are associated with an increased bmi and rising tsh levels which , in turn , are associated with enhanced weight gain . additionally , an increasing amount of evidence indicates that higher levels of tsh are associated with worsening blood pressure ( bp ) and altered lipid levels . a cross - sectional study demonstrated that subjects with sch exhibit increased bp and a population - based study observed positive linear associations between tsh levels and systolic and diastolic bp . similarly , a recent meta - analysis found that sch is associated with higher systolic bp . thyroid hormones exert multiple effects on the regulation , absorption , and metabolism of lipid synthesis as well . previous studies have consistently demonstrated that elevated levels of serum total cholesterol ( tc ) , low density lipoprotein cholesterol ( ldl - c ) , apolipoprotein b , lipoprotein(a ) , and possibly triglycerides ( tg ) are reversed in individuals with overt hypothyroidism following levothyroxine ( t4 ) therapy . a number of studies have reported significant increases in serum tc , ldl - c , and tg in patients with sch and the findings of a meta - analysis found that the tc , ldl - c , and tg levels of sch subjects are significantly increased relative to euthyroid individuals . similarly , a korean study demonstrated that sch subjects with an intermediate - to - high risk of cvd had higher coronary calcium scores than euthyroid subjects and marfella et al . identified enhanced inflammatory activity in the asymptomatic carotid plaques of sch subjects compared to euthyroid subjects . sch can also lead to vascular problems characterized by increased vascular stiffness and endothelial dysfunction . studies of euthyroid individuals have shown that ft4 and triiodothyronine ( t3 ) levels , even within the normal range , are inversely associated with the presence and severity of coronary and carotid atherosclerosis as well as carotid artery intima media thickness . other possible explanations for the increased cv risk in persons with sch include hypercoagulability , insulin resistance , oxidative stress , and endothelial dysfunction . metabolic syndrome ( mets ) is a condition characterized by a cluster of metabolic disorders including abdominal obesity , insulin resistance / glucose intolerance , dyslipidemia , and hypertension . it has been well documented that mets is associated with increased risks of all - cause mortality and cvd . a systemic review by iwen et al . found convincing evidence supporting the major impact of sch on all components of mets and a cross - sectional analysis of cohort studies highlighted the fact that the odds of having mets are positively associated with tsh levels within the reference range . additionally , cross - sectional and longitudinal follow - up studies from japan observed strong associations between sch and mets . in korea , high normal tsh levels were significantly associated with the increased prevalence of mets in 7,270 euthyroid men and women and another study of 2,760 young korean female volunteers with normal tsh levels found that the high tsh group had a 2-fold greater risk of mets than subjects in the low tsh group . it is well known that thyroid hormones act on the heart and vasculature and , as a result , the impact of subclinical thyroid dysfunction on the cv system has recently become an important topic of research . sch can lead to impaired systolic and diastolic cardiac function and symptomatic sch patients often complain of reduced exercise tolerance during effort . this same study found that a slowed rate of left ventricular relaxation might critically impair ventricular filling during exercise and result in left ventricular systolic dysfunction . older people with sch and a high cv risk appear to be at an increased risk of incident heart failure and sch was found to affect left ventricular dysfunction , although this abnormality may be reversed by t4 substitutive therapy . likewise , a recent cardiac magnetic resonance spectroscopic study demonstrated that early cardiac bio - energetic impairments in sch patients are reversible with t4 therapy . a pooled analysis from six prospective cohorts also found that the risk of heart failure events increases in conjunction with higher tsh levels . studies of euthyroid individuals observed a positive association between tsh levels and mortality due to coronary heart disease ( chd ) . tsh levels within the normal range are inversely associated with all - cause mortality and adult taiwanese subjects with sch were shown to have an increased risk for all - cause mortality and cvd death over a 10-year period . however , other studies did not find any associations with all - cause or cv mortality rates . these discrepant findings may be due to differences in sample size and power , the inconsistent age and sex ratios of the study populations , or the varied iodine intakes of different regions . two meta - analyses investigating this matter demonstrated that sch may be associated with a modest increase in the risks of chd and mortality , particularly in subjects with higher tsh levels . a more recent review of the clinical consequences of variations in thyroid function within the normal reference range found that even modest elevations of tsh may have substantial health outcomes , including cv mortality . in a korean cohort that was followed for 10 years , elevated serum tsh levels significantly increased the risk of cvd by approximately 20% per one standard deviation in males . more recently , ft4 and ft3 levels within the normal range were inversely associated with the risks of all - cause and cancer mortality in a korean cohort of euthyroid men and women ( n=212,456 ) . a number of placebo - controlled studies have assessed the effects of t4 replacement therapy on the symptoms of patients with sch . however , the reported benefits of various sch treatments are limited and evidence regarding the association of sch with cvd remains controversial . an intervention study of 56 women with sch identified elevated systolic and diastolic bp and increased serum cholesterol and homocysteine levels compared to healthy controls ; these factors were normalized after 18 months of t4 therapy . a systematic review of 13 heterogeneous studies concluded that t4 treatment leads to a reduction in serum tc and ldl - c levels in patients with sch ; this finding has been confirmed in subsequent randomized controlled trials ( rcts ) . however , to date , clinical trials have not consistently shown the beneficial effects of t4 treatment on serum lipid levels in sch patients . nonetheless , reductions in carotid intima - media thickness and improvements in brachial artery endothelial function have been observed in individuals with sch following t4 replacement . thus , the negative effects of sch on cv function might be improved or reversed by replacement doses of t4 . although only a few studies investigating the cv effects of t4 have been double - blinded and placebo - controlled , these trials concur that t4 replacement therapy improves systolic and diastolic heart function , endothelial function , and carotid intima - media thickness . similarly , in a small rct , the normalization of tsh levels following t4 replacement therapy led to an improvement in cardiac function . moreover , there are new data supporting improved outcomes in younger ( < 70 years ) chd patients treated with t4 . on the other hand , a cochrane review from 2007 assessed 12 rcts of t4 replacement in patients with sch and concluded that replacement therapy for sch did not result in improved survival or decreased cv morbidity . the indications for the screening of sch and the determination of tsh threshold levels for the treatment of sch have remained a clinical controversy for over many years . additional observational data are unlikely to adequately address the uncertainties associated with the risks of sch or the potential benefits and risks of replacement therapy . these concerns will be best addressed in an appropriately powered rct that assesses clinical outcomes . the european commission has now funded such a trial ; the thyroid hormone replacement for untreated older adults with subclinical hypothyroidism ( trust ) study . the trust study is a multicenter , double - blinded , placebo - controlled randomized trial designed to assess the multimodal effects of thyroid hormone replacement in 3,000 adults 65 years of age and older with persistent sch ( clinicaltrials.gov : nct01660126 ) . in addition to cv events such as chd , stroke , and heart failure , this trial will evaluate disease - specific and health - related quality of life , muscle function , and cognitive function . this study should definitively clarify the populations that may benefit from t4 replacement by assessing multiple clinical outcomes as well as whether these benefits may be offset by the potential adverse effects of replacement therapy , such as atrial fibrillation and osteoporosis . the significance of sch is now widely studied and the data continue to accumulate . in summary , the present review has highlighted the fact that sch is associated with an increased risk of developing a wide range of adverse health outcomes and that sch might represent a potentially modifiable risk factor of cvd and mortality . therefore , understanding the prevalence and risk factors of sch could aid in the prevention of cvd in this population . however , until the results of the trust study are published and current clinical recommendations are updated , the decision to treat or not treat an sch patient will be based on clinical judgment , clinical practice guidelines , and expert opinion .

subclinical hypothyroidism ( sch ) is a common disorder that is characterized by elevated thyroid - stimulating hormone levels in conjunction with free thyroxine concentrations within the normal reference range . thyroid hormones are known to affect the heart and vasculature and , as a result , the impact of sch on the cardiovascular ( cv ) system has recently become an important topic of research . strong evidence points to a link between sch and cv risk factors such as alterations in blood pressure , lipid levels , and atherosclerosis . additionally , accumulating evidence indicates that sch is associated with metabolic syndrome and heart failure . the present review proposes that sch may be a potentially modifiable risk factor of cv disease and mortality . however , large - scale clinical trials with appropriate power investigating the risks and benefits of sch treatment are required to determine whether these benefits can be achieved with levothyroxine therapy .

pubmed

the anaerobic gram - positive bacillus clostridium difficile is a leading cause of antibiotic - associated diarrhea . antimicrobial drugs disrupt the normal intestinal flora , allowing c. difficile to flourish and release c. difficile toxin a and toxin b that induce c. difficile - associated diarrhea ( cdad).1,2 although cdad is the common health - care - associated infectious disease , treatment or infection countermeasures are not applied to patients who do not present with diarrhea , including the tens of percents of asymptomatic adults who carry c. difficile in their intestines.35 cdad diagnosis widely uses an enzyme immunoassay ( eia ) kit to detect glutamate dehydrogenase ( gdh ) and toxins a and b ( cd toxins ) released by the c. difficile cells.6 however , the low detection sensitivity of eia for the cd toxins has been reported.7,8 compared to the 90% detection sensitivity of polymerase chain reaction ( pcr ) and cytotoxigenic culture , eia detection sensitivity is usually thought to be ~70% and has been reported to be as low as 50% or below.7,8 cdad diagnosis is based on eia results in japan , because pcr has not been clinically available . if eia testing is performed on c. difficile isolates cultured from the same specimen , an eia gdh - positive and toxin - negative stool specimen may give a toxin - positive result . bacterial content is higher in specimens containing colonies in solution compared to stool ; therefore , toxin concentration may also be markedly higher . if a specimen is gdh positive , follow - up testing with a nucleic acid amplification test , such as pcr , is recommended to compensate for the low sensitivity of eia.911 pcr detection of tcda or tcdb genes is the evidence for the presence of toxigenic c. difficile . while pcr findings have been reported to correlate well with clinical symptoms,12 bacterial toxin proteins are not detected ; therefore , positive results may also include patients carrying c. difficile without cdad.13 there is currently no definite method of determining whether diarrhea is caused by cd toxins present at concentrations below the detection sensitivity of eia in patients with eia toxin - negative stool or whether diarrhea is due to a cause other than cd toxins , despite the presence of c. difficile in the stool . patients with toxin - negative stool may be dismissed as non - cdad and thus overlooked in terms of treatment and infection countermeasures . the present study investigated patients who had submitted stool specimens for cdad diagnosis using eia . differences in patient characteristics , diarrhea duration , antibiotics usage , and severity were compared among patients with toxin - positive stool , toxin - negative stool but toxin - positive isolates , and toxin - negative stool and isolates . the aim of the study is to investigate clinical history and need for the treatment of patients with gdh - positive and toxin - negative stool specimens . the present study retrospectively investigated patients ( age 2 years ) whose stool specimens were submitted to the microbiology laboratory at our institution for cd toxin testing between march 2013 and june 2014 . patients with c. difficile growth on stool culture and stool specimens confirmed to be gdh positive on eia were included . patients were divided into three groups based on the eia cd toxin results in stool specimens and isolates obtained from bacterial culture : a toxin - positive stool group ( positive stool group ) , a toxin - negative stool / toxin - positive isolate group ( discrepant negative / positive group ) , and a dual toxin - negative stool and isolate group ( dual negative group ) . c. diff , blacksburg , va , usa ) eia was performed according to the manufacturer s instructions to determine stool specimen positivity or negativity for gdh and cd toxins . samples of toxin - negative stool specimens were inoculated onto cycloserine - cefoxitin mannitol agar ( ccma ; nissui pharmaceutical co. , ltd . , eia was performed on isolates from colonies using the same method as for the stool specimens to confirm the presence or absence of gdh and cd toxins . bowel movement status was investigated using the electronic medical records ( emrs ) of bowel movement frequency and characteristics . patients with diarrhea or muddy stool passed once per day or loose stool passed four times per day were identified as symptomatic . clinical data relating to white blood cell ( wbc ) count , c - reactive protein levels , serum albumin ( alb ) levels , and body temperature on the day the specimens were submitted to the microbiology laboratory or , if measurements were unavailable for that day , on the day before or after , were also obtained from the emr . one - way analysis of variance was performed to compare the continuous variables among the three groups , and the tukey the ethics committee of the university of tokyo graduate school of medicine approved this study and waived the need for obtaining written informed consent from each patient . the present study retrospectively investigated patients ( age 2 years ) whose stool specimens were submitted to the microbiology laboratory at our institution for cd toxin testing between march 2013 and june 2014 . patients with c. difficile growth on stool culture and stool specimens confirmed to be gdh positive on eia were included . patients were divided into three groups based on the eia cd toxin results in stool specimens and isolates obtained from bacterial culture : a toxin - positive stool group ( positive stool group ) , a toxin - negative stool / toxin - positive isolate group ( discrepant negative / positive group ) , and a dual toxin - negative stool and isolate group ( dual negative group ) . c. diff quik chek complete ( techlab , inc . , blacksburg , va , usa ) eia was performed according to the manufacturer s instructions to determine stool specimen positivity or negativity for gdh and cd toxins . samples of toxin - negative stool specimens were inoculated onto cycloserine - cefoxitin mannitol agar ( ccma ; nissui pharmaceutical co. , ltd . , eia was performed on isolates from colonies using the same method as for the stool specimens to confirm the presence or absence of gdh and cd toxins . bowel movement status was investigated using the electronic medical records ( emrs ) of bowel movement frequency and characteristics . patients with diarrhea or muddy stool passed once per day or loose stool passed four times per day were identified as symptomatic . clinical data relating to white blood cell ( wbc ) count , c - reactive protein levels , serum albumin ( alb ) levels , and body temperature on the day the specimens were submitted to the microbiology laboratory or , if measurements were unavailable for that day , on the day before or after , were also obtained from the emr . cary , nc , usa ) . one - way analysis of variance was performed to compare the continuous variables among the three groups , and the tukey the ethics committee of the university of tokyo graduate school of medicine approved this study and waived the need for obtaining written informed consent from each patient . a flow chart of the study design and patient cohort is shown in figure 1 . during the study period , 127 specimens were excluded for the following reasons : second of multiple specimens submitted during the same diarrhea episode ( n=54 ) , no record of eia results for isolates obtained from stool culture ( n=47 ) , patient age < 2 years ( n=4 ) , and bowel movement status unclear from emr or fever chart ( n=22 ) . all specimens underwent eia , identifying 39 toxin - positive and 29 toxin - negative stool specimens . eia performed on agar - cultured isolates from toxin - negative stool specimens identified 14 toxin - positive and 15 toxin - negative isolates . no significant differences were observed in clinical characteristics such as the number of comorbidities , presence of malignant tumors , recent history of transplant , and use of tube feeding . no significant differences were observed among the three groups with regard to number and respective administration frequencies of antimicrobial drugs received by patients within the 30 days prior to diarrhea onset ( table 2 ) . a significant difference was observed in mean diarrhea duration between the 39 patients in the positive stool group and the 29 patients with toxin - negative stool specimens ( 10.6 days vs 5.6 days ; p=0.0179 ) . among the 29 patients with toxin - negative stool specimens , mean diarrhea durations were 7.9 days and 3.4 days for the discrepant negative / positive group and the dual - negative group , respectively . a significant difference was observed in diarrhea duration among the three groups ( p=0.0395 ; figure 2 ) . comparison between each group found a significant difference in diarrhea duration between the positive stool group and the dual - negative group ( p=0.0312 ) . no significant difference was observed between the positive stool group and the discrepant negative / positive group ( p=0.6006 ) ; however , diarrhea duration tended to be longer in the positive stool group . no difference was observed in diarrhea duration between either of the groups with toxin - negative stool specimens ( p=0.3952 ) . a significant difference was observed among the groups in the proportion of patients who received vancomycin or metronidazole treatment for cdad : positive stool group , 82.1% ( n=32 ) ; discrepant negative / positive group , 7.1% ( n=1 ; vancomycin 500 mg three times daily for 13 days ) ; and dual - negative group , 0% ( n=0 ) ; p<0.001 ( figure 3 ) . in positive stool group , 27 patients received oral vancomycin , four received oral metronidazole , and one received oral metronidazole ( 250 mg four times daily for 11 days ) followed by oral vancomycin ( 125 mg four times daily for 7 days ) . of the patients receiving oral vancomycin alone in positive stool group , 70.4% received 125 mg four times daily , 18.5% received 500 mg four times daily , 7.4% received 500 mg three times daily , or 3.7% received 250 mg four times daily . of the patients receiving oral metronidazole alone in positive stool group , 25% received 250 mg four times daily and 75% received 500 mg three times daily . the present findings demonstrated that , although significantly fewer patients in the discrepant negative / positive group received treatment compared to the positive stool group , diarrhea duration tended to be shorter . no significant differences were observed among the groups with regard to inflammation findings , specifically wbc count , c - reactive protein levels , alb levels , and body temperature , on the day stool specimens were submitted ( table 3 ) . of the 68 patients investigated in the present study , 39 ( 57.4% ) had cd toxin - positive stool specimens , 14 ( 20.6% ) had toxin - negative stool specimens but toxin - positive isolates , and 15 ( 22.1% ) had both toxin - negative stool specimens and isolates . quinn et al14 investigated 174 stool specimens and found that 133 ( 76.4% ) were negative for both gdh and cd toxins , 18 ( 10.3% ) were positive for both gdh and toxins , and 23 ( 13.2% ) were positive for gdh but negative for toxins . in the present study , similar rates of gdh positivity were found in both toxin - positive and toxin - negative patients with regard to stool specimens . these gdh - positive but toxin - negative patients represent the proportion of cases in which diagnosis and treatment requirements are unclear . symptom duration was significantly shorter in patients with toxin - negative rather than toxin - positive stool specimens , despite the lack of cdad drug treatment in all but one of the 29 toxin - negative stool specimen patients . furthermore , diarrhea duration also tended to be shorter in patients with toxin - negative stool specimens but toxin - positive isolates compared to those with toxin - positive stool specimens , despite significantly fewer discrepant negative / positive group patients receiving treatment . this suggests that patients who tested positive for c. difficile on culture but negative for toxins in their stool had milder symptoms , despite having toxin - positive isolates . polage et al15 found that eia toxin - negative patients had significantly milder diarrhea , shorter diarrhea duration , and a lower mortality rate than toxin - positive patients . patel et al16 also reported that an infection with an eia toxin - negative strain of c. difficile presents less severe clinical course , such as lower white blood cell counts and shorter length of hospital stay , than infection with eia - positive c. difficile . meanwhile , baker et al17 demonstrated that eia toxin - negative patients present with milder clinical symptoms than toxin - positive patients . conversely , other studies have reported no difference in clinical symptoms between toxin - negative and toxin - positive patients,18 and a consensus has yet to be reached . the present analysis supported the reports of polage et al,15 patel et al,16 and baker et al,17 finding that mean diarrhea duration was significantly shorter in patients with toxin - negative ( 5.6 days ) than toxin - positive ( 10.6 days ) stool specimens , even with similar gdh positivity . furthermore , 13 of 14 patients with toxin - negative stool specimens but toxin - positive isolates did not receive cdad treatment , and diarrhea duration in this group did not differ significantly from that in the dual - negative group . the group with toxin - negative isolates may have been infected with nontoxigenic c. difficile , while the toxin concentration in the stool and intestines of the group with toxin - positive isolates but toxin - negative stool specimens was likely too low to be detected by eia , despite c. difficile toxigenicity . akerlund et al19 found that toxin concentrations in vitro from cultured c. difficile did not correlate with the toxin concentration in stool , which correlated with diarrhea frequency and abdominal pain . in addition to low toxin concentration , various intestinal factors , such as primary bile salt and human immunity , may contribute to lack of toxin detection in stool specimens in patients with discrepant negative / positive findings,2026 because primary bile salts inhibit the growth of vegetative cells of c. difficile and human alpha - defensins cause loss of cytotoxicity of c. difficile toxin b. however , whatever the cause , diarrhea is comparatively mild in patients with toxin - negative stool , suggesting that severe symptoms are unlikely to develop at toxin concentrations below detection sensitivity . in this study toxin - negative patients with gdh positivity may be simply colonized with another cause of diarrhea , even if toxigenic c. difficile is isolated from stool . some classes of antibiotics , such as clindamycin , broad - spectrum cephalosporins , vancomycin , carbapenems , -lactamase inhibitors combination antibiotics , and fluoroquinolones , are widely known risk factors for cdad.5,2729 on the other hand , linezolid has been reported to have a potential protective role against the development of c. difficile infection30 and a good in vitro activity against c. difficile isolates in a human gut model of c. difficile infection.31 in this study , 48% patients received -lactamase inhibitors combination antibiotics and 25% patients received carbapenems . although cephamycin and fourth - generation cephalosporin tend to be more frequently used in toxin ( + ) stool group than other groups , there were no significant differences among the groups in antimicrobial drug use within the 30 days prior to toxin testing . furthermore , no clear correlation was observed between type of antimicrobial drug administered prior to cdad onset and the presence of toxins . in the majority of patients , stool testing was conducted within several days of diarrhea onset , but no significant differences were observed among the groups with regard to clinical inflammation during the same period , suggesting no strong correlation between toxin detection and inflammation at the time of symptom onset . these findings indicate that it is difficult to predict toxin negativity or positivity prior to toxin testing based on drug use status or other test results . the sensitivity of eia for toxin detection is far inferior to that of pcr , which amplifies the toxin coding regions.14,32 however , pcr detects the presence of bacteria with toxin genes rather than toxin protein production ; therefore , patients who are identified as toxin - positive on pcr may include carriers with diarrhea due to a cause other than toxin.13 eia directly measures the toxin proteins in stool and is thus more likely to reflect the patient s actual disease status . while eia is often associated with low sensitivity , it has a high positive predictive value,14 making it an important test in determining treatment approach . limitations of the present study include the retrospective nature of the study , the small sample size , and the absence of molecular biological testing . potential differences in the gene sequences related to toxin production between patients with toxin - positive stool and those with toxin - positive isolates were also not investigated . hyper - virulent c. difficile strain , such as bi / nap-1/027 strain , has emerged and causes severe and recurrent diseases . in japan , the nap-1 strain is low prevalence.33 recently , polage et al34 reported that patients with a positive molecular test result and a negative toxin immunoassay test result had outcomes that were comparable to patients without c. difficile by either method and that the prevalence of hypervirulent strain was 107 of 293 isolates . however , further large number analysis is required to determine whether the patient groupings in the present study reflect bacterial genetic characteristics . in this study , around half with toxin - negative stool specimens among the patients with eia gdh - positive stool specimens had toxin - positive isolates . since toxigenic c. difficile was thus present in their stool , these patients are at risk of transferring the bacteria to other patients and require care from the perspective of preventing hospital infection . however , from the perspective of treatment for individual patients , diarrhea duration is shorter in patients with toxin - negative stool specimens compared to those with toxin - positive stool specimens even without treatment . nucleic acid amplification tests are recommended to detect toxinogenic c. difficile in stool for a diagnosis of cdad ; however , toxin production is not evaluated . these results may suggest a limited need for cdad treatment for patients with gdh - positive and toxin - negative stool specimens .

