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as clearly shown in previous articles within this special issue , dentin hypersensitivity ( dhs ) is a well - known patient complaint which is patent in middle - aged people and most probably will increase with aging . besides the importance of correct diagnosis [ 2 , 3 ] , appropriate treatment recommendations for patients are needed . it is important to emphasize once more that the basic requirements for having dhs is exposed cervical dentin ( ecd ) and open dentin tubules . in this respect , it is interesting to realize that dentists should be aware of the presence of ecd . most of the patients are not aware themselves of the present harm or the physiologic alteration of the dentition . moreover , patients who experience dhs wait to mention until the next recall visit , and most of them do not specifically seek treatment for this problem , most likely because they do not view it as a significant dental health problem . however , it is clearly shown that dhs can significantly be related to substantially impaired oral health - related quality of life [ 79 ] . as with all conditions or diseases , management strategies that include treatment are usually more successful than treatment alone ; therefore , the following approach was proposed by addy about 10 years ago .ensure the correct diagnosis of dhs , which is based on history and examination and compatible with the definition s clinical descriptor.consider a differential diagnosis , as suggested by the definition of dhs , which alone may explain the symptoms or identify the presence of other conditions contributing to the pain of dhs.treat any and all secondary conditions that induce symptoms similar to dhs ( differential diagnosis).identify etiological and predisposing factors , particularly with respect to erosion and abrasion . consider detailed , written , dietary histories and oral hygiene habits ( frequency , duration and timing of brushing , brushing frequency , estimation of brushing force , frequency of brush change , and appearance of brush at change ) . some of these aspects of tooth brushing behavior are best appraised by observing the patient brushing in the dental practice.remove or modify identified etiological or predisposing factors . offer dietary advice to minimize erosion and oral hygiene instruction to minimize abrasion and to distinguish abrasion from erosion.recommend or provide treatments appropriate to the individual needs of the sufferer . the number of teeth involved and the severity of the pain are important variables and should influence the treatment options . ensure the correct diagnosis of dhs , which is based on history and examination and compatible with the definition s clinical descriptor . consider a differential diagnosis , as suggested by the definition of dhs , which alone may explain the symptoms or identify the presence of other conditions contributing to the pain of dhs . treat any and all secondary conditions that induce symptoms similar to dhs ( differential diagnosis ) . consider detailed , written , dietary histories and oral hygiene habits ( frequency , duration and timing of brushing , brushing frequency , estimation of brushing force , frequency of brush change , and appearance of brush at change ) . some of these aspects of tooth brushing behavior are best appraised by observing the patient brushing in the dental practice . remove or modify identified etiological or predisposing factors . offer dietary advice to minimize erosion and oral hygiene instruction to minimize abrasion and to distinguish abrasion from erosion . the number of teeth involved and the severity of the pain are important variables and should influence the treatment options . the aim of this review was to design a decision tree which can be useful as soon as a patient present with ecd or suffers from dhs . the gaba forum group on dentin hypersensitivity was composed by:- bloch - zupan a. ( strasbourgh , france)- schmalz g. ( regensburg , germany)- gernhardt c. ( halle , germany)- schmidlin p. ( zurich , switzerland)- gillam d. ( london , uk)- sixou ( rennes , france)- hellwig e. ( freiburg , germany)- splieth c. ( greifswald , germany)- bekes k. ( halle , germany)- trp jc . ( basle , switserland)- lussi a. ( bern , switserland)- van loveren c. ( amsterdam , the netherlands)- martens l. ( gent , belgium)- west n. ( bristol , uk)- petersson lg . a pubmed literature search on dentin hypersensitivity was performed focussing on english written manuscripts using the mesh terms review ( 273 hits ) and management ( 12 hits ) . within the reviews , most manuscripts focussed on the etiology or summarized the current treatment strategies . only a few reports could be selected regarding treatment strategies including a decision tree . in addition , some websites of manufacturers were visited . finally , the adelaide university special topic nr 6 on dentin hypersensitivity was consulted . the best developed report came from the canadian advisory board on dentin hypersensitivity ( cabdhs ) who