purposethe enzyme immunoassay ( eia ) has lower sensitivity for clostridium difficile toxins a and b than the polymerase chain reaction in the diagnosis of c. difficile - associated diarrhea ( cdad ) . furthermore , toxin positivity with eia performed on c. difficile isolates from stool cultures may be observed even in patients with eia glutamate dehydrogenase ( gdh)-positive and toxin - negative stool specimens . it is unclear whether such patients should be treated as having cdad.methodsthe present study retrospectively compared patient characteristics , treatment , and diarrhea duration among three groups of patients who underwent stool eia testing for cdad diagnosis : a toxin - positive stool group ( positive stool group ; n=39 ) ; a toxin - negative stool / toxin - positive isolate group ( discrepant negative / positive group , n=14 ) ; and a dual toxin - negative stool and isolate group ( dual negative group , n=15 ) . all cases included were confirmed to be gdh positive on eia test.resultspatients backgrounds and comorbidities were not significantly different among three groups . no difference was observed among the three groups with regard to antimicrobial drug use before diarrhea onset . treatment was received by 82.1% of the positive stool group compared to 7.1% of the discrepant positive / negative group and 0% of the dual negative group , while mean diarrhea duration was 10.6 days compared to 7.9 days ( p=0.6006 ) and 3.4 days ( p=0.0312 ) , respectively.conclusioneven without treatment , patients with toxin - negative stool specimens had shorter diarrhea duration than those with toxin - positive stool specimens even with toxin - positive isolates . these findings may suggest a limited need for cdad treatment for gdh - positive patients and toxin - negative stool specimens .

pubmed

the design and principal findings of the dream trial have been presented in previous publications ( 17 ) . briefly , the dream trial was a large , international , multicenter , double - blind , randomized controlled trial designed to determine whether ramipril and/or rosiglitazone could prevent or delay the development of type 2 diabetes in people with ifg or igt , metabolic states that indicate very high risk for eventual progression to diabetes ( 17 ) . eligibility for the dream trial included a diagnosis of ifg , igt , or both ifg and igt based on a screening 75-g oral glucose tolerance test ( ogtt ) ( 17 ) . a total of 5,269 participants with these disorders were recruited and randomized to either ramipril and/or rosiglitazone using a two - by - two factorial design and followed for a median of 3 years after randomization . participants were assessed at regular intervals to ascertain the occurrence of the primary outcome , which included new - onset diabetes or all - cause mortality . as part of a substudy , 982 dream trial participants attending canadian study centers had ogtts at baseline , after 2 years , and at the end of the study , with blood samples drawn fasting as well as 30 and 120 min after the glucose challenge . the primary outcome variable in the present study was change in -cell function over the course of follow - up . -cell function was assessed using two measures : the insulinogenic index ( igi ) and proinsulin ( pi ) concentration , with igi defined as ( 30-min insulin fasting insulin)/(30-min glucose fasting glucose ) . both indexes have previously been validated against gold - standard measures of insulin secretion ( 18,19 ) and have been shown to be significant predictors of incident diabetes in large epidemiological studies . to account for the compensatory response of insulin secretion in relation to background insulin resistance , igi was divided by the homeostasis model assessment of insulin resistance index ( homa - ir ) ( defined as fasting glucose fasting insulin/22.5 ) ( igi / ir ) for univariate analysis or adjusted for homa - ir in multivariate analysis . similarly , pi concentration was divided by c - peptide concentration ( i.e. , the pi / c - peptide ratio [ pi / c ] ) for univariate analysis or adjusted for insulin secretion using c - peptide as a covariate in multivariate analysis . although the pi - to - insulin ratio is often used to identify disproportionate elevations in pi , c - peptide offers advantages over insulin as a denominator for pi because it is cosecreted with insulin in an equimolar ratio , but unlike insulin it is not extracted by the liver and thus it has a constant peripheral clearance . glucose concentration was determined using an enzymatic reference method on a roche hitachi 917 instrument and a roche reagent kit ( roche diagnostics , indianapolis , in ) . serum insulin and c - peptide were measured on the roche elecsys 2010 immunoassay analyzer using an electrochemiluminescence immunoassay . the insulin assay had a sensitivity of 1.39 pmol / l , an interassay coefficient of variation ( cv ) of < 4.6% at all levels , and < 0.05% cross - reactivity with human c - peptide and pi . the c - peptide assay had a sensitivity of 3.0 pmol / l , an interassay cv of < 3% at all levels , and < 0.005% cross - reactivity with human insulin . pi concentration was measured using a sandwich enzyme - linked immunosorbent assay manufactured by linco research ( linco research , st . this assay had a sensitivity of 2.0 pmol / l , an interassay cv of < 9% at all levels , and no cross - reactivity with human insulin or des ( 31,32 ) split pi , although this assay does cross - react with human des ( 64,65 ) split pi . statistical analyses were conducted using sas version 9.1 ( sas institute , cary , nc ) , and p values < 0.05 were considered statistically significant . the distributions of continuous variables were assessed for normality , and transformations of skewed variables were used in the analysis as appropriate . means and sds for primary -cell function measures ( igi / ir and pi / c ) were calculated for each time point ( baseline , at 2 years , and final visit ) , according to the marginal treatment group . p values for change were based on a t test of the average change being different from zero , while p values for treatment difference were calculated using the wilcoxon 's rank - sum test . similar analyses were conducted within subgroups of isolated ifg ( iifg ) , isolated igt ( iigt ) , and combined ifg and igt ( ifg+igt ) . as there was no significant interaction between ramipril and rosiglitazone 's effect on -cell function , main - effects analyses were conducted according to marginal randomization groups ( i.e. , rosiglitazone versus placebo , ramipril versus placebo ) . longitudinal analyses of the associations between treatments and changes over time in -cell function measures were examined using random - effects models in proc mixed , which provides appropriate options for handling the covariance structure of the repeated - measures ( longitudinal ) data . specifically , we ran four models , which assessed the impact of treatment group on 1 ) igi , with age and homa - ir as covariates ; 2 ) igi / homa - ir , with age as a covariate ; 3 ) pi , with age and c - peptide as covariates ; and 4 ) pi / c , with age as a covariate . finally , using cox proportional hazards regression , we assessed whether the impact of ramipril and/or rosiglitazone treatment on diabetes incidence was independent of baseline levels and changes over the course of the trial in -cell function . the outcome variable in these analyses was diabetes status at the final visit , and the primary exposures were the marginal treatment groups ( rosiglitazone , ramipril ) . in separate models , we assessed the impact of treatment on diabetes incidence , adjusting for either baseline -cell function ( including baseline igi and homa - ir or baseline pi and c - peptide as covariates ) or change over the course of the trial in -cell function ( including changes in the above - mentioned covariates ) . baseline models were also adjusted for age and baseline waist circumference , fasting glucose , triglycerides , and hdl , while change models were also adjusted for age and changes in these covariates . statistical analyses were conducted using sas version 9.1 ( sas institute , cary , nc ) , and p values < 0.05 were considered statistically significant . the distributions of continuous variables were assessed for normality , and transformations of skewed variables were used in the analysis as appropriate . means and sds for primary -cell function measures ( igi / ir and pi / c ) were calculated for each time point ( baseline , at 2 years , and final visit ) , according to the marginal treatment group . p values for change were based on a t test of the average change being different from zero , while p values for treatment difference were calculated using the wilcoxon 's rank - sum test . similar analyses were conducted within subgroups of isolated ifg ( iifg ) , isolated igt ( iigt ) , and combined ifg and igt ( ifg+igt ) . as there was no significant interaction between ramipril and rosiglitazone 's effect on -cell function , main - effects analyses were conducted according to marginal randomization groups ( i.e. , rosiglitazone versus placebo , ramipril versus placebo ) . longitudinal analyses of the associations between treatments and changes over time in -cell function measures were examined using random - effects models in proc mixed , which provides appropriate options for handling the covariance structure of the repeated - measures ( longitudinal ) data . specifically , we ran four models , which assessed the impact of treatment group on 1 ) igi , with age and homa - ir as covariates ; 2 ) igi / homa - ir , with age as a covariate ; 3 ) pi , with age and c - peptide as covariates ; and 4 ) pi / c , with age as a covariate . finally , using cox proportional hazards regression , we assessed whether the impact of ramipril and/or rosiglitazone treatment on diabetes incidence was independent of baseline levels and changes over the course of the trial in -cell function . the outcome variable in these analyses was diabetes status at the final visit , and the primary exposures were the marginal treatment groups ( rosiglitazone , ramipril ) . in separate models , we assessed the impact of treatment on diabetes incidence , adjusting for either baseline -cell function ( including baseline igi and homa - ir or baseline pi and c - peptide as covariates ) or change over the course of the trial in -cell function ( including changes in the above - mentioned covariates ) . baseline models were also adjusted for age and baseline waist circumference , fasting glucose , triglycerides , and hdl , while change models were also adjusted for age and changes in these covariates . the average age and bmi were 54 years and 31.5 kg / m , respectively , and 60% of participants were female . the majority ( 81% ) were of european origin , and substantial proportions had a family history of diabetes or a history of gestational diabetes ( 61 and 16% , respectively ) , characteristics that were consistent with the recruiting strategy for the dream trial ( 17 ) . there were no significant differences between the marginal randomization groups for any of the baseline characteristics other than family history of diabetes in the ramipril versus placebo marginal group ( p = 0.02 ) ( table 1 ) . baseline characteristics of participants with measures of -cell function overall and by allocation data are means sd or n ( % ) . no significant differences between rosiglitazone versus placebo or ramipril versus placebo other than family history of diabetes in the ramipril versus placebo group ( p = 0.02 ) . changes in markers of -cell function in marginal randomization groups are presented in table 2 . participants receiving rosiglitazone versus placebo had a significant increase in igi / ir during the study ( 25.59 vs. 1.94 , p < 0.0001 ) and a significant decrease in pi / c ( 0.010 vs. 0.006 , p < 0.0001 ) . in contrast , there were no significant changes in igi / ir or pi / c in participants receiving ramipril versus placebo ( 11.71 vs. 18.15 , p > 0.05 , and 0.007 vs. 0.008 , p > 0.05 , respectively ) ( table 2 ) . in the rosiglitazone group , changes in the -cell function measures were more substantial between the baseline and 2-year visits compared with changes that occurred between the 2-year and final visits ( table 2 ) . the impact of rosiglitazone on igi / ir and pi / c was similar within subgroups of iigt and ifg + igt ( iigt , igi / ir : 27.74 vs. 2.76 , p < 0.0001 ; pi / c : 0.009 vs. 0.008 , p < 0.001 ; ifg + igt , igi / ir : 27.39 vs. 0.70 , p < 0.0001 ; pi / c : 0.014 vs. 0.002 , p < 0.0001 ) , although effects were more modest in those with iifg ( igi / ir : 8.95 vs. 2.13 , p = 0.03 ; pi / c : 0.003 vs. 0.001 , p > 0.05 ) ( supplemental table , available at http://care.diabetesjournals.org/cgi/content/full/dc09-1579/dc1 ) . changes in markers of -cell function data are means sd and are reported for marginal treatment groups . p value for change were based on a t test of the average change being different from zero . we further assessed the impact of treatment on markers of -cell function by utilizing longitudinal data from multiple study time points in mixed - model analyses . compared with placebo , rosiglitazone significantly increased igi after adjustment for age and homa - ir ( p = 0.015 ) ( table 3 ) . in contrast , ramipril did not significantly affect adjusted igi ( p > 0.05 ) . similar findings were seen using pi concentration as a measure of -cell function . specifically , rosiglitazone significantly reduced pi concentrations over time after adjustment for age and c - peptide concentration ( p = 0.0064 ) ( table 3 ) . in contrast , ramipril did not significantly change adjusted pi concentrations ( p > 0.05 ) . longitudinal changes in markers of -cell function in dream trial : analysis of slopes using mixed - model analysis analysis in table are based on full data ; results essentially unchanged when analysis repeated on subjects with information from all visits . we assessed whether the impact of ramipril and/or rosiglitazone on diabetes incidence was independent of baseline levels or changes in indexes of -cell function with time ( fig . 1 ) . after accounting for baseline -cell function as measured by either igi or pi in models that adjusted for insulin resistance and other covariates , rosiglitazone significantly reduced the risk of developing diabetes ( hazard ratio 0.32 [ 95% ci 0.220.45 ] , p < 0.001 , and 0.33 [ 0.230.47 ] , p < 0.001 , in igi and pi models , respectively ) . adjusting for the change in -cell function as measured by igi attenuated the preventive effect of rosiglitazone on incident diabetes ( 0.53 [ 0.280.99 ] , p = 0.046 ) . such attenuation was not noted when the change in -cell was measured using pi ( 0.28 [ 0.180.42 ] , p < 0.0001 ) ( fig . association of treatment allocation with risk of diabetes : impact of baseline levels and changes in -cell function . for igi / ir models : model 1 adjusted for age and baseline igi , homa - ir , fasting glucose , waist circumference , triglyceride , and hdl ; model 2 adjusted for age and changes in igi , homa - ir , fasting glucose , waist circumference , triglyceride , and hdl . for pi / c models : model 1 adjusted for age and baseline pi , c - peptide , fasting glucose , waist circumference , triglyceride , and hdl ; model 2 adjusted for age and changes in pi , c - peptide , fasting glucose , waist circumference , triglyceride , and hdl . rosiglitazone , but not ramipril , improved measures of -cell function over time in people with ifg and/or igt . specifically , rosiglitazone increased igi / ir and reduced pi / c by 86 and 42% , respectively . these findings were consistent across glucose tolerance subgroups ( iifg , iigt , and ifg+igt ) , although there was the suggestion of a more modest effect in the subgroup with iifg . finally , rosiglitazone 's effect on diabetes prevention persisted after adjustment for baseline -cell function . the demonstration that an agent that reduces diabetes incidence also improves -cell function invites the hypothesis that measures of change in -cell function in response to therapy are indexes of that therapy 's ability to reduce the incidence of diabetes . the modest attenuation of rosiglitazone 's effect after accounting for the change in -cell function suggests that some but not all of the effect of rosiglitazone on diabetes prevention / delay is mediated through its effects on -cell function . whereas a number of previous studies ( 911 ) have documented improvements in -cell function with tzd treatment in people with diabetes , only four studies ( 1215 ) have investigated this question in people with ifg and/or igt . the results of these studies have been inconsistent , with two studies ( 12,15 ) indicating a significant improvement in measures of -cell function with tzd treatment and two studies ( 13,14 ) reporting no change . these studies all had small sample sizes ( n 30 ) , and the methods used to assess -cell function varied widely from intensive approaches ( insulin responses to glucose infusion ) to simple fasting - based indexes ( homa - b ) . interestingly , the complexity of the method used to assess -cell function did not appear to explain the differences in findings of these previous studies ( 12,14 ) . finally , in a diabetes prevention trial among hispanic women with previous gestational diabetes , 70% of whom had igt at enrollment , treatment with troglitazone resulted in significant improvements in the frequently sampled intravenous glucose tolerance our results suggest a significant improvement in -cell function with tzd treatment in pre - diabetic subjects . first , the sample size of our study ( n = 982 ) was much larger that previous investigations . second , we demonstrated improvements in -cell function under tzd treatment using two validated , widely used proxy measures of -cell function , namely igi and pi . third , in our analysis we accounted for the compensatory impact on -cell function of background insulin resistance . specifically , in analyses of igi we used homa - ir either as a covariate in multivariate analysis or as the denominator in the igi to homa - ir ratio . accounting for background insulin resistance is crucial to the interpretation of -cell function measures in this study because tzds improved insulin sensitivity , thus reducing pancreatic -cell demand . the exact mechanisms responsible for the increases in igi / ir and the reductions in pi / c documented in the present study are not known , although a number of possibilities exist . the reduction in insulin resistance with tzds would be expected to reduce insulin secretory demand on the -cells and thus reduce -cell stress . in addition , tzds such as rosiglitazone may improve -cell function indirectly by ameliorating a number of pathogenic processes that have been shown to be detrimental to the -cells , including lipotoxicity , glucotoxicity , and inflammation ( 7,8 ) . tzds lower free fatty acids ( 22 ) , elevated levels of which result in excess deposition of lipid within -cells , which in turn leads to increases in ceremide and the stimulation of nitric oxide mediated -cell apoptosis . as well , the glucose - lowering effect of tzds may reduce the impact of reactive oxygen species on the -cells , which are known to be especially susceptible to oxidative damage ( 23 ) . finally , tzds have been demonstrated to reduce levels of inflammatory cytokines ( 24 ) , which may induce -cell apoptosis when chronically elevated . tzds may also impact -cell function directly by maintaining -cell proliferation and/or reducing -cell apoptosis ( 25 ) . in the hope study , treatment with ramipril was shown to reduce the incidence of diabetes in middle - aged individuals with vascular disease . in the dream trial , although ramipril did not significantly reduce the incidence of diabetes in people with ifg and/or igt , it did significantly increase regression to normoglycemia . the mechanism by which ramipril might reduce glucose levels and/or prevent / delay diabetes is unknown , although vascular and metabolic effects on the muscle and pancreatic -cell have been proposed ( 16 ) . the results of the present study suggest that ramipril does not have significant effects on -cell function compared with placebo in people at high risk for diabetes , and thus its glucose - lowering effects may operate through other metabolic mechanisms . the improvement in -cell function in the placebo arm of the ramipril marginal group analysis may be explained by the fact that under the factorial design of the dream trial , half of the participants on ramipril placebo were receiving active rosiglitazone . the major strengths of the present study include the large sample size , the randomized , double - blind design , and the completeness of follow - up ( 92.6% ) . further , participants were thoroughly characterized in terms of glucose tolerance status and were all in the pre - diabetic range ( either iifg , iigt , or ifg+igt ) . the major limitation of the present study is the lack of detailed measures of -cell function , such as those obtained from the hyperglycemic clamp technique or the frequently sampled intravenous glucose tolerance test . notwithstanding , we used two proxy measures of -cell function that have been extensively validated and used in previous studies including the diabetes prevention program and the american diabetes association genetics of type 2 diabetes study ( in the case of igi / ir ) and the iras study ( in the case of pi ) . in conclusion , rosiglitazone , but not ramipril , resulted in significant improvements in measures of -cell function over time . these findings demonstrate that the diabetes preventive effect of rosiglitazone may in part be a consequence of improved -cell function .

objectivethe objective of this study was to determine the degree to which ramipril and/or rosiglitazone changed -cell function over time among individuals with impaired fasting glucose ( ifg ) and/or impaired glucose tolerance ( igt ) who participated in the diabetes reduction assessment with ramipril and rosiglitazone medication ( dream ) trial , which evaluated whether ramipril and/or rosiglitazone could prevent or delay type 2 diabetes in high - risk individuals.research design and methodsthe present analysis included subjects ( n = 982 ) from dream trial centers in canada who had oral glucose tolerance tests at baseline , after 2 years , and at the end of the study . -cell function was assessed using the fasting proinsulin to c - peptide ratio ( pi / c ) and the insulinogenic index ( defined as 300 min insulin/300 min glucose ) divided by homeostasis model assessment of insulin resistance ( insulinogenic index [ igi]/insulin resistance [ ir]).resultssubjects receiving rosiglitazone had a significant increase in igi / ir between baseline and end of study compared with the placebo group ( 25.59 vs. 1.94 , p < 0.0001 ) and a significant decrease in pi / c ( 0.010 vs. 0.006 , p < 0.0001 ) . in contrast , there were no significant changes in igi / ir or pi / c in subjects receiving ramipril compared with placebo ( 11.71 vs. 18.15 , p = 0.89 , and 0.007 vs. 0.008 , p = 0.64 , respectively ) . the impact of rosiglitazone on igi / ir and pi / c was similar within subgroups of isolated igt and ifg + igt ( all p < 0.001 ) . effects were more modest in those with isolated ifg ( igi / ir : 8.95 vs. 2.13 , p = 0.03 ; pi / c : 0.003 vs. 0.001 , p = 0.07).conclusionstreatment with rosiglitazone , but not ramipril , resulted in significant improvements in measures of -cell function over time in pre - diabetic subjects . although the long - term sustainability of these improvements can not be determined from the present study , these findings demonstrate that the diabetes preventive effect of rosiglitazone was in part a consequence of improved -cell function .

pubmed

microbial communities differ from monocultures in terms of the toxins they degrade ( pelz et al , 1999 ) , the natural products they produce ( pettit , 2009 ) , and even their capacity to grow at all in certain environments ( schink , 2006 ) . a complete understanding of microbial metabolism must extend from the properties of individual strains in pure culture to the combinatorial interactions supported by complex communities . such strains may exhibit naturally complementary metabolism ( kim et al , 2008 ; rozen et al , 2009 ; hillesland and stahl , 2010 ) , or may be genetically engineered to interact ( shou et al , 2007 ; balagadd et al , 2008 ; gore et al , 2009 ) . interacting partners share metabolites , such as hydrogen ( hillesland and stahl , 2010 ) , acetate ( rozen et al , 2009 ) , amino acids ( shou et al , 2007 ) , fixed nitrogen ( kim et al , 2008 ) or glucose ( kim et al , 2008 ; gore et al , 2009 ) . in each case , it has been possible to extrapolate interactive dynamics from the individual characteristics of the participating strains . a general model of metabolic interaction would account for the variety of metabolites that can be exchanged and the genotypic diversity of the strains that exchange them . ideally , such a model would not require detailed biochemical characterization of each individual participant to be predictive . optimization - based techniques of flux balance analysis have been successful in describing metabolic systems ( feist et al , 2007 ) . these approaches draw on the well - characterized biochemical reaction network of organisms such as escherichia coli ( kanehisa and goto , 2000 ) , and can be easily generalized to more exotic species ( feist et al , 2009 ) . using a model set of e. coli auxotrophs , we extend these approaches to the higher - order system of two strains in concourse . we find that certain pairs of auxotrophs will complement one - another 's growth in minimal media by cross - feeding essential metabolites , a relationship we refer to as synthetic mutualism in trans ( smit ) . the smit interactions constitute a class of synthetic genetic interactions because , similar to other synthetic interactions , they reflect and elucidate the genetic and metabolic network from which they derive . we develop a quantitative and predictive theoretical model that reveals the extent to which complex metabolic interactions emerge from the relatively simple topology of the biochemical reaction network . we selected 46 conditionally lethal auxotrophic e. coli from the keio collection ( baba et al , 2006 ) . each deletion blocks the biosynthesis of an essential metabolite , imparting a strict dependency on an external supply of that metabolite for growth . the selected mutations cover a significant portion of metabolic diversity and include genes required for the biosynthesis of amino acids , nucleotides , and cofactors , as well as genes involved in glycolysis and respiration . each deletion strain exhibits robust growth in rich ( lb ) medium but no measurable growth in minimal ( m9 ) medium . strains were labeled individually with plasmids expressing either venus or mcherry fluorescent proteins and co - cultured in minimal media in all pairwise combinations ( box 1 ) . theoretical and empirical precedents have established that metabolic cooperation is enhanced at intermediate cell densities when the concentration of cross - fed metabolites is sufficiently high to improve cooperative fitness ( shou et al , 2007 ; bull and harcombe , 2009 ) . strains were therefore each inoculated at 10 cells per ml , roughly 1/200th saturation density , and fold growth is reported relative to this initial inoculation level . a subset of mixed pairs showed improved growth relative to either corresponding monoculture ( figure 1 ) . in control cultures of self - same pairs , no strain was observed to grow more than eight - fold . however , 17% of dissimilar pairings showed greater than 50-fold growth , indicating metabolic synergy . growth phenotypes were variable , with alternately one strain , both , or neither benefiting from the interaction . for each strain , we were able to identify at least one partner with which synergistic growth was evident . conversely , no strain exhibited a universally cooperative phenotype , either in the role of nutrient donor or recipient . we first explored how the growth of a given strain relates to its level of cooperation , as inferred from the growth of its partner strain . cooperating strains in this system may show enhanced proliferation through the mechanism of invested benefits ( connor , 1995 ; west et al , 2007 ) . cooperation on the part of strain a will augment a 's growth only if b cooperates in turn . by increasing b 's abundance , a then cultivates the source of a 's own metabolites and therefore a 's own growth . given the relatively low growth rates of the co - cultured strains and the excess availability of nutrients , we do not expect a significant metabolic burden to be associated with the production of shared metabolites . however , we do expect a limit to the invested benefits of cooperative growth . specifically , this should arise when oversharing on the part of a results in b monopolizing the batch culture and therefore necessarily limiting a. the data reflect a significant growth benefit for cooperating strains . we record an overall positive correlation between the growth of strains a and b ( spearman 's =0.2 , p - value : < 10 ) . this correlation was highest when the total growth of a and b was low to moderate . we could also observe a characteristic anti - correlation between the growth of a and b in cultures that approach saturation , when more growth of one strain tended to impede the other ( figure 2a ) . we assign cooperation levels , ca and cb , to quantify cooperation on the part of strain a and b , respectively . growth was allowed to proceed for a fixed and finite amount of time , so our system does not necessarily approach a steady state . although analytical solutions to ( 1 ) are not available , numerically solving the system of two equations for the two variables ca and cb transforms the growth data into biologically relevant parameters that reflect the level of cooperation evidenced by each strain . the measured growth of a increases monotonically with respect to the derived value of cb , indicating that it is always beneficial to be partnered with a cooperating strain . a characteristically also increases with ca for low ca , as strain a reaps the invested benefits of cultivating strain b. however , a declines for high ca , when excess cooperation allows b to dominate the available carrying capacity ( figure 2b ) . we sought to relate the various observed cooperation phenotypes to the properties of individual mutations in the global context of e. coli metabolism . synthetic genetic interactions , the synergistic growth effects of multiple gene deletions , are known to identify epistatic or modular relationships among genes ( segr et al , 2005 ; ooi et al , 2006 ; motter et al , 2008 ) . previous studies emphasize the phenotypic consequences of multiple genetic deletions within the genome of a single strain . we define a new synthetic relationship , the synergistic growth between two otherwise lethal mutants , as smit . similar to other synthetic interactions , smit interactions can be applied to infer properties of the e. coli metabolic network . pairings of strains with metabolic blocks in the same biosynthetic pathway grew less than pairings of strains blocked in distinct pathways . approximately 3.6% of pathway - matched pairs showed growth above 50-fold against 18% of pathway - distinct pairs ( n=55 , n=980 , respectively , p - value : 2 10 ) . however , mutants sharing pathways showed consistency in their set of cooperating partners ( supplementary figure 1 ) . we constructed for each strain a cooperation profile vector consisting of the measured growth of that strain and that of its partner strain in each of the 46 co - cultures . correlations of the cooperation profiles therefore indicate the overall similarity of the cooperative behavior between two strains . we applied an unsupervised hierarchical clustering algorithm to the set of cooperation profiles , using the spearman rank correlation between vectors as a distance metric and a complete linkage function . mutants in the biosynthetic pathways of tryptophan , guanine , histidine , purine , pyramidine , arginine , cysteine , leucine , and pantothenate were recovered as distinct branches in the resulting dendrogram . analysis of smit interactions is therefore capable of identifying biosynthetic modules ( figure 3a ) . more distal correspondence in the metabolic network can also be detected in the analysis of smit interactions . for each pair of interacting mutants , we define a shortest path through the metabolic network connecting the two reactions . the correlation between the cooperation profiles characterizing any two strains declines with the distance spanning their mutations ( =0.34 , p - value : < 10 ) . this suggests that , to a significant extent , the complex emergent phenotype of smit growth is governed by the relatively simple stoichiometries of the metabolic reaction network ( figure 3b ) . metabolic network stoichiometries can be applied to infer biochemical reaction rates through the method of flux balance analysis ( fba ; palsson , 2006 ) . we extend fba modeling from a single e. coli to a system of two interacting strains . beginning with the standard iaf1260 reconstruction ( feist et al , 2007 ) , the stoichiometric matrices and flux bounds for the two strains are concatenated appropriately to produce the joint model , allowing the free exchange of metabolites between the two mutants ( see materials and methods ) . we derived solutions to the joint model by applying the minimization of metabolic adjustment ( moma ) objective function ( segr et al , 2002 ) . the moma methods hypothesize that mutant systems will tend to approximate the wild - type flux distribution , even as mutation precludes an exact match . this objective function is conservative ; it identifies solutions in the joint vector closest , in the euclidean sense , to the wild - type fluxes . total growth of the co - cultures was correlated with predicted growth derived from the joint flux model . we calculated a rank correlation of 0.42 across predictions for all pairs ( p - value : < 10 ) . this model performed less well in comparisons of the subset of pairs with mutations in the nucleotide biosynthesis pathway ( =0.17 , p - value : 0.13 , n=78 ) , and better when both mutants were involved in amino - acid production ( =0.4 , p - value : < 10 , n=378 ) , or in neither of those classes ( =0.72 , p - value : 0.02 , n=10 ) . the joint flux model was effective in reproducing our observation that mutants in shared pathways generally do not supplement one another 's growth ( supplementary figure 2 ) . in general , growth predictions from the joint flux model were higher than those measured . this might be explained by the relatively high levels of cooperation implicit in the objective function . in selecting an optimal joint flux vector , fluxes are weighted equally in their contribution to minimizing the metabolic adjustment of their own strain and of the partner strain . this is theoretically unlike the situation in vivo , in which any flux optimality is expected only within a given strain , and any benefits to the partner strain are only as a byproduct . indeed , our system has no co - evolutionary history and has not been optimized by natural selection in any sense . we reasoned that the likelihood of a given metabolite being shared should depend on the growth value of that metabolite to both the donor and recipient strain . metabolites less valuable to the donor might be more readily shared , and metabolites more valuable to the recipient should effect more growth . in an efficient cooperative interaction , each strain would exchange growth - cheap metabolites for growth - dear metabolites ( boucher , 1988 ) . we can derive explicit predictions for the value of each exchanged metabolite for each mutant strain in our system as shadow prices . a shadow price , in a linear programming model , is defined as the marginal change in the objective function associated with the strengthening or loosening of a particular model constraint . as in standard flux balance models , calculating shadow prices requires only the reaction network stoichiometries and the measured composition of biomass . we solved the iaf1260 model for shadow prices representing the fitness benefit , b , to each strain a in taking up a unit of its required metabolite . for example , a tryptophan auxotroph is associated with a high benefit term because a unit of the rare amino acid tryptophan is sufficient to produce a relatively high amount of biomass . similarly , we derive a cost term , p , representing the loss in growth rate for each strain , b , in secreting a given metabolite . the calculated costs and benefits produced the expected correlation with measured growth . the benefit associated with a metabolite b was positively correlated with the growth of strain a ( =0.11 , p - value : 10 ) , but showed no correlation with the growth of the partner strain . conversely , the production cost of metabolites p , incurred by strain b , was negatively correlated with a 's growth ( =0.26 , p - value:<10 ) but was uncorrelated with b 's growth . thus , strains tended to show the most growth when predicted to require only small quantities of metabolites that were cheap for their partner to produce . we define the predicted efficiency of cooperation , , as the ratio b / p . we observed that was more predictive than either b or p alone in determining the growth of strain a ( : 0.31 , p - value : < 10 ) . we also determined an overall positive correlation between the growth of strain a and , the efficiency term as calculated for the partner strain , consistent with the invested benefits a receives from the growth of b ( : 0.14 , p - value : 10 ) . indeed , in the regime of low a growth , was more strongly correlated with a 's growth than , emphasizing the strong fitness advantage for efficient cooperators . the cooperation efficiency terms reproduce the characteristic pattern in their correlation with strain growth , predicted in our invested benefits model of cooperation ( figure 3c ) . the term is positively correlated with a 's growth across all growth levels , as it is always beneficial to be paired with an efficient cooperator . the correlates positively to a 's growth for low growth , then negatively as the strains approach saturation . only in this narrow range would the less generous show more growth . cooperation efficiencies derive purely from stoichiometric information , and so can be calculated for strains without detailed biochemical characterization . they are independent of the specific physiological mechanisms that effect metabolite production , secretion , and exchange , which in the case of this system remain to be described . intracellular metabolites represent valuable resources , and should therefore be retained by cell membranes in the absence of a specific mechanism for secreting them . such reasoning can explain the behavior of most co - cultures , in which essential metabolites are retained and no cross - feeding occurs . e. coli are known to secrete a number of amino acids , particularly when starved or otherwise stressed ( burkovski et al , 1995 ; kaderbhai et al , 2003 ; valle et al , 2008 ) . alternately , the intracellular metabolite pool also undergoes significant changes during starvation ( tweeddale et al , 1998 ) . the results presented here reflect only cooperation before co - evolution , and do not reveal the evolutionarily stable steady state that may arise for cross - feeding organisms . to persist in natural systems , cooperative behavior must be robust to variations of environment , and to the emergence of exploitative mutants . many natural syntrophic systems seem to be cases of byproduct cooperation , in which one strain feeds on the waste products of another ( marx , 2009 ; rozen et al , 2009 ) . we find that the metabolites most readily exchanged are those of little value to the secreting strain . this is consistent with proposals that byproduct cooperation evolves more readily than other forms , because it requires no active investment of one partner in the other ( connor , 1995 ) . the fitness costs and benefits of biological interactions are often difficult or impossible to quantify ( boucher , 1988 ) , but are required for the application of population dynamic models ( nowak , 2006 ) . the a priori prediction of such parameters in an experimentally tractable model organism brings a new degree of quantitative resolution to theories of metabolic interaction . deletion strains from the keio collection were grown to saturation in rich ( lb ) medium with appropriate antibiotics , washed twice in pbs , and re - suspended in m9 minimal medium at a 1:100 dilution ( 6.8 g / l na2hpo4 , 3 g / l kh2po4 , 0.5 g / l nacl , 1 g / l nh4cl , 20 g / l glucose , 100 m cacl2 , 1 mm mgso4 , 50 g / l ampicillin , and 100 g / l kanamycin ) . strains were cultured overnight in minimal medium to allow depletion of residual nutrients , then re - diluted to the initial 1:100 level . viable cell counts were determined by plating for all strains both before and after starvation , and were observed to be linear with od and similar for all strains . this indicated that total cell density could be accurately measured by od , despite the unique genetic background and metabolic conditions of each co - culture . growth was measured by optical density at 600 nm using a perkin elmer envision plate reader . cell density for a strain a was calculated as (odmodbkg)afrac , where odm is the measured od , odbkg was the background absorbance of blank medium , is the linear scaling factor from od to cell density , calculated by plating standard curves , and afrac is the proportion of strain a as measured by flow cytometry . growth experiments were conducted in duplicate and with label - switched pairs , a total of four replicates for each of 1035 possible experimental pairs . growth measurements were reproducible in both biological replicates ( =0.92 ) and label - switched replicates ( =0.9 ) . following data acquisition , a random sample of 50 growing mixed cultures were struck out on rich medium and minimal medium . in no case were single colonies observed to grow on minimal plates , indicating that observed growth was not due to the restoration of prototrophy in a single lineage from any genetic event . metabolic network distances were calculated excluding metabolites with very high connectivities ( > 20 reactions ) , because of their effect of radically shrinking the network radius . a standard metabolic optimization problem with a moma - type objective function identifies a flux vector j in the mutant flux space , which minimizes the euclidean distance to the wild - type flux vector . thus , it satisfies the following : where j denotes a wild - type flux vector , derived from a standard fba formulation ( segr et al , 2002 ) . bounds on the exchange fluxes , corresponding to the media conditions , are set to u and l . finally , js and jx are the subsets of internal and exchange fluxes of j. for the joint problem , we concatenate the flux vectors , bounds , and stoichiometric matrices appropriately to define a joint flux space , thus solving : superscripts a and b denote fluxes , matrices and bounds associated with strain a or b. the exchange matrix , x , is identical for all mutants . the third row of the joint stoichiometric matrix allows the exchange of metabolites between strains . the upper bounds of the exchange reactions , u , are modified to allow the uptake of all shared metabolites . the term 2xu transposes the unmodified bounds from the individual fluxes on problem 2 to apply to the sum of the joint fluxes , maintaining conservation of mass between the two strains . the mean marginal production cost of a metabolite m , pm , we define as the reciprocal of the solution to a linear programming problem : this represents the minimum flux value , that is , the maximum secretion level , which can be attained consistent with a growth rate of at least 90% wild type . the reciprocal of this solution , 1/jm , is proportional to the mean shadow price associated with the bound on the exchange flux of metabolite m , jm , taken over the interval 10090% of wild - type growth rate . we allow pm to take the value of infinity for those cases when jm=0 , meaning a given metabolite is stoichiometrically blocked from being secreted and is therefore not available at any cost . the mean marginal benefit , bm , which a given strain , , receives from a metabolite , m , is the reciprocal of the solution to a similar problem : note that although secretion costs are calculated with respect to the wild - type strain background , uptake benefits are defined with respect to a specific mutant strain that can not grow without supplemented metabolites . problem 4 identifies the minimum supplemental flux consistent with growth of at least 10% of the wild - type level . most metabolites are not capable of rescuing growth of most mutant strains , therefore the solution to problem 4 is undefined and we allow bm=0 . otherwise . as above , the benefit is proportional to the mean shadow price for the uptake bound on metabolite m over the interval 010% of wild - type growth rate . in cases , in which pm and bm are defined for more than one m , because strain a can be rescued by more than one metabolite , we take the values p and b to be the mean values over all m for which they are defined . the efficiency of cooperation for a strain b , , for a strain b as the ratio b / p . in cases , in which a can be rescued by more than one metabolite , we take to be the mean ratio for all such metabolites . deletion strains from the keio collection were grown to saturation in rich ( lb ) medium with appropriate antibiotics , washed twice in pbs , and re - suspended in m9 minimal medium at a 1:100 dilution ( 6.8 g / l na2hpo4 , 3 g / l kh2po4 , 0.5 g / l nacl , 1 g / l nh4cl , 20 g / l glucose , 100 m cacl2 , 1 mm mgso4 , 50 g / l ampicillin , and 100 g / l kanamycin ) . strains were cultured overnight in minimal medium to allow depletion of residual nutrients , then re - diluted to the initial 1:100 level . viable cell counts were determined by plating for all strains both before and after starvation , and were observed to be linear with od and similar for all strains . this indicated that total cell density could be accurately measured by od , despite the unique genetic background and metabolic conditions of each co - culture . growth was measured by optical density at 600 nm using a perkin elmer envision plate reader . cell density for a strain a was calculated as (odmodbkg)afrac , where odm is the measured od , odbkg was the background absorbance of blank medium , is the linear scaling factor from od to cell density , calculated by plating standard curves , and afrac is the proportion of strain a as measured by flow cytometry . growth experiments were conducted in duplicate and with label - switched pairs , a total of four replicates for each of 1035 possible experimental pairs . growth measurements were reproducible in both biological replicates ( =0.92 ) and label - switched replicates ( =0.9 ) . following data acquisition , a random sample of 50 growing mixed cultures were struck out on rich medium and minimal medium . in no case were single colonies observed to grow on minimal plates , indicating that observed growth was not due to the restoration of prototrophy in a single lineage from any genetic event . metabolic network distances were calculated excluding metabolites with very high connectivities ( > 20 reactions ) , because of their effect of radically shrinking the network radius . a standard metabolic optimization problem with a moma - type objective function identifies a flux vector j in the mutant flux space , which minimizes the euclidean distance to the wild - type flux vector . thus , it satisfies the following : where j denotes a wild - type flux vector , derived from a standard fba formulation ( segr et al , 2002 ) . bounds on the exchange fluxes , corresponding to the media conditions , are set to u and l . finally , js and jx are the subsets of internal and exchange fluxes of j. for the joint problem , we concatenate the flux vectors , bounds , and stoichiometric matrices appropriately to define a joint flux space , thus solving : superscripts a and b denote fluxes , matrices and bounds associated with strain a or b. the exchange matrix , x , is identical for all mutants . the third row of the joint stoichiometric matrix allows the exchange of metabolites between strains . the upper bounds of the exchange reactions , u , are modified to allow the uptake of all shared metabolites . the term 2xu transposes the unmodified bounds from the individual fluxes on problem 2 to apply to the sum of the joint fluxes , maintaining conservation of mass between the two strains . the mean marginal production cost of a metabolite m , pm , we define as the reciprocal of the solution to a linear programming problem : this represents the minimum flux value , that is , the maximum secretion level , which can be attained consistent with a growth rate of at least 90% wild type . the reciprocal of this solution , 1/jm , is proportional to the mean shadow price associated with the bound on the exchange flux of metabolite m , jm , taken over the interval 10090% of wild - type growth rate . we allow pm to take the value of infinity for those cases when jm=0 , meaning a given metabolite is stoichiometrically blocked from being secreted and is therefore not available at any cost . the mean marginal benefit , bm , which a given strain , , receives from a metabolite , m , is the reciprocal of the solution to a similar problem : note that although secretion costs are calculated with respect to the wild - type strain background , uptake benefits are defined with respect to a specific mutant strain that can not grow without supplemented metabolites . problem 4 identifies the minimum supplemental flux consistent with growth of at least 10% of the wild - type level . most metabolites are not capable of rescuing growth of most mutant strains , therefore the solution to problem 4 is undefined and we allow bm=0 . otherwise . as above , the benefit is proportional to the mean shadow price for the uptake bound on metabolite m over the interval 010% of wild - type growth rate . in cases , in which pm and bm are defined for more than one m , because strain a can be rescued by more than one metabolite , we take the values p and b to be the mean values over all m for which they are defined . the efficiency of cooperation for a strain b , , for a strain b as the ratio b / p . in cases , in which a can be rescued by more than one metabolite , we take to be the mean ratio for all such metabolites .