published in 2003 their consensus - based recommendations for the diagnosis and management of dentin hypersensitivity . since then , an interesting flowchart for the clinical management of dhs and only some reports dealing with the concept and methodology for its objective evaluation or reviewing diagnosis and treatment procedures of dentinal hypersensitivity were published . most recently , the academy of general dentistry published a dhs management document which was mainly based on the cabdhs report . from the cabdhs report , it became clear that there is a knowledge gap within the dentist and dental hygienist populations . to illustrate , the survey found that fewer than half of the 542 respondents ( 331 dentists and 211 dental hygienists ) considered a differential diagnosis for dhs , even though it is by definition a diagnosis of exclusion [ 2 , 3 ] . the survey also revealed that many respondents ( 64 % of dentists and 77 % of hygienists ) incorrectly cited bruxism and malocclusion as triggers for dhs , while only a small percentage of the respondents ( 7 % of dentists and 5 % of dental hygienists ) could correctly identify erosion as a primary cause . in this respect , it is clear that abrasion and erosion may be implicated here , but acid erosion seems to be predominant . furthermore , 17 % of dentists and 48 % of hygienists were unable to identify the accepted hydrodynamic theory of dhs . approximately half of the respondents reported that they lacked the confidence to manage a patient s pain caused by dhs . also , only half of the respondents reported that they would try to modify the patient s predisposing factors to control the pain . a total of 14 knowledge gaps were identified , and they were classified as related to the causes and diagnosis or the management of the condition ( see table 1).table 1summary of knowledge gaps regarding the etiology , diagnosis and management of dentin hypersensitivity as proven by the canadian advisory board on dentin hypersensitivity gaps related to etiology / diagnosisgaps related to management1 most of the respondents reported incorrectly that fluoride compounds are the most popular desensitizing ingredients4 . only 10% of respondents correctly thought that desensitizing toothpastes disrupt pain transmission by preventing repolarisation within the nerve.5 . a number of respondents did not believe that desensitizing toothpastes were effective in preventing caries even though most contain fluoride.6 . a number of respondents believed that desensitizing toothpastes were effective in preventing dentine hypersensitivity despite the lack of data 7 . most of the dentists and hygients incorrectly cited toothbrush abrasion as a reason for continued tubule exposure7 . although the latter gap , a number of respondents did not believe that desensitizing toothpastes relieved dentine hypersensitivity summary of knowledge gaps regarding the etiology , diagnosis and management of dentin hypersensitivity as proven by the canadian advisory board on dentin hypersensitivity the most striking gaps related to management were the lack in knowledge on the working mechanisms of the longest used and best known active ingredients in toothpastes : strontium chloride ( occluding the dentinal tubules ) and potassium nitrate ( decrease of excitability of nerves that transmit pain sensation ) . the following was not taken into account : having ecd without painhaving sensitive teeth without decrease of a patient s quality of lifean order of priority or hierarchy in treatment options having ecd without pain having sensitive teeth without decrease of a patient s quality of life an order of priority or hierarchy in treatment options furthermore , the recommendation to initiate for dhs management can be extended and should include also recommendations for the dental professionals and not for patients only as suggested by chu et al . based on drisko et al . . in the present review , an overall approach of the management of ecd and dhs will be given . illustrates how to start the thinking and managing process when ecd is occasionally found during routine check ups . as soon as ecd is seen , one should initiate a further screening asking some specific questions ( cfr fig . 2 ( aa)):fig . 1a treatment decision tree for patients with exposed cervical dentine ( ecd ) ( qol = quality of life , dhs = dentin hypersensitivity)fig . 