mixed microbial communities exhibit emergent biochemical properties not found in clonal monocultures . we report a new type of synthetic genetic interaction , synthetic mutualism in trans ( smit ) , in which certain pairs of auxotrophic escherichia coli mutants complement one another 's growth by cross - feeding essential metabolites . we find significant metabolic synergy in 17% of 1035 such pairs tested , with smit partners identified throughout the metabolic network . cooperative phenotypes show more growth on average by aiding the proliferation of their conjugate partner , thereby expanding the source of their own essential metabolites . we construct a quantitative , predictive , framework for describing smit interactions as governed by stoichiometric models of the metabolic networks of the interacting strains .

pubmed

brook - spiegler syndrome ( bss ) is a rare genodermatosis characterized by the progressive formation of adnexal skin tumors in the scalp and face , mainly trichoepiteliomas , cylindromas and spiradenomas . it is due to mutations in the tumor suppressor gene cyld ( cylindromatosis gene ) on chromosome 16q12q13 . around 93 mutations the study of cyld gene in patients and their relatives can provide the molecular diagnosis and appropriate genetic counselling . there is a low risk of malignancy and patients require long - term follow - up . brooke spiegler syndrome ( bss ) is a rare genodermatosis due to mutations in the tumor suppressor gene cylindromatosis ( cyld gene ) on chromosome 16q12q13 . there is a low risk of malignancy and patients require long - term follow - up . a 71-year - old woman was evaluated due to the progressive development of asymptomatic lesions in her head during the last 4 years . the physical examination showed multiple rounded skin - colored or erythematous papules , 28 mm in diameter , located in scalp and forehead [ figure 1 ] . we performed several excisional biopsies and the histopathological diagnosis was spiradenoma in all of the lesions , except for one cylindroma . multiple rounded skin - colored or erythematous papules in scalp and forehead , compatible with spiradenomas and cylindromas regarding her family history , she referred the presence of lesions at the scalp and forehead in her mother , her mother 's brother , and two of her three daughters , some of them very similar to our patient s . next , we performed a physical examination of her two affected daughters showing multiple papules and nodules of 13 cm , some of them painful [ figures 2 and 3 ] . once all the information was collected , we elaborated the family pedigree , which suggested an autosomal dominant inheritance pattern of the disease [ figure 4a ] . above : rounded pink nodule with superficial telangiectasias at the scalp of one of the patient 's daughters . below : multiple skin - colored or yellowish millimetric papules at the forehead and periocular area , suggesting multiple trichoepitheliomas multiple firm pink nodules of variable size in scalp of the second daughter ( a ) family pedigree . ( b ) exon 8-cylindromatosis gene sequencing that shows ( a ) heterozygous mutation c. 1628_1629delct in our patient and her two affected daughters . ( b ) wild - type genotype in the unaffected daughter suspecting a genetic syndrome , we requested the study of the cyld gene in our patient and her two affected offsprings . the exon 8 sequencing of the cyld gene revealed the heterozygous mutation c. 1628_1629delct [ figure 4b ] . it consists of a two base - pair deletion that originates a new frameshift , the first codon is now the stop codon p. ser543xfs*1 . therefore , to complete the familiar study , we performed the genetic test on our patient 's daughter presenting no signs or symptoms suggesting bss . the result was negative , showing the wild - type genotype [ figure 4b ] . her 11 and 5-year - old children did not present any clinical data either . finally , we offered genetic counseling to her first - grade relatives . we provided concise information about the main characteristics of sbs and the 50% probability of the descendants to develop this syndrome , due to its autosomal dominant inheritance . it is characterized by the progressive development of adnexal skin tumors in scalp and face . clinical manifestations start since puberty and its incidence is higher in females without distinction of race . bss has also been associated with increased risk of salivary glands neoplasms and other tumors although with very little evidence . cylindromas appear at the scalp reaching a considerable size , often forming the turban tumor . spiradenomas are also multiple and appear in extremities , trunk and less frequently in the scalp ( this is a rare location of single sporadic spiradenomas ) . recent studies demonstrate a common apocrine origin of spiradenomas and cylindromas , with an expression of hair follicle stem cells specific markers , mainly cd200 and cytokeratin . thus , we prefer to avoid the adjective eccrine traditionally applied to spiradenomas . this syndrome is due to mutations in the tumor suppressor gene cyld localized on chromosome 16q12q13 . it is a tumor suppressor gene that codifies a protein of the deubiquitinating enzymes family . it negatively regulates activation of factors such as nuclear factor - kappa - b , jnk , and wnt , involved in inflammation and cellular division . the loss of deubiquitinating activity of cyld correlates with tumorigenesis ( apoptosis resistance , uncontrolled cell division ) . germ - line mutations predispose to the disease , and sometimes , a second acquired somatic mutation determines the final phenotype . around 93 different mutations its phenotypic expression has been found so far . the genetic study in our case revealed the existence of the heterozygotic mutation c. 1628_1629delct . there is only one report of this mutation in 2008 found in a family with multiple trichoepitheliomas ; therefore , our case confirm the association of this genetic mutation with bss . two entities have been recognized as phenotypic variations of bss ( mim#605041 ) due to mutations in the cyld gene . multiple familial trichoepithelioma , also known as trichoepithelioma adenoides cystica ( mim#601606 ) , presents with numerous trichoepitheliomas since puberty predominantly located in the face . in familial cyld ( mim#132700 ) , multiple cylindromas appear in face and scalp of the patients . on the other hand , there have been described some cases of multiple familial spiradenomas , predominantly located in the scalp . no genetic study was performed in these cases ; therefore , it could not be confirmed the existence of brss or one phenotypic variation . alarm signs are ulceration , bleeding , fast growth , blue nodules , or pain . malignant spiradenoma is a very aggressive neoplasm with a risk of metastasis around 50% and a mortality of 39% if left untreated . we present a new case of bss in a family due to the heterozygous cyld mutation c. 1628_1629delct . particularities of this case were the late onset in our patient and the relatively low number of cylindromas and trichoepitheliomas found in comparison with the cases found in the literature . thus , our case may support the hypothesis about the influence of the concrete germline mutation and the variable presence of a somatic mutation in the development of the final phenotype . for this reason , it is imperative to perform a correct diagnosis and periodic follow - up of the patients . in addition , the genetic study should be considered individually with the possibility of genetic counseling to the family members . the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s ) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed . we describe a new case of bss due to the rare heterozygous cyld gene mutation c.1628_1629delct in three women of the same family . the particularities of the clinical manifestations ( late onset , relatively low number of tumors ) may support the hypothesis about the relationship between the germline mutation , variable presence of somatic mutation and the final phenotype . the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s ) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed . we describe a new case of bss due to the rare heterozygous cyld gene mutation c.1628_1629delct in three women of the same family . the particularities of the clinical manifestations ( late onset , relatively low number of tumors ) may support the hypothesis about the relationship between the germline mutation , variable presence of somatic mutation and the final phenotype . the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s ) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed . we describe a new case of bss due to the rare heterozygous cyld gene mutation c.1628_1629delct in three women of the same family . the particularities of the clinical manifestations ( late onset , relatively low number of tumors ) may support the hypothesis about the relationship between the germline mutation , variable presence of somatic mutation and the final phenotype .

brooke spiegler syndrome ( bss ) is a rare genodermatosis characterized by the progressive formation of adnexal skin tumors in the scalp and face , mainly trichoepitheliomas , cylindromas , and spiradenomas . it has also been associated with salivary glands neoplasms . it is due to mutations in the tumor suppressor gene cylindromatosis ( cyld gene ) localized on chromosome 16q12q13 . around 93 mutations have been described . the study of cyld gene in patients and their relatives is of vital importance to establish the molecular diagnosis and offer appropriate genetic counseling . there is a low risk of malignancy and patients require long - term follow - up . a case of bss in a family is described . the existence of the genetic mutation at the cyld gene c. 1628_1629delct in three of the women affected was demonstrated . this mutation has only been described once in a previous study .