2a treatment decision tree for patients with dentin hypersensitivity ( dhs ) and a decreased quality of life ( qol ) ( modified from the canadian advisory board on dentin hypersensitivity-2003)probing for lifestyle habits / practices , intrinsic and extrinsic acid ( citrus juices and fruits , carbonated drinks , wines , and ciders),obtaining detailed dietary information including the dietary intake relevant to medical problems that may have an impact on the oral cavity , probing for gastric acid reflux and excessive vomiting . a treatment decision tree for patients with exposed cervical dentine ( ecd ) ( qol = quality of life , dhs = dentin hypersensitivity ) a treatment decision tree for patients with dentin hypersensitivity ( dhs ) and a decreased quality of life ( qol ) ( modified from the canadian advisory board on dentin hypersensitivity-2003 ) probing for lifestyle habits / practices , intrinsic and extrinsic acid ( citrus juices and fruits , carbonated drinks , wines , and ciders ) , obtaining detailed dietary information including the dietary intake relevant to medical problems that may have an impact on the oral cavity , probing for gastric acid reflux and excessive vomiting . along with this specific screening , it is important to distinguish between a localized and a generalized problem which may be mild , moderate , or severe in nature . the latter will influence the future management which usually will involve a combination of at - home and in - office therapies . in practice , the regimen adopted will depend on the perceived severity of the condition and the number of teeth involved . the above - mentioned screening needs to be followed by the initiation of prevention of dhs in two directions : education of patients and self education of practitioners and care givers . in fig . 2 ( ba and bb ) if the patient presents with ecd combined with a complaint of dhs , one has to point out if this pain sensation affects the patient s quality of life ( qol ) . in this respect , the patient can be questioned on : pain and discomfortprobable limitations in dietary choices ( drinks?)the effectiveness of oral hygieneprobable negative esthetics probable limitations in dietary choices ( drinks ? ) the effectiveness of oral hygiene probable negative esthetics if qol is not affected , it is recommended to start the prevention program of dhs . if qol is decreased , it is strongly recommended to start a further management program of dhs as illustrated in fig . 2 . when a patient presents with dhs expressed by sharp pain sensations and with a negative influence on the patients qol , an additional screening obtaining a complete history of the patient especially focussed on nutritional habits ( fig . 2 ( aa ) ) and the promoted diagnosis by exclusion ( fig . 2 ( ab ) ) has to be performed . if there is no consistency between history and examination , other causes than dhs must be sought for . 2 ( ba ) ) as well as for dentists and caregivers ( fig . 2 ( bb ) ) . regarding the patients , dietary counseling and non - harmful oral hygiene habits are very important . therefore , a combination of individualized instructions on rather oral health behaviors , use of at - home products , and additional professional treatment may be required to manage the problem . while at - home treatment can be the first choice for generalized dhs , localized to one or two teeth practitioners may elect to use an in - office method as the first choice of treatment for dhs . regarding the dentists and caregivers , non - harmful professional dental care this must result in a well - considered choice and use of instruments and additional tools performing restorative dentistry . if , during follow up , symptoms are relieved or disappeared , improving the patient s qol , no further treatment is required . a well - maintained preventive management program must be followed . besides eliminating etiological factors , it is strongly recommended to advise a regimen of brushing with desensitizing toothpastes twice a day . regarding desensitizing toothpastes , two treatment approaches are well known : occluding dentinal tubules ( plugging ) or blocking the neural transmission to the pulp . these mechanisms will not further be discussed within this manuscript as other authors did this in the same issue of this journal . if necessary , a less traumatic brushing method may also be introduced . if symptoms are confirmed , no pain relief present , or a further decrease of the patients qol is present , treatment for dhs must be initiated . in this perspective , the non - invasive and invasive approach can be considered ( fig . 2 ( ca cb ) ) . it is recommended to start with minimal invasive in - office procedures such as the use of topical fluorides and dentin bondings , which were discussed in the corresponding reports within this issue or laser therapy . regarding the latter , apparently no significant reductions in sensitivity could be found . as a consequence when there is no long - term benefit of these minimal invasive procedures , more invasive procedures can be carried out after advanced diagnosis . mainly mucogingival surgery and/or pulpectomy will then be the most appropriate choices . if treatment is carried out successfully , one should maintain and review the therapy on a regularly basis . if treatment procedures did not result in pain relief , one should again start an advanced diagnosis based on exclusion ( fig . 2 ( e ) ) and especially focussing on : periodontal painreferred painneuropathic painchronic pain syndrome chronic pain syndrome depending on the results , a decision must be made to continue dhs management and treatment or the patient must be referred to an appropriate dental or medical specialist . a more handy flowchart [ 11 , 13 ] for daily use is also proposed ( fig . 