pubmed

biological scaffold materials derived from dermis , pericardium , and small intestine submucosa of human , bovine , and porcine origin have been utilized over the last decade for soft tissue repair applications such as hernia repair , breast reconstruction , staple - line reinforcement , and orthopedic applications . these scaffold materials are particularly useful in clean - contaminated or contaminated settings due to their rapid revascularization and clearance of bacteria [ 5 , 6 ] . permanent , synthetic polymer mesh materials are not typically utilized in these settings due to risk of infection . biological scaffolds are also utilized as an alternative to fascial closure when there is excessive tension on the wound , when tissue loss makes closure especially difficult , or in damage - control/abdominal compartment settings in which the abdomen must be left open until the patient is stabilized . in these settings , scaffolds are utilized to protect the abdominal contents , typically until granulation of the wound occurs and a split - thickness skin graft can be applied . although biological scaffolds present many attractive advantages , these materials are also extremely expensive , exhibit substantial scaffold variability , and provoke patient - specific immunological responses . early designs of absorbable polymer scaffolds included materials such as dexon ( covidien , mansfield , ma ) and vicryl ( ethicon , inc . , dexon is comprised of polyglycolic acid ( pga ) , and it degrades in vivo via hydrolysis . full resorption of the pga is expected to be complete in approximately 6090 days ( 2 - 3 months ) . animal studies have shown that dexon is quickly resorbed and associated with hernia formation after just 14 days due to central mesh failure . by 90 days , greater than 80% of the scaffolds were fully resorbed in that particular animal study . clinical studies have shown that dexon has primarily been utilized in damage control situations to cover the open abdomen until granulation tissue is present and a skin graft can be applied . however , subsequent development of a ventral hernia has also been observed in these patients . in one study , the authors concluded that the dexon mesh was useful for providing a temporary support for the abdominal wall but would likely require a ventral hernia repair with placement of a permanent mesh once the contamination was resolved , making this material a less than ideal long - term solution . vicryl is a copolymer of glycolide and lactide which also degrades in vivo through hydrolysis . according to its instructions for use , vicryl loses 77% of its strength in the first 14 days ( 0.5 months ) in a rat model , and it is fully resorbed in approximately 6090 days ( 2 - 3 months ) . clinically , vicryl has been utilized primarily in damage - control applications as a buttress to the abdominal wall or as a prophylactic measure to prevent incisional hernia formation . animal studies have shown that the tensile strength of the rat abdominal wall at 30 and 60 days is equivalent whether vicryl mesh or biological scaffolds were utilized ( surgisis and alloderm ) . however , collagen deposition and neovascularization were greater for the biological scaffolds than for vicryl . this could be explained by the decreased ph and associated increase in inflammation observed at the wound site as the vicryl mesh degrades . pronounced inflammation , reduced tissue ingrowth , and greater angiogenesis were observed for vicryl mesh compared to permanent prolene and ultrapro meshes in another animal study involving a hamster model . the authors of that study concluded that the more aggressive foreign body response incited by the resorbable vicryl mesh did not lead to better tissue incorporation as predicted . in a similar study , tissue ingrowth was also unsatisfactory for the absorbable vicryl mesh when compared to permanent synthetic materials such as polypropylene and expanded polytetrafluoroethylene in a rabbit model . overall , both dexon and vicryl scaffolds lose mechanical strength and are resorbed fairly quickly , making them less than ideal for hernia repair applications which require more long - term support of the repair site until tissue remodeling is complete . thus , more recent absorbable scaffold designs have been developed which utilize long - lasting polymers that degrade more slowly . scaffolds such as gore bio - a ( w.l . gore and associates , inc . , flagstaff , az ) , tigr matrix ( novus scientific , uppsala , sweden ) , and phasix mesh ( c. r. bard , inc./davol inc . , gore bio - a is a copolymer of poly(glycolide : trimethylene carbonate ) that degrades in vivo through both hydrolytic and enzymatic mechanisms and is fully resorbed within approximately 180 days ( 6 months ) according to the instructions for use . in a rat model with methicillin - resistant staphylococcus aureus ( mrsa ) contamination , bacteria were cleared from the gore bio - a mesh more effectively than either vicryl or tigr matrix at the 10 inoculum . overall , all three scaffolds exhibited reduced tensile strength and increased rate of mesh failure regardless of scaffold composition . gore bio - a has also been utilized in a number of clinical applications including amyand hernia repair , open elective hernia repair , paraesophageal / hiatal hernia repair , suture line reinforcement , pelvic floor reinforcement , and breast reconstruction . the outcomes thus far have been promising with low rates of recurrence , infection , and pain . however , the majority of these studies have been case reports or very small series ( i.e. , less than 10 patients ) . larger , more comprehensive clinical trials are needed to fully understand the long - term capabilities of this scaffold . tigr matrix is knitted from two fibers having different resorption rates . according to the instructions for use , the first fiber makes up approximately 40% of the overall mesh by weight and is a copolymer of polyglycolide , polylactide , and polytrimethylene carbonate . this fiber degrades in vivo through hydrolysis , loses substantial mechanical strength in the first 14 days ( 0.5 months ) , and is fully resorbed in approximately 120 days ( 4 months ) . the second fiber makes up approximately 60% of the overall mesh by weight and is a copolymer of polylactide and polytrimethylene carbonate . this fiber also degrades in vivo through hydrolysis , but it retains its mechanical strength longer than the first fiber . it begins to demonstrate loss of mechanical strength after approximately 270 days ( 9 months ) and is fully resorbed in approximately 1095 days ( 36 months ) . tigr matrix has been evaluated in a long - term animal model , and a clinical trial is currently underway . in the animal study , tigr matrix was compared to permanent polypropylene mesh in sheep with full thickness abdominal wall defects over the course of 4 , 9 , 15 , 24 , and 36 months . the results showed a typical long - term inflammatory response to the permanent polypropylene contrasted with a gradual resorption of the tigr matrix until it was fully resorbed at 36 months . the tigr matrix also exhibited collagen deposition at the repair site that increased over time and eventually resembled native connective tissue . in the clinical trial , forty subjects were enrolled ( n = 40 ) and followed for 1 year after placement of tigr matrix to repair a primary inguinal hernia . pain and recurrence were evaluated at 0.5 , 1 , 3 , 6 , and 12 months , and pain scores were reduced from an average of 17.4 before surgery to 0.3 after just 6 months . p4hb is a natural polymer from the class of polyhydroxyalkanoates . in nature , these polymers are produced by microorganisms for the purpose of regulating energy metabolism . in the case of phasix mesh , p4hb is a naturally derived , fully absorbable polymer produced by escherichia coli k12 bacteria via transgenic fermentation techniques . p4hb has a chemical structure very similar to many of the synthetic polyester polymers , but because it is biologically derived rather than chemically synthesized , p4hb does not contain any residues from metal catalysts that are typically utilized during chemical synthesis of other polyester polymers . p4hb degrades in vivo through both hydrolysis and a hydrolytic enzymatic digestive process and is fully resorbed in approximately 365545 days ( 1218 months ) according to the instructions for use . the resulting by - products ( carbon dioxide and water ) are metabolized very quickly via the krebs cycle and beta - oxidation . unlike absorbable scaffolds such as dexon and vicryl , whose by - products decrease the ph at the wound site , degradation of p4hb is not as acidic , which may reduce the inflammatory response associated with these materials . in addition , p4hb has been shown to degrade more slowly than pga , yielding more gradual loss of mechanical strength . this is advantageous in applications such as hernia repair in which the rate of degradation should ideally match the rate of remodeling and neotissue deposition at the repair site . a gradual change in mechanical properties is also advantageous because it leads to a gradual transfer of the load from the scaffold back to the tissue , which may help to prevent hernia recurrence . p4hb has been evaluated in a number of animal studies over the past decade , particularly those investigating cardiovascular applications such as tissue engineered trileaflet heart valves , artery augmentation patches , and small diameter vascular grafts , as well as development of p4hb as a suture material . however , this is the first study to evaluate this material specifically for hernia repair applications . thus , the purpose of this study was to determine the mechanical properties , resorption profile , and histological characteristics of phasix mesh and p4hb plug compared to the native abdominal wall when utilized to bridge a surgical defect in a porcine model . phasix mesh is comprised of a fully resorbable polymer monofilament ( poly-4-hydroxybutyrate , p4hb ) that is knitted into a flat sheet configuration as shown in figure 1(a ) . the p4hb plug design is also comprised of p4hb monofilament , but it is preformed into a three - dimensional shape with a fluted outer layer and inner layers or petals attached at the tip as shown in figure 1(b ) . twelve specimens ( n = 12 ) measuring 2.5 5.1 cm ( 1 2 in ) were prepared from phasix mesh and subjected to suture retention testing at time zero , t0 ( i.e. , prior to implantation ) . six of the specimens ( n = 6 ) were oriented such that the load applied during testing was parallel to the longest dimension of the mesh interstices . the other six ( n = 6 ) specimens were oriented such that the load applied during testing was perpendicular to the longest dimension of the mesh interstices . a custom test fixture was utilized in which the mesh specimen was loaded vertically in the instron machine ( instron , norwood , ma ) with a gauge length of 2.5 cm ( 1 in ) and clamped along the upper edge using pneumatic grips set to 60 psi . a stainless steel wire with a diameter of 0.36 mm ( simulating 0 polypropylene suture material ) was passed through the mesh 1.0 cm from the bottom edge of the mesh . each specimen was tested in tension at a rate of 300 mm / min ( 12 in / min ) until the suture pulled through the mesh . the suture retention strength was recorded as the maximum load sustained by the mesh in units of newtons ( n ) . tear resistance testing was based on the american society for testing and materials ( astm ) specification # d2261 - 07a . for this type of testing , twelve mesh specimens ( n = 12 ) measuring 2.5 7.6 cm ( 1 3 in ) were prepared from phasix mesh and subjected to tear resistance testing at time zero , t0 ( i.e. , prior to implantation ) . a 2.5 cm ( 1 in ) slit was cut from the center of the 2.5 cm edge of the specimen toward the center of the mesh to form two tabs . the left tab was clamped in the upper grip of the instron machine using a pneumatic grip set to 60 psi , and the right tab was clamped in an identical fashion in the lower grip . six of the specimens ( n = 6 ) were oriented such that the load applied during testing was parallel to the longest dimension of the mesh interstices . conversely , the other six ( n = 6 ) specimens were oriented such that the load applied during testing was perpendicular to the longest dimension of the mesh interstices . the test was conducted in tension at a rate of 300 mm / min ( 12 in / min ) until the specimen tore in half . the tear strength was recorded as the maximum load sustained by the mesh in units of newtons ( n ) . six circular specimens ( n = 6 ) measuring 7.5 cm in diameter ( 3 in diameter ) were prepared from phasix mesh and subjected to ball burst testing at time zero , t0 ( i.e. , prior to implantation ) . for this reason , only one set of mesh specimens was prepared with no indication of the orientation of the mesh interstices during testing . two circular stainless steel rings were utilized to clamp the mesh specimen to prevent slipping during the test . then , a 2.5 cm diameter ( 1 in ) stainless steel ball was applied in compression at a rate of 300 mm / min ( 12 in / min ) until it burst through the mesh . the ultimate tensile stress and the strain at a stress of 16 n / cm were recorded in units of n / cm and percent , respectively . the institutional animal care and use committee ( iacuc ) of the cbset , inc . facility ( lexington , ma ) where the study was conducted approved the experimental protocol prior to the start of the study , and standard operating procedures were followed at all times . twenty ( n = 20 ) castrated male , yucatan minipigs weighing 33.741.8 kg ( at surgery ) were acquired for the study and acclimated to the facility for a minimum of 21 days . animals were fasted for at least 12 hours prior to surgery . on the morning of surgery , buprenorphine ( 0.03 mg / kg , i m ) and rimadyl ( 2.2 mg / kg , po ) were administered . following sedation , the animals were intubated and maintained under anesthesia with 0.55% , isoflurane inhalant anesthetic , to effect . the animals were placed in dorsal recumbency , and the ventral abdomen was prepared for aseptic survival surgery by shaving the entire abdominal region , cleaning the operative area with three alternating scrubs of povidone - iodine solution and 70% isopropyl alcohol solution , and applying sterilized surgical drapes over the entire field . following preparation of the abdomen , a midline laparotomy ( ~30 cm ) was performed . two , 3 cm diameter ( 1.2 in diameter ) bilateral muscular defects were created in the anterior abdominal wall using a preperitoneal approach ( i.e. , the peritoneum remained intact ) . the surgical defects were not closed and were bridged with a 10.2 cm diameter ( 4 in diameter ) phasix mesh on the right side ( figure 2(a ) ) or a 7.9 cm diameter ( 3.1 in diameter ) p4hb plug on the left side ( figure 2(b ) ) . the phasix mesh was placed in the preperitoneal plane ( bridging the defect ) and fixated circumferentially with approximately n = 16 sorbafix absorbable fixation device constructs comprised of poly(d , l - lactide ) . the p4hb plug was placed in the preperitoneal plane as an onlay mesh bridging the defect and fixated circumferentially with approximately n = 12 permanent prolene ( 2 - 0 ) sutures . the abdominal midline was then repaired via standard closure techniques , and the skin was tattooed to define the cranial , caudal , medial , and lateral aspects of each device to facilitate identification at the time of explant . the animals were recovered from anesthesia and allowed free access to food and water ad libitum . mg / kg , i m ) was administered every 412 hours , and rimadyl ( 2.2 mg / kg , po ) was administered every 24 hours for 72 hours postoperatively . the abdominal region of each animal was examined daily to assess the condition of the wound and the subcutaneous tissues for evidence of herniation , diastasis , seromas , and/or hematomas . animals were survived for 6 , 12 , 26 , or 52 weeks ( n = 5 in each group ) , followed by euthanasia . humane euthanasia was carried out after sedation with telazol ( 4 mg / kg , i m ) and anesthesia via inhalant isoflurane . euthanasia was achieved by administering an overdose of sodium pentobarbital ( 60150 mg / kg , intravenously , to effect ) in accordance with the american veterinary medical association ( avma ) panel of euthanasia and journal of the american veterinary medical association . following euthanasia , the abdominal skin was dissected from the entire abdomen , and the abdominal wall ( including the 2 surgical defects ) was excised , photographed , and placed in saline solution ( 0.9% nacl ) for subsequent mechanical testing . a representative specimen from each phasix mesh and p4hb plug repair one half was placed in 10% neutral buffered formalin for histological analysis , and the other was placed in an empty specimen jar for molecular weight analysis . the repair sites ( i.e. , abdominal wall tissue plus test device as a composite specimen ) were subjected to ball burst testing to evaluate the peak load and relative stiffness of each repair site compared to native abdominal wall . the peritoneum was carefully removed from both of the repair sites and the native abdominal wall sites prior to testing to eliminate the contribution of the peritoneum and to allow assessment of the strength of the repair site alone . the repair sites and native abdominal wall sites were subjected to burst testing using an instron servohydraulic test frame ( instron , norwood , ma ) . a 0.95 cm ( 0.375 in ) diameter ball was applied in compression at a rate of 25.4 mm / min ( 1 inch / min ) until the ball burst through . the peak load ( ball burst force in units of newtons , n ) sustained by each specimen was recorded , and the relative stiffness was calculated from the slope of the line formed when load was graphed versus elongation ( units = n / mm ) . relative stiffness was taken from the slope of the line in the region of 3080% of the peak load . explanted phasix mesh and p4hb plug specimens were enzymatically digested and manually cleaned to remove residual tissue prior to gel permeation chromatography ( gpc ) analysis . gpc was conducted to quantify the molecular weight of the mesh material remaining at each time point . specimens were placed in 50 ml tubes containing 40 ml collagenase ( type i ) solution ( 1.0 mg / ml ) in tesca buffer ( 50 mm tes , 2 mm cacl2 , 10 mm nan3 , ph 7.4 ) . the tubes were placed on a shaker ( 50 rpm ) and incubated at 37c to digest the tissue . after overnight incubation ( ~17 hours ) , the specimens were removed from the buffer and manually cleaned of tissue . the cleaned specimens were rinsed in distilled water , followed by 70% ethanol , and dried prior to gpc analysis . cleaned mesh specimens were dissolved at 1 mg / ml in chloroform , filtered using a 0.45 m filter to remove undigested particulates , and 95 l of this solution was then injected onto a gpc column for analysis . gpc was performed in chloroform at 1 ml / min using a polymer labs , plgel column ( 5 micron , mixed c , 300 7.5 mm ) with an agilent 1100 series hplc with ri detector . all histological assessments were conducted by a board - certified veterinary pathologist ( cbset , lexington , ma ) . the explanted specimens were immersion - fixed in 10% neutral buffered formalin , cut into cross - section , and paraffin - embedded for further processing . paraffin - embedded sections were microtomed ( 4 - 5 m ) , mounted onto glass slides , and stained with hematoxylin and eosin ( h&e ) , masson 's trichrome , and picrosirius red to characterize the host inflammatory / fibrotic response , collagen deposition / remodeling , and absorption properties associated with each specimen based on a standardized scoring system . specimens were scored for inflammatory cell infiltrates ( neutrophils , eosinophils , macrophages , lymphocytes , giant cells ) , neovascularization , fibroplasia ( granulation tissue ) , hemorrhage , necrosis , and fibrosis using the following scoring system as described previously : 0 = absent / no response 1 = minimal / barely detectable 2 = mild / slightly detectable 3 = moderate / easily detectable 4 = marked / very evident . 0 = absent / no response 1 = minimal / barely detectable 2 = mild / slightly detectable 3 = moderate / easily detectable 4 = marked / very evident . newly deposited , type iii collagen appeared green , while mature , type i collagen appeared red or orange under these conditions . a score of 1 was assigned when green ( type iii collagen ) predominated , a score of 2 was assigned when there was a mixture of green , yellow , and yellow - orange , and a score of 3 was assigned when red - orange ( type i collagen ) predominated . san jose , ca ) was utilized to perform all statistical analyses . for continuous data in which three or more groups of data were compared , ( i.e. , peak burst load , relative stiffness , molecular weight ) a one - way analysis of variance ( anova ) was performed followed by a fisher 's lsd post - test as appropriate . for scores such as histological parameters in which three or more groups of data were compared , a nonparametric test ( kruskal - wallis ) was performed followed by a dunn 's posttest as appropriate . all data are reported as mean standard error of the mean ( sem ) except the histological parameters in which the median is reported . when phasix mesh was evaluated in the direction parallel to the longest dimension of the interstices , the suture retention strength was 59.16 5.7 n , which was greater than the 20 n suture retention strength suggested for hernia repair applications [ 29 , 30 ] . similarly , when tested in the direction perpendicular to the longest dimension of the interstices , the suture retention strength of phasix mesh was again greater than 20 n at 49.10 2.3 n. when phasix mesh was evaluated in the direction parallel to the longest dimension of the interstices , the tear resistance strength was 30.33 3.1 n , which was greater than the 20 n tear resistance strength suggested for hernia repair applications [ 29 , 30 ] . similarly , when tested in the direction perpendicular to the longest dimension of the interstices , the tear resistance strength of phasix mesh was again greater than 20 n at 29.48 2.4 n. when subjected to ball burst testing , the maximum compressive load sustained by the phasix mesh was 486.97 12.6 n , with a tensile strength of 140.70 5.4 n / cm and a strain at a stress of 16 n / cm of 15.43 0.2% . tensile strength greater than 50 n / cm and strain in the range of 1030% are considered suitable properties for hernia repair applications [ 29 , 30 ] . thus , phasix mesh possessed appropriate tensile strength and strain values at time zero , t0 ( prior to implantation ) . one animal died prior to its expected time point for reasons unrelated to the materials being evaluated in this study ( i.e. , sedation - related complication ) . this animal was replaced with an additional animal in order to maintain n = 5 animals in each group . all other animals survived until their expected time points . as shown in figures 3(a)3(d ) both devices ( i.e. , phasix mesh and p4hb plug ) and both types of fixation ( i.e. , sorbafix absorbable fixation device fasteners and prolene sutures ) remained visible and intact at the repair sites at 6 , 12 , 26 , and 52 weeks . no evidence of hernia or diastasis was documented in any of the animals at any of the time points . as shown in figure 4(a ) , the burst strength of the native abdominal wall tissue remained stable throughout the duration of the study with no significant changes over time . at 6 , 12 , 26 , and 52 weeks , the burst strength of the native abdominal wall was 76.9 6.3 n , 62.8 14.7 n , 58.7 9.4 n , and 69.7 13.6 n , respectively . no significant differences were detected between 6 and 12 weeks ( p = 0.788 ) , between 12 and 26 weeks ( p = 0.938 ) , between 26 and 52 weeks ( p = 0.834 ) , or overall between 6 and 52 weeks ( p = 0.891 ) . the burst strength of the abdominal wall repaired with phasix mesh also remained stable throughout the duration of the study with no significant changes over time . at 6 , 12 , 26 , and 52 weeks , the burst strength of the phasix mesh repair site was 294.0 31.4 n , 277.0 14.8 n , 217.6 20.3 n , and 260.7 93.8 n , respectively . no significant differences were detected between 6 and 12 weeks ( p = 0.746 ) , between 12 and 26 weeks ( p = 0.262 ) , between 26 and 52 weeks ( p = 0.414 ) , or overall between 6 and 52 weeks ( p = 0.527 ) . the burst strength of the abdominal wall repaired with p4hb plug also remained stable throughout the duration of the study with no significant changes over time . at 6 , 12 , 26 , and 52 weeks , the burst strength of the p4hb plug repair site was 215.2 9.3 n , 307.0 36.0 n , 231.0 28.0 n , and 298.5 57.6 n , respectively . no significant differences were detected between 6 and 12 weeks ( p = 0.086 ) , between 12 and 26 weeks ( p = 0.153 ) , between 26 and 52 weeks ( p = 0.204 ) , or overall between 6 and 52 weeks ( p = 0.118 ) . phasix mesh and p4hb plug repairs demonstrated similar burst strengths at 6 , 12 , 26 , and 52 weeks with no significant differences detected between the two devices at any of the time points evaluated ( p = 0.139 , p = 0.568 , p = 0.798 , and p = 0.474 , resp . ) . in addition , porcine abdominal wall sites repaired with phasix mesh or p4hb plug materials both demonstrated significantly greater burst strength compared to the native abdominal wall at 6 , 12 , 26 , and 52 weeks regardless of whether the mesh ( p < 0.001 , p < 0.001 , p = 0.004 , and p = 0.001 , resp . ) or the plug ( p = 0.011 , p < 0.001 , p = 0.002 , and p < 0.001 , resp . ) was utilized to bridge the defect . as shown in figure 4(b ) , the relative stiffness of the native abdominal wall tissue remained stable throughout the duration of the study with no significant changes over time . at 6 , 12 , 26 , and 52 weeks , the relative stiffness of the native abdominal wall was 5.8 1.2 n / mm , 6.3 1.6 n / mm , 5.5 0.7 n / mm , and 6.5 1.8 n / mm , respectively . no significant differences were detected between 6 and 12 weeks ( p = 0.940 ) , between 12 and 26 weeks ( p = 0.910 ) , between 26 and 52 weeks ( p = 0.887 ) , or overall between 6 and 52 weeks ( p = 0.917 ) . the relative stiffness of the abdominal wall repaired with phasix mesh also remained stable throughout the duration of the study with no significant changes over time . at 6 , 12 , 26 , and 52 weeks , the relative stiffness of the phasix mesh repair site was 22.2 4.7 n / mm , 21.9 1.0 n / mm , 28.7 7.8 n / mm , and 30.5 11.1 n / mm , respectively . no significant differences were detected between 6 and 12 weeks ( p = 0.970 ) , between 12 and 26 weeks ( p = 0.329 ) , between 26 and 52 weeks ( p = 0.799 ) , or overall between 6 and 52 weeks ( p = 0.234 ) . the relative stiffness of the abdominal wall repaired with p4hb plug also remained stable throughout the duration of the study with no significant changes over time . at 6 , 12 , 26 , and 52 weeks , the relative stiffness of the p4hb plug repair site was 17.9 4.3 n / mm , 22.5 2.4 n / mm , 24.2 2.2 n / mm , and 33.8 6.3 n / mm , respectively . no significant differences were detected between 6 and 12 weeks ( p = 0.508 ) , between 12 and 26 weeks ( p = 0.804 ) , or between 26 and 52 weeks ( p = 0.170 ) . however , there was a slight trend toward greater stiffness overall at 52 weeks compared to 6 weeks ( p = 0.025 ) . phasix mesh and p4hb plug repairs demonstrated similar relative stiffness at 6 , 12 , 26 , and 52 weeks with no significant differences detected between the two devices at any of the time points evaluated ( p = 0.533 , p = 0.940 , p = 0.512 , and p = 0.635 , resp . ) . in addition , porcine abdominal wall sites repaired with phasix mesh or p4hb plug materials both demonstrated significantly greater relative stiffness compared to the native abdominal wall at 6 , 12 , 26 , and 52 weeks regardless of whether the mesh ( p = 0.021 , p = 0.027 , p = 0.001 , and p = 0.001 , resp . ) or the plug ( p = 0.084 , p = 0.023 , p = 0.009 , and p < 0.001 , resp . ) was utilized to bridge the defect . as demonstrated by a p value of 0.084 , the stiffness of the abdominal wall sites repaired with p4hb plug materials at 6 weeks did not quite reach statistical significance and were only trending toward greater relative stiffness compared to the native abdominal wall . as shown in figure 5 , phasix mesh and p4hb plug repairs demonstrated similar molecular weights at 0 , 6 , 12 , 26 , and 52 weeks with no significant differences detected between the two devices at any of the time points evaluated ( p = 0.804 , p = 0.640 , p = 0.268 , p = 0.150 , and p = 0.936 , resp . ) . the molecular weight of the phasix mesh decreased significantly over time with molecular weights of 240 , 510 2 , 018 da , 204 , 282 4 , 457 da , 118 , 884 2 , 821 da , and 44 , 434 879 da at 6 , 12 , 26 , and 52 weeks , respectively . molecular weight decreased significantly at each time point compared to the preimplantation ( i.e. , 0 weeks ) molecular weight of the phasix mesh ( 300 , 397 972 da ) with p < 0.001 in all cases . furthermore , the molecular weight of the phasix mesh decreased progressively between 0 and 6 weeks , between 6 and 12 weeks , between 12 and 26 weeks , between 26 and 52 weeks , and overall between 0 and 52 weeks with p < 0.001 in all cases . similarly , the molecular weight of the p4hb plug decreased significantly over time with molecular weights of 242 , 190 1 , 259 da , 208 , 288 3 , 420 da , 124 , 126 3 , 219 da , and 44 , 148 644 da at 6 , 12 , 26 , and 52 weeks , respectively . molecular weight decreased significantly at each time point compared to the preimplantation ( i.e. , 0 weeks ) molecular weight of the p4hb plug ( 301 , 543 110 da ) with p < 0.001 in all cases . furthermore , the molecular weight of the p4hb plug decreased progressively between 0 and 6 weeks , between 6 and 12 weeks , between 12 and 26 weeks , between 26 and 52 weeks , and overall between 0 and 52 weeks with p < 0.001 in all cases . as shown in table 2 , median scores for inflammation were reported in the range of 2 - 3 for both the phasix mesh and the p4hb plug at the early 6- and 12-week time points , and both consistently scored 2 at the later 26- and 52-week time points . no significant differences in inflammation scores were detected over time for either the phasix mesh or the p4hb plug , and no significant differences in inflammation scores were detected between the mesh and plug designs at any of the time points ( p > 0.05 in all cases ) . macrophage scores of 2 were consistently reported for the phasix mesh at all time points with no significant changes over time ( p > 0.05 ) . interestingly , macrophage scores for the p4hb plug varied slightly over time with a median score of 3 at the 6-week time point , trending toward a significantly lower score of 1 at the 52-week time point ( p < 0.05 ) . again , no significant differences were identified between the mesh and the plug designs at any of the time points evaluated ( p > 0.05 in all cases ) . giant cell scores of 1 were consistently observed for both the phasix mesh and the p4hb plug at all of the time points . no significant differences in giant cell scores were detected over time for either the phasix mesh or the p4hb plug , and no significant differences in giant cells scores were detected between the mesh and plug designs at any of the time points ( p > 0.05 in all cases ) . fibrosis / encapsulation scores for the phasix mesh varied slightly over time with a score of 4 at the 6-week time point that trended toward a significantly lower fibrosis / encapsulation score of 2 at the 12- and 52- week time points ( p < 0.05 ) . no significant changes in fibrosis / encapsulation scores were observed for the p4hb plug with scores of 2 - 3 observed at all time points . in addition , no significant differences in fibrosis / encapsulation scores were detected between the mesh and plug designs at any of the time points . granulation tissue / vascularization scores ranged from 2 - 3 for both the phasix mesh and the p4hb plug at all time points evaluated . the phasix mesh scores alternated between 3 and 2 at 6 and 12 weeks and again at 26 and 52 weeks , respectively , while the p4hb plug scores were 2 at the early 6- and 12-week time points and 3 at the later 26- and 52-week time points . however , no significant differences in granulation tissue / vascularization scores were detected over time for either the phasix mesh or the p4hb plug , and no significant differences were detected between the mesh and plug designs at any of the time points ( p > 0.05 in all cases ) . collagen morphology scores of 2 were consistently reported for the phasix mesh at all time points with no significant changes over time ( p > 0.05 ) . similarly , collagen morphology scores for the p4hb plug were 1 at the early 6-week time point and 2 at all subsequent time points . as with the mesh design , no significant changes in collagen morphology were detected over time for the plug design ( p > 0.05 ) , and no significant differences were detected between the mesh and plug designs at any of the time points ( p > 0.05 in all cases ) . poly-4-hydroxybutyrate ( p4hb ) is a naturally derived , fully absorbable polymer produced by bacteria via transgenic fermentation techniques . this material has been manufactured into a number of configurations including the phasix mesh and p4hb plug , which are designed for soft tissue repair applications such as hernia repair . over the last decade , p4hb materials have been evaluated in a number of animal studies , particularly those investigating cardiovascular applications such as tissue engineered trileaflet heart valves , artery augmentation patches , and small diameter vascular grafts , as well as development of p4hb as a suture material . however , the current study is the first of its kind to evaluate this material specifically for hernia repair applications . the objective of this study was to determine the mechanical properties , resorption profile , and histological characteristics of phasix mesh and p4hb plug designs when utilized to bridge a surgical defect in the porcine abdominal wall over a period of 6 , 12 , 26 , or 52 weeks . to accomplish this , abdominal wall sites repaired with phasix mesh and p4hb plug materials were harvested at the end of each implantation period and subjected to mechanical testing , gel permeation chromatography , and histological analysis to assess the strength of the repair , the amount of p4hb material remaining at the repair site , and the host response to the p4hb material , respectively . mechanical testing revealed that the burst strength and relative stiffness of the phasix mesh and p4hb plug repaired sites were similar to each other at all four time points evaluated with no significant differences detected between the two devices for either of these parameters throughout the course of the study ( p > 0.05 in all cases ) . in addition , the sites repaired with phasix mesh and p4hb plug materials also exhibited significantly greater burst strength and relative stiffness compared to the native abdominal wall at all time points regardless of which device was utilized to bridge the defect ( p < 0.05 in all cases ) . these results indicate that the p4hb material is capable of augmenting the strength of the native porcine abdominal wall regardless of its configuration as a mesh or plug design . it should also be noted that burst strength and relative stiffness of the phasix mesh and p4hb plug repaired sites remained stable and did not change significantly between 6 and 52 weeks , suggesting that scaffolds derived from the p4hb material are durable and capable of maintaining support at the repair site over a 52-week period in a porcine model . gel permeation chromatography was conducted to quantify the molecular weight of the p4hb material remaining at the repair site at each time point . the data showed that phasix mesh and p4hb plug possessed similar molecular weights at all time points with no significant differences detected between the two devices at any time ( p > 0.05 in all cases ) . these results correspond with the mechanical testing data and demonstrate that the configuration of the p4hb material does not significantly impact its resorption profile . in addition , the molecular weight of both the phasix mesh and the p4hb plug decreased significantly at each time point compared to the corresponding preimplantation ( i.e. , 0 weeks ) value and also decreased progressively between each time point ( i.e. , between 0 and 6 weeks , between 6 and 12 weeks , between 12 and 26 weeks , between 26 and 52 weeks , and overall between 0 and 52 weeks ) . these results indicate that the p4hb material was significantly resorbed over time , but as indicated by the mechanical testing data , significant material resorption did not correspond with a significant drop in mechanical strength at the repair site for either the phasix mesh or the p4hb plug design . in addition to mechanical testing and gel permeation chromatography , histological analyses were also performed in order to assess the host response to the p4hb material comprising the phasix mesh and the p4hb plug designs . no statistically significant changes were observed over time for either the phasix mesh or the p4hb plug with respect to histological parameters such as inflammation , granulation tissue / vascularization , giant cells , or collagen morphology . inflammation scores and granulation tissue / vascularization scores ranged from 2 - 3 for both the mesh and the plug design indicating a mild / slightly detectable to moderate / easily detectable inflammatory response and presence of granulation tissue and vascularization for both devices at all time points . in addition , giant cells scores were consistently reported as 1 , indicating a minimal / barely detectable presence of giant cells for both devices at all time points . similarly , collagen morphology scores were consistently reported as 2 , indicating a mixture of both mature ( type i , red and orange - red ) and immature ( type iii , green and yellow - green ) collagen at all time points . it should be noted that a few differences were observed with respect to time of implantation . for instance fibrosis / encapsulation scores for the phasix mesh varied slightly over time with a score of 4 at the 6-week time point that trended toward a significantly lower fibrosis / encapsulation score of 2 at the 12- and 52- week time points ( p < 0.05 ) . these scores correspond to marked / very evident fibrosis / encapsulation at the 6-week time point that was ultimately improved to mild / slightly detectable fibrosis / encapsulation at the later time points . some degree of fibrosis / encapsulation is expected in the presence of an implanted material such as the phasix mesh , and this response is expected to vary over time with the tissue remodeling / wound healing process . similarly , the macrophage scores for the p4hb plug varied slightly over time with a score of 3 at the 6-week time point , trending toward a significantly lower score of 1 at the 52-week time point ( p < 0.05 ) . these scores correspond to moderate / easily detectable presence of macrophages at the 6-week time point that ultimately improved to only minimal / barely detectable presence of macrophages at the 52-week time point . in general , no statistically significant differences were detected between the histological scores reported for the phasix mesh design versus the p4hb plug design for any of the parameters evaluated . these results suggest that the host tissue response was most influenced by the chemical composition of the device ( i.e. , poly-4-hydroxybutyrate ) rather than the structure of the device ( i.e. , mesh versus plug ) . inflammatory infiltrates associated with both devices were predominantly mononuclear with an overall mild response commonly associated with the implantation of a foreign material . similarly , both devices resulted in comparable , mild to moderate granulation tissue / vascularization that is expected in association with wound healing and tissue remodeling , suggesting that the p4hb material performed appropriately in terms of overall foreign body response regardless of its configuration as a mesh or a plug . phasix mesh and p4hb plug provided durable scaffolds for soft tissue repair in a porcine model . both repairs demonstrated significant mechanical strength compared to native abdominal wall over a 52-week period , which remained elevated despite significant material resorption over time with no evidence of herniation and/or diastasis . in addition , histological assessment revealed a comparable and mild inflammatory response and mild to moderate granulation tissue / vascularization associated with the p4hb material regardless of its configuration as a mesh or a plug .

purpose . poly-4-hydroxybutyrate ( p4hb ) is a naturally derived , absorbable polymer . p4hb has been manufactured into phasix mesh and p4hb plug designs for soft tissue repair . the objective of this study was to evaluate mechanical strength , resorption properties , and histologic characteristics in a porcine model . methods . bilateral defects were created in the abdominal wall of n = 20 yucatan minipigs and repaired in a bridged fashion with phasix mesh or p4hb plug fixated with sorbafix or permanent suture , respectively . mechanical strength , resorption properties , and histologic characteristics were evaluated at 6 , 12 , 26 , and 52 weeks ( n = 5 each ) . results . phasix mesh and p4hb plug repairs exhibited similar burst strength , stiffness , and molecular weight at all time points , with no significant differences detected between the two devices ( p > 0.05 ) . phasix mesh and p4hb plug repairs also demonstrated significantly greater burst strength and stiffness than native abdominal wall at all time points ( p < 0.05 ) , and material resorption increased significantly over time ( p < 0.001 ) . inflammatory infiltrates were mononuclear , and both devices exhibited mild to moderate granulation tissue / vascularization . conclusions . phasix mesh and p4hb plug demonstrated significant mechanical strength compared to native abdominal wall , despite significant material resorption over time . histological assessment revealed a comparable mild inflammatory response and mild to moderate granulation tissue / vascularization .

pubmed

glycopeptide resistance was detected in enterococci more than 25 years ago , ; since then , vancomycin - resistant enterococci ( vre ) have become important nosocomial pathogens associated with high mortality and costs , , , . glycopeptide resistance in enterococci is mediated by van genes , with vana and vanb being the most prevalent . several european countries have reported a rising incidence of vre in recent years , , . in denmark , a dramatic increase since 2012 has been due to multiclonal outbreaks of vana - positive enterococcus faecium , . it has been estimated that for every patient with a vre infection detected in a clinical sample , up to ten patients are intestinal carriers of vre . therefore , current guidelines recommend screening of patients at risk and implementation of isolation precautions for vre carriers , . screening relies on selective culturing and/or detection of the resistance genes vana or vanb by pcr . however , substantial indirect savings can be achieved by rapidly available results , as highlighted by a report from france , which compared the management of two simultaneous vre outbreaks on two different wards . several studies have examined the diagnostic accuracy of various assays to detect vana and vanb with or without a previous enrichment step , , , , , , , , . however , sensitivities ranging from 43.5% to 100% and specificities from 79.2% to 99.6% have been reported . in contrast , the specificity of the detection of the vanb gene is considerably lower , , , which is attributed to the presence of vanb - positive anaerobic bacteria , . in our laboratory , we have used a culture - based method using overnight enrichment and subsequent plating on chromogenic agar to screen for vre . in autumn 2014 , prompted by long - lasting vre outbreaks , we implemented the xpert vana / vanb assay performed directly on rectal swabs . this fully automated system integrating dna extraction and real - time pcr provides results within an hour . we used the vana part of the assay to guide rapid implementation of infection control measures . here , we evaluate the diagnostic accuracy of the xpert vana / vanb assay and the impact on infection control decision making . our laboratory serves the primary and secondary sectors of a population of 800 000 inhabitants in the northern part of the capital region of denmark , including four university hospitals with a total number of 1577 beds and covering all internal medicine subspecialties and three intensive care units . the infection control units at these four hospitals are part of the clinical microbiology department and employ six infection control nurses . diagnosis of vre in a clinical sample results in roommate screening and isolation precautions are implemented until results are available . if any roommate is found positive , an outbreak is declared and all patients in the ward are screened weekly until no new patient is diagnosed with vre . during weekly screenings , isolation precautions are implemented as soon as a positive vre result is available . for roommate screening and weekly screening of wards , the routine culture - based screening is supplemented by vana pcr . before transfer of patients from an outbreak ward , pcr is performed and the implementation of isolation precautions is based on the pcr result . for vre - positive patients readmitted within 6 months after the last positive screening sample , isolation precautions are implemented at admission independently from screening results . in the case of readmission > 6 months after the last positive sample , screening is advised and the implementation of isolation precautions is individualized based on screening results and risk factors . in these cases , for phenotypic screening , eswab transport medium was transferred to an enrichment broth ( brain heart infusion supplemented with 4 mg / l vancomycin and 60 mg / l aztreonam ) either by shaking the flocked swab several times in the enrichment medium or by pipetting 100 l . after overnight incubation , 100 l of the enrichment medium was plated on selective chromogenic agar plates ( chromagartm vre , chromagar , paris , france ) and read after 1 and 2 days of incubation . for vana pcr - positive , but culture - negative samples , the culture protocol was thoroughly repeated . species identification was performed by matrix - assisted laser desorption / ionization time - of - flight mass spectrometry ( biotyper , bruker , bremen , germany ) and a preliminary positive result was given if there was abundant growth of e. faecium on the selective chromogenic agar . susceptibility testing for vancomycin and teicoplanin was performed by the eucast disc diffusion method and determination of the mic used a gradient strip ( etest , biomrieux , solna , sweden ) to confirm the vana or vanb phenotype . pcr - based screening was undertaken by the automated system genexpert using the xpert vana / vanb assay ( cepheid ab , solna , sweden ) . one hundred microlitres of the eswab transport medium was added to the sample reagent and further handled as recommended by the manufacturer . in case of invalid or error results ( pcr inhibition or technical error ) , the assay was repeated once . only vana pcr results were reported to the requesting wards as a preliminary result and used for guidance of infection control measures . pcr was performed on the same day if the sample arrived in the laboratory before 16.00 h , all days of the week . sample arrival date , laboratory results , dates of preliminary and final results and sample indications were prospectively collected . for final analysis the impact of the vana pcr result on infection control measures was estimated by calculation of isolation days saved ( number of days from negative pcr result to final negative culture result ) and transmission risk days saved ( number of days from positive pcr result to preliminary or final positive culture result ) . the computer program , version 3.0.2 ( r foundation for statistical computing , vienna , austria ; http://www.r-project.org/ ) was used for statistical analysis . for comparison of categorical data , fisher s exact test was used . for comparison of numerical data , unpaired groups , sensitivity , specificity , and positive and negative predictive values with 95% ci were calculated using the r package dtcompair . 3 - 3013 - 1217/1/ ) and the danish data protection agency ( record no . 2012 - 58 - 0004 ; heh-2015 - 066 ) . from 30 december 2014 to 15 august 2015 , 1110 vre screening samples from 804 patients were examined both by the culture - based method and by pcr . the samples were from 24 different departments from four different hospitals . in 1067 ( 96.1% ) samples , pcr was performed and reported the same day , the sample was received in the laboratory . the median turnaround time for culture results was 3 days ( mean 3.2 ; range 39 days ) . in 35 ( 3.2% ) samples , invalid or error results were obtained after the first pcr analysis and for 11 ( 1.0% ) samples , repeated analysis did not give a result either . of the 1099 samples where a pcr result was obtained , 152 ( 13.8% ) were positive for the vana gene and 167 ( 15.2% ) were culture - positive for an enterococcus species ( all e. faecium ) with the vana phenotype ( vancomycin - resistant with mic 256 mg / l and teicoplanin - resistant with mic 8256 mg the vana pcr results in relation to culture results are given in table 1 . the vanb pcr was positive in 247 ( 22.5% ) samples , but only one was culture - positive for enterococcus sp . ( enterococcus faecalis ) with the vanb phenotype ( vancomycin - resistant with mic 128 mg / l and teicoplanin - susceptible with mic 0.5 discrepant results between culture and vana pcr were further investigated . in 12 of the 22 pcr - negative samples , from which a vana phenotype e. faecium was cultured , pcr was repeated on the eswab transport medium and two were found positive with high circles of threshold ( ct ) of 36.9 and 38.2 . pcr was also performed on the isolates from the 20 remaining samples and they were all vana pcr - positive . of the 145 culture - positive samples that had been vana pcr - positive , most ( n = 135 ) were culture positive after 1 day of incubation of the chromogenic agar , whereas the remaining ten samples ( 6.9% ) were only positive after 2 days of incubation . in comparison , eight of the 22 culture - positive samples that had been pcr - negative ( 36.4% ) only turned positive on the second day of incubation ( fisher 's exact test , p < 0.001 ) . on the other hand , ct values of the culture - negative sample were significantly higher ( median 32.9 ; range 24.138.3 ) than ct values of the culture - positive samples ( median 22.9 ; range 12.837.8 ) ( wilcoxon rank sum test ; p < 0.001 ) . to calculate the test accuracy of the xpert vana / vanb assay , culture results complemented with information from the patient s earlier and later samples were used as a reference standard . for the analysis of vana pcr , four of seven true positives : three samples from the same patient from which phenotypically vancomycin - susceptible ( mic 1 mg / l ) , but vana - positive e. faecium was isolated by non - selective culturing ( unpublished data , holzknecht bj ) and one sample taken under linezolid therapy from a patient with vre culture - positive samples 15 days earlier and 28 days later . the sensitivity of the vana pcr assay was 87.1% ( 95% ci 82.1%92.2% ) , the specificity was 99.7% ( 95% ci 99.3%100% ) and the positive and negative predictive values were 98.0% ( 95% ci 95.8%100% ) and 97.7% ( 95% ci 96.7%98.6% ) , respectively . the specificity and positive predictive value of the vanb part of the assay were low at 77.6% ( 95% ci 75.1%80.1% ) and 0.4% ( 95% ci 0.00%1.2% ) , respectively . the majority of the samples ( n = 1033 ; 93.1% ) were taken as weekly screening samples from 12 different outbreaks during the study period . thirty - seven ( 3.3% ) were roommate screening samples , 14 ( 1.3% ) were taken at readmission , 13 ( 1.2% ) at transferral from outbreak wards and 13 ( 1.2% ) for other indications ( fig . 1 ) . a total of 141 isolation days could be saved as a consequence of faster available negative pcr results . the saving of time due to rapidly available positive pcr results corresponded to a total of 292 saved transmission risk days . on the other hand , false - negative or false - positive pcr resulted in six additional transmission risk days and 13 additional isolation days ( fig . 1 ) . this study describes the performance and impact of the xpert vana / vanb assay , when performed directly on rectal swabs , in the setting of vana e. faecium outbreaks . of the 1099 samples with available pcr and culture results , only 29 showed discordant results : seven were vana pcr - positive , but culture - negative and 22 were culture - positive , but vana pcr - negative . vancomycin - resistant isolates cultured from samples that had been pcr - negative , were vana pcr - positive , which confirms that false - negative pcr results , when performed directly on the sample , were not due to alterations of the primer binding sites . ct - values of vana pcr - positive , culture - negative samples were significantly higher than ct - values in culture - positive samples . likewise , the culture - positive , but vana pcr - negative samples were significantly more often positive after 2 days of incubation of the chromogenic plate than pcr - positive samples , which might also indicate a lower bacteria count . in the implementation phase of the xpert vana / vanb assay , investigation of vana pcr - positive , culture - negative samples had actually led to a change in the culture protocol from plating 10 l to 100 l of enrichment broth on the chromogenic plates to increase sensitivity ( data not shown ) . using culture results complemented with information from patients follow - up samples as a reference standard , the diagnostic accuracy of the vana part of the xpert vana / vanb assay was excellent and in accordance with previous studies , , . however , one previous , but far smaller , study found both a lower sensitivity and a lower specificity of the assay . as there was only one sample that was culture - positive for vanb - positive vre , we could not fully analyse the diagnostic accuracy of the vanb part of the assay . however , the large proportion ( 22.3% ) of false - positive vanb pcr results is in accordance with previous studies , , , , . first , investigation of vana pcr - positive , culture - negative samples from the same patient led to the isolation of phenotypically vancomycin - susceptible , but vana pcr - positive e. faecium . an outbreak with a similar strain has been described and infection control management similar to vre seems prudent , . the strengths of the diagnostic accuracy study are the large sample size and the prospective and thorough comparison of the xpert vana / vanb assay with the selective culture method , including further investigation of discordant samples by follow - up samples to define a robust reference standard . however , it has been shown that several outbreak clones and multilocus sequence types are circulating in our region . a systematic study of genetically diverse strains would though be necessary to finally conclude on the generalizability of our results . using the xpert vana / vanb assay , 96.1% of screening results were available within 1 day , whereas the median turnaround time for selective culturing was 3 days . based on the time interval between available pcr and culture results , we calculated saved isolation days and saved transmission risk days as a consequence of earlier suspension or implementation of isolation measures . however , these numbers might be overestimated , as patients might have been isolated for other reasons or dismissed from the hospital before results were available . besides saved transmission risk days , earlier available positive screening results from roommates also led to faster outbreak declaration and implementation of additional infection control measures such as screening of all patients , enhanced cleaning , enforcement of hand hygiene and staff teaching . we have seen a faster control of vre outbreaks after the implementation of pcr - based screening . however , as this is part of a bundle approach we can not determine the isolated value of the pcr assay ( mogensen and midttun , infection prevention and control canada 2016 national education conference , poster 84 ) . detailed health economics calculations are beyond the scope of this study . however , considering the assay s official price in denmark of 661 dkk ( approx . 89 ) per sample and estimated additional costs per isolation day of 3350 ddk ( approx . 451 ) , we feel that saving 141 isolation days and 292 transmission risk days as a result of 1110 pcr analyses is a considerable impact and worth the costs . this is more important when the psychosocial consequences of a vre outbreak situation and isolation precautions for patients and staff members are also taken into account . the vana pcr of the xpert vana / vanb assay had a high diagnostic accuracy and considerable impact on infection control decision making in our setting . ln received non - financial support from roche diagnostics a / s and from msd , outside the submitted work . bjh contributed to conception and design of the study , acquisition and analysis of data and drafting of the manuscript . dsh and joj contributed to conception and design of the study and critical revision of the manuscript . ak contributed to conception of the study , acquisition of data and critical revision of the manuscript .