3flowchart for the diagnosis and management of dentin hypersensitivity ( dhs ) in daily practice ( based on ochardsen and gillam 2006 and on the special topic nr 6 on sensitive teeth by colgate and the adelaide university ) flowchart for the diagnosis and management of dentin hypersensitivity ( dhs ) in daily practice ( based on ochardsen and gillam 2006 and on the special topic nr 6 on sensitive teeth by colgate and the adelaide university ) dhs is a problematic clinical entity that may become an increasing clinical problem for dentists to treat as a consequence of patients retaining their teeth throughout life and improved oral hygiene practices . for that , it is strongly recommended to screen routinely all dentate patients for ecd and dhs . in this respect , underdiagnosis of the condition will be avoided and the preventive management can be initiated early . active management of dhs usually will begin with at - home therapy of which brushing with desensitizing toothpastes is the most important . complete management will usually involve a combination of at - home and in - office therapies .	introductiondentin hypersensitivity ( dhs ) is a problematic clinical entity that may become an increasing clinical problem for dentists to treat as a consequence of patients retaining their teeth throughout life and improved oral hygiene practices.objectivesthe aim of this review was to develop a decision tree for the management of exposed cervical dentin ( ecd ) and dhs.material and methodsa brief pubmed literature search was performed on dentin hypersensitivity using mesh terms , review , and management . in addition , some websites and local guidelines were screened.resultsfrom this review , it became clear that all dentate patients should routinely be screened for ecd and dhs . in this respect , underdiagnosis of the condition will be avoided and the preventive management can be initiated early.conclusiona decision tree process and a flowchart for daily practice were designed which should be started up as soon as a patient present with ecd or suffers from dhs . this approach takes into account the possible improved quality of life of the patient and is further based on a hierarchy of treatment options . in this respect , active management of dhs will usually involve a combination of at - home and in - office therapies . starting with the use of desensitizing toothpastes is strongly recommended .
initiation and early growth of primary epithelial cancers , and acquisition of invasiveness is thought to herald the advanced stages of this multistep process that eventually leads to metastatic dissemination , with life - threatening consequences . the genetic control and biochemical mechanisms underlying the acquisition of the invasive phenotype are mediated by the activation of an epithelial mesenchymal transition ( emt ) program . emt is a biologic process that allows an epithelial cell to acquire the properties of a mesenchymal cell , thereby promoting invasion into the surrounding stroma and intravasation of tumor cells . a number of distinct molecular markers are engaged in order to initiate emt and enable its completion . one such marker is the s100a4 , a flament - associated calcium - binding protein , which is associated with the cells of mesenchymal origin , and deemed to play a significant role in emt . s100a4 expression levels strongly correlate with motility , which is a central element of the metastatic cascade . in oral squamous cell carcinoma ( oscc ) , metastasis results in 90% of cancer associated mortality . in this work , we studied the role of s100a4 in oral cancer as a biomarker of metastasis by assessing its expression in primary tumors from all the clinical stages of oral cancer and determined the association of s100a4 expression with aggressive clinico - pathological parameters including the pattern of invasion , distant metastasis and recurrence . assessment of s100a4 in oral cancer may be related to the phenotypic alteration and thereby predict its metastatic potential . the material for present study included a total of 57 formalin fixed paraffin embedded tissue sections retrieved from the departmental archives . out of these , 47 were patients with carcinoma of the bucco - alveolar mucosal complex and of the foor of the mouth . fhe remaining 10 were healthy mucosal tissues , which were obtained at the time of extraction of impacted teeth . only surgically treated cases of oral squamous cell carcinoma ( oscc ) with a minimum follow - up period of 5 years were included in the study . institutional ethics committee approval ( iec 36/2012 ) was obtained from kasturba hospital , manipal , prior to carrying out the study . the patient demographics and other details of the cases included in the study are presented in table 1 . a chart analysis was carried out to study the expression of s100a4 in all these cases by immunohistochemistry ( ihc ) . nt- no tobacco immunohistochemical staining was carried out using indirect