vancomycin - resistant enterococci ( vre ) are increasingly important nosocomial pathogens and screening for colonization status is a mainstay in infection control . we implemented pcr - based screening during vana - positive enterococcus faecium outbreaks in four university hospitals in copenhagen , denmark . xpertvana / vanb was performed directly on rectal swabs and the vana pcr result was used to guide infection control measures . concurrently , all samples were selectively cultured including an overnight enrichment step . diagnostic accuracy was calculated as well as turnaround time and the impact of the earlier available pcr results on infection control decision making . in all , 1110 samples were analysed . the vana pcr positivity rate was 13.8% and culture positivity rate was 15.2% . the diagnostic accuracy of the vana part of the assay was high with a sensitivity of 87.1% , a specificity of 99.7% , and positive and negative predictive values of 98.0% and 97.7% , respectively . the vanb pcr had a considerably lower specificity of 77.6% and a positive predictive value of 0.4% . in 1067 ( 96.1% ) samples , pcr results were reported within 1 day , whereas median culture turnaround time was 3 days . the saving of time to available results corresponded to 141 saved isolation days and 292 saved transmission risk days . false - negative or false - positive pcr results led to six additional transmission risk days and 13 additional isolation days , respectively.the vana pcr had high diagnostic accuracy and the prompt availability of results gave a considerable benefit for infection control decision making .

pubmed

in addition to the circulatory renin - angiotensin - aldosterone system ( raas ) , there is now a significant body of evidence supporting the concept of a local tissue or cellular raas that has important roles in the pathology of cardiovascular diseases . the local production of aldosterone and the discovery of mineralocorticoid receptor ( mr ) expression in the heart have led to a greater understanding of the role of aldosterone / mineralocorticoid receptor activation in the cardiovascular diseases , including hypertension and heart failure [ 2 , 3 ] . aldosterone activates its mineralocorticoid receptor ( mr ) in the nondiabetic heart and can cause structural and electrical remodelling , fibrosis , oxidative stress , inflammation , and arrhythmias [ 1 , 47 ] . mr antagonists have shown significant benefit in patients with left ventricular dysfunction and myocardial infarction . for example , the recent eplerenone in mild patients hospitalization and survival study in heart failure ( emphasis - hf ) study has shown that eplerenone , an mr antagonist , has beneficial effects in patients with moderate heart failure ( nyha class ii ) . however , beneficial effects of mr blockade in pathological states such as diabetes are unclear . it is well established that signaling network alterations in diabetes are such that potential therapies will need to be tailored for this pathological state [ 1012 ] . inhibitors of the renin - angiotensin - aldosterone system ( raas ) , such as angiotensin converting enzyme inhibitors ( aceis ) and angiotensin type-1 receptor ( at1 ) blockers ( arbs ) , have been shown to protect against hypertension and/or diabetes - induced end - organ damage [ 13 , 14 ] . for example , in a group of 99 patients with severe heart failure , van de wal et al . demonstrated that 45% had elevated plasma angiotensin ii ( ang ii ) levels independent of serum ace activity despite long - term ace inhibitor use . acei and/or arbs do not completely block end - organ damage in diabetes and/or hypertension , and clinical trials of acei and arbs in combination have generally shown that they do not offer added benefits but rather lead to greater adverse effects such as electrolyte imbalance and renal complications [ 1 , 15 ] . in addition , inhibition of ang ii does not reliably suppress aldosterone production , with the aldosterone escape phenomenon occurring in up to 40% of patients with heart failure [ 17 , 18 ] . thus , the need for additional raas inhibition in these individuals would be a logical consequence such as combinations of acei and/or arbs with mr antagonist . the randomized aldactone evaluation study ( rales ) and the eplerenone postacute myocardial infarction heart failure efficacy and survival study ( ephesus ) suggested that an mr antagonist on top of an acei or an arb can reduce mortality in patients with severe congestive heart failure and left ventricular dysfunction after mi [ 15 , 18 ] . thus , there is an important need to identify the best possible treatment option for normal and diabetic patients with cardiac dysfunction . important issues that still need to be addressed include identifying the optimal combination of drugs to use and their timing , either administration before or after ischemic injury , in both normal and diabetic states . hence , the aim of the present study was to characterize and compare the effects of ru28318 ( ru , a selective mr antagonist ) , captopril ( capt , an acei ) , and losartan ( los , an arb ) , alone or in double and triple therapy combinations administered either before or after ischemia on ischemia / reperfusion- ( i / r- ) induced cardiac dysfunction in isolated hearts obtained from normal and diabetic rats . 17-week - old male wistar rats were divided into 2 groups ( n = 6 per studied group ) . group 1 was control animals and group 2 was streptozotocin- ( stz- ) treated diabetic animals . all animal experiments in this study were approved by the research administration at kuwait university and conformed to their ethics guidelines for the care and use of laboratory animals that are based on those published by the us national institute of health ( nih publication no . diabetes was induced by a single intraperitoneal injection of 55 mg / kg body weight stz . basal glucose levels were determined prior to stz injection and 48 h after stz injection . rats with a blood glucose concentration above 250 mg / dl were declared diabetic and any not meeting this criterion were excluded from the study . the animals ' diabetic state was re - assessed after 4 weeks just before sacrificing the animals . rats were anesthetized with intraval sodium ( 40 mg / kg body weight ) , and hearts were rapidly removed after intravenous heparinization ( 1000 u / kg body weight ) . the excised hearts were immediately mounted on the langendorff perfusion assembly ( hugo sachs electronics , freiburg , germany ) and were perfused initially with a constant pressure perfusion of 50 mmhg with the oxygenated ( 95% o2 + 5% co2 ) krebs - henselit buffer ( 37c ) of the following composition ( in mm ) : nacl 117 ; kcl 4.39 ; cacl2 2.5 ; nahco3 20.0 ; kh2po4 1.21 ; mgcl26h2o 1.2 ; glucose 12.0 ; osmolarity 300 mosm / l , ph 7.35 . a water - filled balloon was introduced into the left ventricle and connected to a statham pressure transducer ( p23db ) and balloon volume was adjusted to give the baseline end - diastolic pressure of 5 mmhg . left ventricular developed pressure ( pmax ) and its positive and negative derivatives ( + dp / dt and dp / dt , resp . ) and left ventricular end - diastolic pressure ( lvedp ) were continuously monitored . coronary flow ( cf ) was measured by means of an electromagnetic flow probe positioned in the inflow tubing immediately above the aortic perfusion cannula . this system permits accurate adjustment of perfusion pressure between 5 and 300 mmhg to an accuracy of 1 mmhg . hearts removed from animals were perfused for 30 min and then subjected to 30 min of global ischemia ( i ) followed by a period of 30 min of reperfusion ( r ) . drugs were administered for 30 min either during perfusion before ischemia or during reperfusion after ischemia . drug regimens tested were ru28318 ( ru ; 10 m ) , captopril ( capt ; 3.6 10 m ) , losartan ( los ; 3 10 m ) , ru + capt , ru + los , capt + los , and ru + capt + los ( triple ) . the doses used gave maximal responses in preliminary experiments and were similar to those used in previous studies [ 2123 ] . percent recovery ( % r ) was calculated using the following formula : ( reperfusion value)/(baseline value ) 100 . the baseline value is the value recorded at 30 min perfusion before exposure to global ischemia . mean values were compared using analysis of variance followed by bonferroni 's post hoc test . hyperglycaemia persisted throughout the study period and was 306 18 mg / dl at four weeks in stz - treated animals compared to 96 4 mg / dl in the nondiabetic ( control ) animals . there were no significant differences in body ( 275 17 gm ) and heart weights ( 0.95 0.03 gm ) corrected to tibia length in these animals . the effects of various acute drug treatments ( ru , capt , los , capt + los , capt + ru , and los + ru , capt + los + ru ) administered during perfusion ( before ischemia ) or during reperfusion ( after ischemia ) on recovery of each cardiac function parameter following i / r in normal and diabetic hearts were recorded . the percent ( % ) recovery in pmax , lvedp , + dp / dt , dp / dt , and cf as a function of reperfusion time for nondiabetic control hearts is shown in figure 1 ( before ischemia ) and figure 2 ( after ischemia ) and for diabetic hearts in figure 3 ( before ischemia ) , and figure 4 ( after ischemia ) . in general , drug treatments resulted in a very rapid rate of cardiac function recovery within the first 10 min of reperfusion followed by a more gradual rate of recovery ( figures 14 ) . ru resulted in the most rapid recovery in cf within the first 10 min of reperfusion for cf when given before ischemia and in + dp / dt and pmax when administered after ischemia in control hearts ( figures 1 and 2 ) . in diabetic hearts , ru , ru + los , and triple therapy similarly gave the most rapid recovery within 10 mins of reperfusion for lvedp when given before ischemia . interestingly , cap + los therapy given before ischemia in diabetic hearts resulted in a very rapid improvement in pmax and cf within the first 10 min of reperfusion that was almost the maximal recovery obtained ( figures 3(a ) and 3(e ) ) . ru + capt and triple therapy given before ischemia to diabetic hearts resulted in the most rapid recovery in + dp / dt . ru + los yielded the most rapid recovery in lvedp when given before ischemia in nondiabetic hearts . other significantly rapid recoveries in function were observed for + dp / dt for capt alone given after ischemia and for dp / dt when ru was given after ischemia in diabetic hearts ( figures 4(c ) and 4(d ) ) . in untreated controls , % recovery ( % r ) of pmax at the end of the 30 min reperfusion period was around 50% . administration of ru either before ischemia ( during perfusion ) or after ischemia ( during reperfusion ) led to a similar but significant improvement in pmax of between 14 and 18% ( see figure 5(a ) ) . of the individual ( single ) drug treatments administered before ischemia , the improvement ( % change in function relative to untreated controls ) in pmax with ru was the highest compared to capt alone ( 8% ) , whereas los showed no significant improvement ( figure 5(a ) ) . however , when single drugs were administered after ischemia , the % improvement in pmax was the highest for capt at around 25% , and again the least improvement was seen with los at around 14% . when drugs were administered as combinations , triple therapy ( capt + los + ru ) yielded the best recovery in pmax of about 74%an improvement over controls of 48%irrespective of whether it was administered before or after ischemia ( figure 5 ) . of the double combinations , only capt + ru was significantly ( p < 0.05 ) better over either of the single agents alone when administered either before or after ischemia ( figure 5 ) . recovery in pmax of diabetic hearts ( % r of 9 3 mmhg ) from i / r was significantly ( p < 0.05 ) impaired compared to nondiabetic controls ( % r of 50 2 mmhg ) . administration of all drug treatments to diabetic hearts before ischemia led to significant improvement in pmax of between 122 and 222% ( see figure 5(c ) ) . again in general , the triple therapy appeared to be better than double therapies which appeared to be better than single therapies with some notable exceptions . capt + los was equally as effective as the triple combination , and capt + ru double combination was not significantly ( p < 0.05 ) better than any of the single therapies ( figure 5 ) . however , when single drugs were administered after ischemia in diabetic hearts , interestingly , there was no significant improvement in pmax with ru ( figure 5(d ) ) . this is in marked contrast to what was observed in the nondiabetic controls ( figures 5(a ) and 5(b ) ) . however , all other drug treatments gave comparable improvements in pmax with capt + los being the best treatment option ( figures 5(c ) and 5(d ) ) . % r in lvedp of around 250% was observed in nondiabetic controls that could be significantly reduced ( p < 0.05 ) ( i.e. , improved ) by all drug treatments ( figures 6(a ) and 6(b ) ) . in the case of single therapies for control ( nondiabetic ) hearts , acute treatment with capt gave the best improvement in lvedp when given before ischemia ( figure 6(a ) ) and was joint best with ru when given after ischemia ( figure 6(b ) ) . double therapies gave no advantage in lvedp improvement over single therapies , whereas triple therapy ( capt + los + ru ) yielded the best recovery in lvedp of about of 27% and 38% , respectively , when given before or after ischemia ( figures 6(a ) and 6(b ) ) . capt given before ischemia gave similar improvement of about 23% to the triple therapy ( i.e. , % improvement values were not significantly different ; p < 0.05 ; see figure 6(a ) ) . in diabetes , lvedp was significantly ( p < 0.05 ) elevated compared to nondiabetic control hearts by over 3-fold . capt + los double therapy yielded the best improvement of around 30% in lvedp when given either before or after ischemia ( figures 6(c ) and 6(d ) ) . capt alone was equally effective as capt + los when administered after ischemia ( figure 6(d ) ) . in the case of lvedp , all other therapies gave similar % improvements in function ( figures 6(c ) and 6(d ) ) . interestingly , los given alone in diabetic hearts was relatively more effective at improving lvedp than in nondiabetic control hearts ( figures 5(a ) and 5(b ) ; figures 6(c ) and 6(d ) ) . in contrast to pmax where greater improvements in function were observed for diabetic hearts ( figure 5 ) , in the case of lvedp , drug - induced improvements appeared similar for diabetes and controls when presented as % change ( figure 6 ) . the reasons for this apparent anomaly are that in diabetes lvedp significantly ( p < 0.03 ) increases by about 3-fold , and thus in calculating a % change , the denominator is now a large number and thus yields modest changes as a percentage . however , when considering the actual numerical changes induced by the different drug treatments in terms of % r for lvedp , this suggests that the degree of change induced by drugs was considerably higher ( about 25 fold ) in diabetic hearts compared to control hearts . in untreated controls , % recovery of + dp / dt following i / r was around 47% and similar to that observed for pmax ( figure 5 ) . drug treatments significantly ( p < 0.02 ) improved function when administered either before or after ischemia ( figures 7(a ) and 7(b ) ) . in general , triple therapy ( around 60% improvement ) was better than double therapy which in turn was more effective than single therapies in improving recovery of + dp / dt ( figures 7(a ) and 7(b ) ) . of the single therapies , los appeared to be the least effective in improving + dp / dt ( figures 7(a ) and 7(b ) ) . ru was the most effective when given before ischemia whereas capt was the best when given after ischemia in improving + dp / dt ( figures 7(a ) and 7(b ) ) . diabetes ( 8 2% ) led to about a 6-fold reduction in % r for + dp / dt as compared to controls ( 47 2% ) . drug treatments generally led to marked and significant improvement in function . when given before ischemia , drugs gave similar improvements in function at the end of perfusion , but triple therapy as well as ru + capt gave the most rapid improvement within the first 10 min of perfusion . in the case of capt + los , approximately 250% improvement was attained which was similar to that obtained with triple therapy when given before ischemia . when given after ischemia , surprisingly capt alone ( approx 300% improvement ) was the best therapy ( figure 7 ) . all drug treatments generally showed greater % improvement in diabetes compared to controls where less than 60% improvement in + dp / dt was noted ( figure 7 ) . in untreated controls , % recovery ( % r ) of drug treatments significantly ( p < 0.05 ) improved dp / dt by about 40100% when administered either before or after ischemia ( figures 8(a ) and 8(b ) ) . all the single therapies when administered before ischemia gave similar improvements in recovery of dp / dt ( figure 8(a ) ) . with the exception of los + ru , double therapies were better than single therapies and showed similar effectiveness as the triple therapy ( figure 8(a ) ) . when drugs were administered after ischemia , with the exception of los alone which exhibited the least improvement of about 40% , all other treatment regimens were similarly effective exhibiting improvements in the range 6080% ( figure 8(b ) ) . diabetes ( 11 3% ) led to a about a 3.4-fold reduction in % r for dp / dt as compared to controls ( 37 2% ) . drug treatments in diabetic hearts generally led to marked and significant improvement in function from about 100% to 200% and this was generally higher compared to that seen following drug treatments in control hearts ( see figures 8(c ) and 8(d ) ) . all drug treatments were equally effective in improving dp / dt when administered after ischemia ( figure 8(c ) ) , whereas combination therapies was more effective than single therapies when administered before ischemia ( figure 8(d ) ) . administration of capt + ru before ischemia and ru alone after ischemia were the most effective treatment in improving dp / dt in diabetic hearts ( figure 8(c ) ) . in untreated controls , % recovery ( % r ) of cf following i / r was around 43% . drug treatments significantly ( p < 0.05 ) improved function by about 2060% when administered either before or after ischemia ( figures 9(a ) and 9(b ) ) . when drugs were administered before ischemia , ru gave the most rapid recovery within the first 10 mins of reperfusion . triple therapy was generally better than double therapies which in turn were more effective than single agents ( figure 9(a ) ) . triple therapy ( 60% improvement ) was also more effective when administered after ischemia , and with the exception of los alone ( around 10% improvement ) , all other treatments yielded similar improvements in the range 3040% ; see figures 9(a ) and 9(b ) . diabetes ( 12 3% ) led to about a 3.8-fold reduction in % r for cf as compared to controls ( 46 3% ) . drug treatments in diabetic hearts generally led to marked and significant improvement in function from about 100% to 200% and this was generally higher ( figures 9(c ) and 9(d ) ) , compared to that seen following drug treatments in control hearts ( figures 9(a ) and 9(b ) ) . all other drug treatments were equally effective in improving cf when administered before ischemia except for capt + los which was the best treatment ( about 200% improvement ) ; see figures 3(c ) and 9(c ) . given after ischemia , capt , los , capt + los , and triple therapy were similarly effective in improving cf in diabetic hearts , whereas ru alone was the least effective ( figure 9(d ) ) . a major goal of this study was to identify the optimal treatment strategy of raas blockade to prevent or treat ischemia - reperfusion injury in normal and diabetic hearts [ 2426 ] . although the use of acei or arbs has been well studied , the effects of aldosterone antagonism alone or in combination with these other raas blockers especially in diabetes are not well understood . this study showed that treatment with ru was generally better in preventing or reversing ischemia - induced cardiac dysfunction in normal hearts compared to treatment with an acei ( capt ) or arb ( los ) alone . in the case of diabetic hearts , ru was generally similarly effective as capt or los treatment . also , dual therapies involving ru were similarly effective as capt + los therapy , whereas triple combination was generally equal to or the most effective strategy in preventing or reversing ischemia - induced cardiac dysfunction in normal or diabetic hearts . for example , irrespective of whether drugs were administered before or after ischemia , combination therapies appeared more effective , whereby triple > double > single therapy in most of the study scenarios ( i.e. , 18 out of the 20 experimental scenarios of looking at 5 different cardiac parameters studied for both normal and diabetic hearts for when drugs were given either before or after ischemia ) . in contrast to nondiabetic hearts , capt + los dual therapy was the most effective therapy ( alongside triple therapy ) in diabetic hearts which is consistent with the known diabetes - induced overactivity of the ace / ang ii / at1 signaling cascade [ 16 , 27 , 28 ] . in this study , looking at an mr antagonist in diabetic hearts , we show that indeed the diabetic pathology leads to altered cardiac response to treatments with ru as well as arbs and acei . in general , with the exception of pmax for ru given after ischemia , the relative improvement in cardiac function or effectiveness of raas blockers was greater in diabetes than in control hearts . thus , in nondiabetic hearts , ru alone was highly effective at preventing and treating i / r - induced cardiac dysfunction , whereas in diabetes , in terms of pmax , it was only effective at preventing ( i.e. , when given before ischemia ) i / r - induced cardiac injury . it was ineffective , in terms of pmax , when given after ischemia in diabetes implying a differential role of aldosterone mr signaling in the diabetic pathology . these data suggest that mr signaling is detrimental for all cardiac parameters in nondiabetic hearts during ischemia as well as reperfusion phases , whereas in the diabetic hearts , at least for pmax , it may be detrimental only during the ischemia - induced phase of i / r injury . the reasons for the differential response in pmax in diabetes are not understood but clearly require further study . indeed , the effectiveness of drugs generally appeared to be cardiac parameter specific with some drugs being better at improving one cardiac function parameter over others . for example , although ru was ineffective at improving pmax , it was the most effective at improving dp / dt when administered to diabetic hearts after ischemia . this may imply that ru may not improve systolic function but may show marked improvements in diastolic function in diabetic hearts when given after ischemia . this assertion is further supported by the fact that the ru significantly ( p < 0.03 ) improved lvedp in diabetic hearts ( figures 3 , 4 , and 6 ) . ru exhibited greater beneficial effects in lvedp for diabetic compared to control hearts when considering the actual numerical changes in terms of % r for lvedp . although ru - mediated improvement in cardiac function generally appears to be as good as arbs and aceis , differences exist between ru and other drugs as to effectiveness when given before or after ischemia . in nondiabetic hearts , ru was the best or one of the best single agents when given before or after ischemia , whereas in diabetes , it generally provided minimal or least benefit when given after ischemia with the exception of dp / dt where ru was the best treatment option . this finding may be clinically relevant as diabetes patients generally present with compromised diastolic function as an early indicator of cardiac dysfunction and is detected in about 75% of asymptomatic diabetic patients [ 29 , 30 ] . our results show that ru importantly improves diastolic function ( as measured by dp / dt ) in diabetes especially when given after ischemia and thus early - stage ru treatment may represent a novel strategy for treating diabetes - induced diastolic dysfunction . our study also showed that irrespective of whether drugs were administered before or after ischemia , their effectiveness in recovering cardiac function appeared to be time dependent ; typically , drugs rapidly improved cardiac function within the first 10 min of reperfusion followed by a period of steady improvement thereafter up to the 30 min study period ( figures 14 ) . however , there were some interesting differences as to which cardiac function parameters were improved during this initial rate of recovery . for example , significantly rapid recoveries in function were observed for + dp / dt for therapy with captopril alone when administered after ischemia and for dp / dt when ru was given after ischemia in diabetic hearts ( figures 4(c ) and 4(d ) ) . these data further imply that in patients with diabetes where diastolic function is often the only early indicator of cardiac dysfunction , these treatments offering rapid improvements in dp / dt should be considered as potential therapeutic options . the possible mechanisms by which ru may be acting beneficially in normal and diabetic hearts subjected to i / r are not known but these may include blockade of aldosterone - induced oxidative stress , endothelial dysfunction , and inflammation in the heart and in the coronary vasculature that generally contribute to abnormal calcium homeostasis and cardiac dysfunction . thus , ru - mediated blockade of these important processes likely also improves calcium handling and overload in hearts subjected to i / r . this assertion is supported by previous findings that aldosterone / mr activation induces cardiomyocyte ionic remodelling by modulating potassium and l - type calcium channel activity [ 31 , 32 ] , t - type calcium channel expression , and ryanodine receptor activity . changes in these calcium handling proteins have been reported to lead to important consequences in the control of calcium signaling , modulation of calcium transients , sarcoplasmic reticulum diastolic leaks , and promotion of rhythm disorders that can be corrected by aldosterone / mr antagonism [ 3537 ] . aldosterone is also known to induce increased na / h exchanger-1 ( nhe-1 ) activity via transactivation of the epidermal growth factor receptor ( egfr ) and subsequent reactive oxygen species ( ros ) formation which is thought to be an important signaling cascade in the genesis of many cardiac pathologies . hence , another possible mechanism by which mr antagonism with ru may be exerting its beneficial effects is via inhibition of the detrimental cardiac egfr / nhe-1 signaling network . however , we have recently shown that egfr signaling via pathways involving erk1/2 , p38 map kinase , and akt / foxo appears to be an important beneficial survival mechanism , and its inhibition leads to worsening of cardiac function following i / r . thus , targeting egfr inhibition with mr antagonists such as ru may have beneficial and detrimental consequences in the heart and thus their net therapeutic advantage will depend on the relative importance or contributions of these different egfr - driven mechanisms in a given pathological state . indeed , we have recently shown that by removing the egfr inhibitory effects of los , which blocks ang ii - mediated transactivation of egfr , by coadministering an egfr ligand , significant improvement in cardiac function over that achieved by los alone was attained . whether the beneficial effects of ru may be via antagonism of aldosterone - mediated effects in cardiac muscle and/or vasculature is not clear but recent studies suggest the involvement of both . it appears that cardiomyocyte aldosterone / mr participates in the crosstalk between cardiomyocytes and coronary blood vessels , such as increased aldosterone synthesis by the cardiomyocytes resulting in coronary dysfunction [ 1 , 41 ] . interestingly , signaling by egfr , the transactivation target of aldosterone , is known to be elevated in the diabetic vasculature and is detrimental to vascular function [ 40 , 4244 ] . thus , ru - mediated blockade of aldosterone - induced egfr transactivation may be beneficial in improving diabetes - induced vascular dysfunction in the heart but this requires further study . in diabetic patients , in addition to cardiac dysfunction , there is also significant risk of renal damage where ang ii blockade is contraindicated as it will lead to attenuation of ang ii - driven glomerular filtration rate ( gfr ) and renal shutdown particularly in patients with renal artery stenosis . however , ru which does not affect gfr may be particularly useful for such patients . further , addition of ru as a combination strategy with acei and arbs may allow for a reduction in the dose of the latter agents such that they have minimal disruption on gfr and electrolyte balance whilst retaining their ability to reduce diabetes - induced proteinuria and their beneficial effects on cardiac function as highlighted in the present study . importantly , mr antagonists when combined with acei but not arbs have shown significant reduction in total mortality in patients with chf . but here in i / r injury , we show generally that combination approaches are better where capt + los + ru ( triple ) therapy is mostly the best treatment option in normal and diabetes . however , combination therapies with acei and arbs are rarely employed in the clinic possibly due to fear over accumulating adverse effects in patients with heart disorders [ 15 , 20 , 46 ] . whether the inclusion of ru in the highly effective triple therapies described herein can lead to dose reduction of acei and/or arbs to minimize or eliminate these adverse effects requires further clinical study . in diabetic patients , therapies involving aldosterone / mr antagonists they may oppose aldosterone - mediated detrimental effects on structural and functional integrity of the pancreatic [ beta]-cell resulting from islet cell inflammation and oxidative stress as well as aldosterone - induced insulin resistance [ 47 , 48 ] . if our findings reported here are reproduced in clinical studies , our study may have important clinical implications in the way these drugs should be administered in cardiac dysfunction . firstly , our data implies that in normal patients , ru alone could be an effective therapy for prevention of cardiac dysfunction because as a single agent , it yields the best improvements in cardiac function when given before ischemia . furthermore , for postischemic injury , although ru appears to be an effective therapy , aceis and/or arbs appear to be the drugs of choice for diabetics as they yielded the best improvements in cardiac function when administered after ischemia . we also suggest that mr antagonists , since they act through a non - ace / ang ii / at1r pathway , may represent a novel class of raas inhibitor that potentially could overcome the limitations observed with the acei and arb combinations of raas inhibitors . our study by selecting to administer drugs acutely in isolated hearts is advantageous in that it examines the effects of these therapeutic agents directly on the heart and avoids noncardiac contributions of these agents . furthermore , this study highlights that in addition to the benefits observed by mr antagonists when administered systemically , these agents can also be beneficial when administered locally . thus , our data implies that in the clinic ru treatment locally might be considered as an effective therapy or preventative measure in cardiac i / r injury for susceptible patients and possibly also preoperatively for patients undergoing aortic cross - clamping or other cardiac surgeries such as cardiopulmonary bypass or coronary artery bypass grafting . our study also suggests that optimal usage of drug(s ) alone or in combination may require their selection based on several criteria including their relative benefit in the normal versus pathological state , whether being considered for prevention or treatment strategy , on the specific cardiac parameter that might need to be improved ( e.g. , diastolic function in diabetes ) and on the optimal rate of cardiac function recovery required for a given condition .