streptavidin biotin immunoperoxidase technique . sections of 4 m thickness taken on amino propyl triethoxysilane ( apes ) coated slides were incubated overnight at 48c in a slide warmer to ensure complete adhesion of sections . following tissue sections deparaffinization and hydration through descending grades of alcohol , sections were placed in phosphate buffered saline ( pbs ) for 30 minutes . the slides were immersed in a microwave resistant plastic staining jar containing antigen retrieval solution ( 10 mm sodium citrate buffer , ph 6.0 ) and maintained at 700 w power for 10 minutes . subsequently , the solution was allowed to cool at room temperature for at least 20 minutes . the slides were incubated in 3% hydrogen peroxide for 5 minutes at room temperature , following which slides were washed with pbs . to decrease the background staining due to nonspecific binding of antibodies , the slides were incubated with 5% bovine serum albumin ( bsa ) for 10 minutes at room temperature and washed with pbs for 5 minutes . the researchers applied 100 l of the primary antibody ( rabbit polyclonal anti - s100a4 antibody , 1:250 ) over the section completely , which was incubated for 60 minutes at 37c in a humidified chamber . the slides were rinsed with pbs and placed in the pbs bath for 5 minutes . after draining and wiping of the slides , sections were incubated in 100 l of biotinylated secondary antibody , anti - rabbit igg ( whole molecule - peroxidase antibody produced in goat ) for 30 minutes at 37c . the slides were washed with pbs and placed in pbs bath for 5 minutes . for visualization , sections were incubated with freshly prepared substrate solution ( sigmafast 3 , 3'-diaminobenzidine tablets ) for 5 - 10 minutes . this was followed by washing the slides in running tap water and counter staining with mayer 's haematoxylin ( mhs1- haematoxylin solution , mayer 's 1 gil certified hematoxylin * ph 2.4 ) for 5 minutes , after which the slides were washed in running tap water for 1 minute . briefy , the sections were dehydrated through ascending grades of alcohol ( 70% , 95% and 100% ) for 2 minutes each , cleared in xylene for 1 minute , mounted in a resinous media and coverslipped . the neoplastic cell with cytoplasmic staining was viewed at 200 magnification , using a light microscope . the distribution of immunolabelling in neoplastic epithelial cells was determined from a minimum of five representative high - power fields at the invasive front . a positive cell demonstrated a diffuse brown signal in the cytoplasm , independent of its intensity . to eliminate any inter - observer bias , the degree of staining for s100a4 was evaluated using a semi - quantitative scale29 . tissues were graded as absent ( - ) when there was no staining , low ( 1 + ) when less than 25% of the tumor cells were positive , moderate ( 2 + ) when 25 to 75% of the tumor cells stained positively and diffuse ( 3 + ) when more than 75% of the tumor cells were positive for s100a4 . s100a4 expression was then correlated with clinicopathological parameters including primary tumor site , stage of tumor , degree of differentiation , lymph node metastasis and mode ( pattern ) of invasion . kendall 's tau - b statistics was applied to assess the measure of agreement between two observers . chi - square test was used to study the association between the s100a4 staining and clinicopathological parameters . the material for present study included a total of 57 formalin fixed paraffin embedded tissue sections retrieved from the departmental archives . out of these , 47 were patients with carcinoma of the bucco - alveolar mucosal complex and of the foor of the mouth . fhe remaining 10 were healthy mucosal tissues , which were obtained at the time of extraction of impacted teeth . only surgically treated cases of oral squamous cell carcinoma ( oscc ) with a minimum follow - up period of 5 years were included in the study . institutional ethics committee approval ( iec 36/2012 ) was obtained from kasturba hospital , manipal , prior to carrying out the study . the patient demographics and other details of the cases included in the study are presented in table 1 . a chart analysis was carried out to study the expression of s100a4 in all these cases by immunohistochemistry ( ihc ) . the antibodies and reagents were obtained from sigma aldrich co. ( st . louis , mo , usa ) . sections of 4 m thickness taken on amino propyl triethoxysilane ( apes ) coated slides were incubated overnight at 48c in a slide warmer to ensure complete adhesion of sections . following tissue sections deparaffinization and hydration through descending grades of alcohol , sections were placed in phosphate buffered saline ( pbs ) for 30 minutes . the slides were immersed in a microwave resistant plastic staining jar containing antigen retrieval solution ( 10 mm sodium citrate buffer , ph 6.0 ) and maintained at 700 w power for 10 minutes . subsequently , the solution was allowed to cool at room temperature for at least 20 minutes . the slides were incubated in 3% hydrogen peroxide for 5 minutes at room temperature , following which slides were washed with pbs . to decrease the background staining due to nonspecific binding of antibodies , the slides were incubated with 5% bovine serum albumin ( bsa ) for 10 minutes at room temperature and washed with pbs for 5 minutes . the researchers applied 100 l of the primary antibody ( rabbit polyclonal anti - s100a4 antibody , 1:250 ) over the section completely , which was incubated for 60 minutes at 37c in a humidified chamber . the slides were rinsed with pbs and placed in the pbs bath for 5 minutes . after draining and wiping of the slides , sections were incubated in 100 l of biotinylated secondary antibody , anti - rabbit igg ( whole molecule - peroxidase antibody produced in goat ) for 30 minutes at 37c . the slides were washed with pbs and placed in pbs bath for 5 minutes . for visualization , sections were incubated with freshly prepared substrate solution ( sigmafast 3 , 3'-diaminobenzidine tablets ) for 5 - 10 minutes . this was followed by washing the slides in running tap water and counter staining with mayer 's haematoxylin ( mhs1- haematoxylin solution , mayer 's 1 gil certified hematoxylin * ph 2.4 ) for 5 minutes , after which the slides were washed in running tap water for 1 minute . briefy , the sections were dehydrated through ascending grades of alcohol ( 70% , 95% and 100% ) for 2 minutes each , cleared in xylene for 1 minute , mounted in a resinous media and coverslipped . the neoplastic cell with cytoplasmic staining was viewed at 200 magnification , using a light microscope . the distribution of immunolabelling in neoplastic epithelial cells was determined from a minimum of five representative high - power fields at the invasive front . a positive cell demonstrated a diffuse brown signal in the cytoplasm , independent of its intensity . to eliminate any inter - observer bias , the degree of staining for s100a4 was evaluated using a semi - quantitative scale29 . tissues were graded as absent ( - ) when there was no staining , low ( 1 + ) when less than 25% of the tumor cells were positive , moderate ( 2 + ) when 25 to 75% of the tumor cells stained positively and diffuse ( 3 + ) when more than 75% of the tumor cells were positive for s100a4 . s100a4 expression was then correlated with clinicopathological parameters including primary tumor site , stage of tumor , degree of differentiation , lymph node metastasis and mode ( pattern ) of invasion . kendall 's tau - b statistics was applied to assess the measure of agreement between two observers . chi - square test was used to study the association between the s100a4 staining and clinicopathological parameters . positive s100a4 expression in the cytoplasm of the tumor cells was observed in 30 out of 47 cases ( 63.82% ) of oscc . kendall 's tau - b test between the two observers showed a very high level of agreement for the expression of s100a4 ( 0.8 , p<0.001 ) . in the control group , none of oral mucosal tissues were positive for s100a4 in the cytoplasm of the epithelium . out of 47 cases studied , 1/6 of stage i , 1/2 of stage ii , 13/17 of stage iii and 15/22 of stage iv cases showed positive s100a4 expression ( table 2 ) . a strong association was noted between the stage of the tumor and the expression score of s100a4 ( p=0.007 ) . s100a4 expression was positive in 12/14 ( 85.7% ) node positive stage iii cases , of which 7/12 were ( 3 + ) and 5/12 were ( 2 + ) for s100a4 expression . among the node negative cases , 1/3 cases showed ( 3 + ) for s100a4 expression . likewise , s100a4 expression was positive in 14/15 ( 93.3% ) stage iv cases , of which 13/14 were ( 3 + ) and 1/14 was ( 2 + ) . among the 7 node negative cases , 1/7 was ( 3 + ) and the remaining 6/7 cases were negative for s100a4 expression . although the cytoplasmic expression of s100a4 was strongly associated with lymph node involvement ( p<0.001 ) , there was no association between s100a4 expression in lymph node positive cases and primary size of the tumor ( p=0.15 ) . association of s100a4 expression with clinical stage , lymph node involvement , tumor size , metastasis , recurrence , grade of tumor and pattern of invasion wdscc - well differentiated squamous cell carcinoma ; mdscc - moderately differentiated squamous cell carcinoma ; pdscc - poorly differentiated squamous cell carcinoma with regard to distant metastasis ( m ) , 11/22 ( 50% ) cases in stage iv were positive for s100a4 expression , out of which 10/11 were ( 3 + ) and 1/11 was ( 2 + ) for s100a4 expression . a strong association could be established between the s100a4 expression in tumor cells and distant metastasis ( p=0.004 ) . likewise , 11/47 ( 23.4% ) cases that recurred after treatment were either in stage iii ( 2/11 ) or stage iv ( 9/11 ) . all these cases showed ( 3 + ) expression for s100a4 . among the stage iii cases that recurred , one was node positive and the other was negative , whereas all the 9 stage iv cases that recurred were node positive . there was a strong positive association between the s100a4 expression