aims . we evaluated the effects of ru28318 ( ru ) , a selective mineralocorticoid receptor ( mr ) antagonist , captopril ( capt ) , an angiotensin converting enzyme inhibitor , and losartan ( los ) , an angiotensin receptor blocker , alone or in combination with ischemia / reperfusion- ( i / r- ) induced cardiac dysfunction in hearts obtained from normal and diabetic rats . methods . isolated hearts were perfused for 30 min and then subjected to 30 min of global ischemia ( i ) followed by a period of 30 min of reperfusion ( r ) . drugs were administered for 30 min either before or after ischemia . drug regimens tested were ru , capt , los , ru + capt , ru + los , capt + los , and ru + capt + los ( triple ) . recovery of cardiac hemodynamics was evaluated . results . recovery of cardiac function was up to 5-fold worse in hearts obtained from diabetic animals compared to controls . treatment with ru was generally better in preventing or reversing ischemia - induced cardiac dysfunction in normal hearts compared to treatment with capt or los alone . in diabetic hearts , ru was generally similarly effective as capt or los treatment . conclusions . ru treatment locally might be considered as an effective therapy or preventative measure in cardiac i / r injury . importantly , ru was the most effective at improving dp / dt ( a measure of diastolic function ) when administered to diabetic hearts after ischemia .

pubmed

modern nasal surgery takes into account the cosmetic appearance of the nose but also seeks to improve the natural functions of the nose . furthermore , in modern respiratory medicine , objective measurement of airway patency is a must . measurement of nasal cavity geometry has proven to be a great challenge for researchers in modern rhinology . the most common method used in these measurements is acoustic rhinometry ( ar ) , which is based on analysis of reflected acoustic impulses [ 3 , 4 ] . it has demonstrated that the cross - sectional area of the nasal airway measured by acoustic rhinometry and the area found by calculations using data from rhinomanometry has close correlation [ 3 , 5 ] . furthermore , several significant associations between nasal cavity dimensions and nasal airflow have been reported . and the most important benefit of acoustic rhinomanometry is that is easy to perform and requires less cooperation from subject . however , nose geometry may be influenced by various factors including race , age , sex , height , weight , and smoking habit . we evaluate the changes in nasal dimensions of healthy iranian volunteered for cosmetic rhinoplasty after surgery using acoustic rhinometry and comparing differences in length , minimal cross sectional area ( mca ) , and volume of each nasal cavity before and after surgery . the cases were all healthy , without any prior history of nose surgery , fracture , allergy or asthma , and sinus disorders , and they had no complaint about nasal obstruction . all cases were iranian aged at least 18 who were visited in ent clinic of rassul - e - akram teaching hospital between 2006 and 2008 . the surgeries according to the structure of the nose were open rhinoplasty with or without septoplasty . t - type of osteotomy ( single versus double ) , placement of columellar strut , or other variations of surgery were dependent on the surgeons preference . all operations were performed by a single experienced surgeon , and septoplasty was performed in the case of moderate - to - severe septal deviation . the device was consists of a sound source ( loudspeaker ) distally positioned in relation to a 24 cm tube equipped with a microphone for acquisition in its proximal portion . a sound pulse was generated with a peak power of 146 db sound pressure level and a 50-ms duration . all measurements were performed after a short period of acclimatization and in a relatively quiet room at normal temperature ( mean = 21.4c ) to minimize artifacts from physical stress , environmental noise , and temperature changes . the measurements were performed during a breathing pause while patients were in a sitting position . the nosepiece used in the measurements was 5 cm in length and was anatomically sculptured . to ensure a tight connection between the nosepiece and tip of the nose , a small amount of ultrasound transmission gel was applied to the edge of the nosepiece . care was taken not to obstruct the nasal vestibule with gel or deform the nose during testing . the angle of the incident acoustic impulse was about 45 with respect to a line joining the base of the piriform aperture of the nose to the tragus . measurement performed in either of two conditions of without a decongestant and with a decongestant , respectively . the first measurement performed before surgery and the second measurement performed 3 months after surgery . the technique we used allowed us to analyze two local minimum points giving us minimum cross - sectional areas ( mca1 and mca2 ) and distance ( d1 and d2 ) to the points from the nostril and volumes of nostrils . to examine the geometrically difference in pre - and postoperative nasal cavities , paired t test used for repeated measurements . mean age ( sd ) of cases were 24.63 ( 4.4 ) years , minimum age was 18 , and maximum age was 40 . none cases had complaints of breathing problems before and after the surgery , and all of our cases were satisfied with their surgery result . mean ( sd ) geometric values of cases preoperatively with and without decongestant are demonstrated in table 1 . by the pair wise comparison of cases without decongestant before and after surgery , right ( r ) d1 , bilateral d2 , and left ( l ) mca2 did not change significantly after surgery . however , pairwise comparison of nasal dimensions with decongestant revealed that bilateral d2 and v did not changed significantly postoperatively . bilateral d1 and both mca2 decreased significantly , while significant increase was observed in mca1 postoperatively ( table 2 ) . in order to assess the result of septoplasty , patients divided into 2 groups of rhinoplasty plus septoplasty and rhinoplasty alone . comparison of the finding showed that cases with septoplasty would experience more increase in mca1 ( increase in mca1 was significant and much more in decongested state of group with septoplasty ) and less constriction in mca2 postoperatively ( significant constriction were observed in both groups . however , amount of this constriction was more in group without septoplasty ) ( table 3 ) . in cases of rhinoplasty without septoplasty , increase in mca1 was significant only in group of single osteotomy without decongestant . although changes in mcv2 were significant in both with and without decongestion groups of double osteotomy , but the net effects were not clinically significant comparing single osteotomies . table 4 demonstrates that cases with rhinoplasty alone would benefit from double osteotomy in mca1 . effect of osteotomy on patency of nose in cases with septoplasty is shown in table 5 ; it is more prominent at the level of mca1 . placing a columellar strut in group of cases of rhinoplasty plus septoplasty has significant effect both at mca1 and mca2 levels ( table 6 ) . in cases of rhinoplasty alone , placing a columellar strut caused greater amount of increase in mca1 . mca2 after decongestion significantly increased in group without columellar strut , but it did not changed in group with replacing a columellar strut . regression analysis of difference after surgery in mca1 , mca2 , and v revealed no fix significant effect for septoplasty , osteotomy , and placing a columellar strut . advanced equipment for these methods is now available , and most devices are reliable provided that care is taken to calibrate the device properly . in the current study , we used an ar to measure the dimensions of the nose . ar evaluation of the nasal airway has been reported to be accurate when compared with mri2 and ct [ 15 , 16 ] . the closest correlations have been noted after decongestant use and in the most anterior 5 cm of the nasal cavity . it is believed that acoustic rhinometry provided less variability of results than those obtained with rhinomanometry . in the study of corey he concludes that the anatomic correlate of csa1 ( cross - sectional area ) is not as clearly delineated ; however , it likely corresponds to an area at or near the nasal valve . furthermore , csa2 correspond to the general areas of the anterior portions of the inferior turbinate . he claimed that in conjunction with a careful clinical exam , ar can provide objective documentation and diagnosis for better treatment of nasal airway blockage . acoustic rhinometry also seems very suitable for evaluation of the nasal cavity in cases where septoplasty and turbinoplasty is considered as well as for the postoperative objective evaluation . studied the changes in nasal airways after le fort i osteotomy and functional rhinosurgery with both ar and rhinomanometry techniques . he declare that rhinomanometry and acoustic rhinometry are reliable and objective methods of determining functional and geometric changes in the nasal cavity after le fort i osteotomy . in this paper we described reference values in normal and decongested noses of normal iranian population with acoustic rhinometry . all of our cases were normal people without significant symptoms of nasal dysfunction in their nose , and surgery was performed just because of aesthetic reasons . comparing our finding with mohebbi et al . , in iranian population , no significant difference was observed in mcas and v. this finding supports the original idea of study to enroll cases with normal nose geometry . by comparing nose geometry before and after surgery in both normal state and decongested state , considering presence of inflammation after surgery and more precise estimation in decongested estate , the main findings were a reduction in d1 , an increase in mca1 , a decrease in mca2 and no change in v and d2 . these findings are consistent with findings of greymer that reported reduction in internal dimensions of the nasal cavity especially the anterior dimensions . this finding highlight the fact that second constriction is a critical place during the surgery and adequate attention should be paid to this point . the anatomic correlate of this point is anterior portion of the inferior turbinate , hence decreasing the resistance in this region could be beneficial for patients . operations on the nasal septum are frequently performed with the intention of improving the nasal airway . for this reason , they form much of the routine workload in most otolaryngology departments . a meta - analysis performed by singh et al . demonstrated that an overall significant improvement following surgery is present in patient undergoing septal surgery . in our study , comparison of cases of rhinoplasty with and without septoplasty revealed that the cases with septoplasty would experience more increase in mca1 and less constriction in mca2 . as a conclusion , even in the cases which mild septal deviation is present , performing the septoplasty may increase patient nose patency . more increase in mca1 and no significant effect on mca2 were observed in patients in double osteotomy group comparing group of single osteotomy . it seems as if double osteotomy positively affects nose function by more increase in mca1 . double osteotomy is positively affecting mca1 considering the cases without septoplasty or cases with septoplasty . in either group of rhinoplasty with and without septoplasty , placing a strut was beneficial for patients . in the study of oeken and kiefer on 52 cases of septorhinoplasty total nasal airflow it should be mentioned that in the above study , all cases suffered from a deviated septum and some degree of obstruction . anselmo - lima et al . in a study of 30 cases of rhinoplasty with lateral osteotomy observed that there is a reduction in nasal valve area ( significant reduction in mca1 ) as well as in anterior volume of the nose . they propose more criteria to indicate rhinoplasty , because the procedure might lead to nasal obstruction . in the study of kemker patients in the septoplasty - only group showed a statistically significant increase in volume as measured by ar . patients who had septoplasty plus other sinonasal procedures showed significant increases in volume and cross - sectional area ( csa ) 3 , whereas csas 1 and 2 increased also , but not significantly . shemen and hamburg studied 24 patients undergone septal surgery , and skouras et al . studied 16 patients , the improvement in nasal patency was seen in both studies . however , cases in both studies have complained of nasal obstruction before surgery [ 25 , 26 ] . in a study by can et al . on 26 children with septal deviation the results showed that the surgery was successful ; however , they claim that surgery should be restricted to only the pathologic area and should be conservative . as we know , ar has some limitations , as it can not measure the columella width in nasal geometry and also differentiate the nostrils shape that has effect on the turbulence of air delivered to nasal cavity . as we know , the cross - sectional area of the nose is a major factor in the determination of airflow , with airflow increasing as the cross - sectional area increases . hence , according to the above findings , cosmetic rhinoplasty may generate a mix effect on nose function . comparison of our finding to other studies shows that condition of the patient before surgery is the principal factor for the prediction of surgery results . putting all together , surgeons should be aware of common pattern of change in nose geometry ; care must be taken in intervention in the region of second constriction . performing osteotomy may better help patients to save nasal patency , septoplasty is beneficial even in mildly deviated septum , and placing a strut may be beneficial in most of the cases .

objective . to evaluate the changes in nasal dimensions of healthy iranian volunteered for cosmetic rhinoplasty after surgery using acoustic rhinometry . methods . pre- and postoperative nasal dimension of 36 cases undergoing cosmetic rhinoplasty were compared using acoustic rhinometry ( ar ) , and the measured variables were distance to first and second constriction ( d1 , d2 ) , first and second minimal cross - sectional area ( mca1 , 2 ) , and volume . results . mean age ( sd ) of cases were 24.63 ( 4.4 ) years . septoplasty was performed in 12 cases ( 33.3% ) . after surgery , bilateral d1 and both mca2 decreased significantly , while significant increase was observed in mca1 postoperatively using decongestant . cases with septoplasty experienced more increase in mca1 and less constriction in mca2 postoperatively . in cases with rhinoplasty alone , they received benefit from double osteotomy in mca1 . in either group of rhinoplasty with and without septoplasty , placing a strut was beneficial for patients . discussion . the cross - sectional area of the nose is a major factor in the determination of airflow . cosmetic rhinoplasty may generate a mix effect on nose function . performing osteotomy may better help patients to save nasal patency , septoplasty is beneficial even in mildly deviated septums , and placing a strut may be beneficial in most of the cases .

pubmed