and rate of recurrence ( p=0.002 ) . with regard to the pattern of invasion among the positive cases , 1/6 cases of stage i showed a type 1 ( figure 1 ) pattern and 1/2 cases in stage ii showed type 2 ( figure 2 ) pattern . out of the 13 stage iii cases that showed positive s100a4 expression , 2 were type 1 , 2 others were type 2 , 3 were type 3 ( figure 3 ) , 3 were type 4c ( figure 4 ) and 3 were type 4d ( figure 5 ) . in the same manner , out of the 15 stage iv cases that showed positive s100a4 expression , 2 were type 3 , 4 were type 4c and 9 were type 4d . there was a strong association ( p<0.001 ) between s100a4 expression and pattern of invasion of oscc . however , there was no association seen between the s100a4 expression and histological grade of the oscc ( p=0.27 ) . oral squamous cell carcinoma ( oscc ) showing type 1 well defined borderline pattern of invasion ( s100a4 , 20x ) oral squamous cell carcinoma ( oscc ) showing type 2 cord like pattern of invasion ( s100 a4 , 20x ) oral squamous cell carcinoma ( oscc ) showing type 3 pattern of invasion with groups of cells with no distinct borderline ( s100a4 , 20x ) oral squamous cell carcinoma ( oscc ) showing type 4c diffuse cord like pattern of invasion ( s100a4 , 20x ) oral squamous cell carcinoma ( oscc ) showing type 4d diffuse widespread pattern of invasion ( s100a4 , 20x ) oral cancer is one of the major cancers worldwide , with a low five - year survival rate that has not significantly changed over the past few decades . metastasis , which is responsible for 90% of the cancer associated mortality , is one of the poorly understood factors in cancer progression . an important step for the invasion and metastasis is the epithelial mesenchymal transition ( emt ) , a complex process induced by modifications of multiple pathways leading to a spectrum of epithelial cellular changes , including loss of polarity and cohesion , increased motility , and the acquisition of mesenchymal phenotype . emt is critical for the invasion , progression , and metastasis in epithelial carcinogenesis . metastasis promoting s100a4 is a typical representative of s100 family of ca2 + -binding multifunctional proteins with dual extra- and intracellular function . the increased expression of the s100a4 in invasive front of the tumor suggests the pivotal role played by s100a4 in invasion of tumor cells into the surrounding matrix . the acquisition of invasive phenotype with altered pattern of adhesion is a result of e - cadherin down regulation by s100a4 , which the tumor cells acquire . further influences cell - matrix adhesions by interacting with liprin [ 1 and masking their protein kinase c - mediated phosphorylation sites . inhibition of the liprin [ 1-lar complex by s100a4 loosens cell adhesion and allows cell invasion . cell invasion is also facilitated by matrix metalloproteinases ( mmps ) , as s100a4 causes metastasis not only by affecting the motility of cells but also by affecting its invasive properties through influencing the expression of mmps and their endogenous inhibitors . a positive correlation of the s100a4 and mmp2 expression in oesophageal squamous cell carcinoma has been reported . s100a4 is located at the leading edge of migrating cells , where it induces the formation of fexible protrusions . the s100a4-myosin ii - a interaction does not only increase cell motility , but also enhances cell polarization and directed migration . s100a4 also binds to the septins 2 , 6 and 7 , which play a central role in cytokinesis , cell polarity determination , cytoskeletal reorganization , and membrane dynamics10 , . s100a4 binds to annexin ii on the endothelial surface , a complex that activates the tissue plasminogen activator , which in turn converts the plasminogen to plasmin , activating prommps . ca - dependent binding of s100a4 to metap2 modulates the metap2 activity , which could promote endothelial growth and angiogenesis . this process of angiogenesis favors the metastasis of the tumor to the regional lymph nodes . elevated s100a4 expression has shown to be the marker of metastasis and poor prognosis in patients with pancreatic ductal adenocarcinoma , prostate adenocarcinoma , colorectal cancer4 , and breast cancer . a higher level of s100a4 expression has shown to correlate with auxillary lymph node metastasis and overall patient survival . evidence suggesting the role for s100a4 in hnscc , especially in oscc , is scarce . elevated s100a4 as a prognostic factor in metastatic cases of oscc was demonstrated by moriyama - kita , et al . ( 2004).our findings showing increased expression of s100a4 in cases with regional lymphnode involvement and metastasis further emphasizes the role of s100a4 in oral cancer patients with poor prognosis . the final step in the metastatic cascade is the migration of the cells to a distant site and secondary colonization . in our study this finding has been elucidated earlier in papillary thyroid carcinoma and pancreatic cancer , in which poor outcome of patients was related to increased s100a4 expression coupled with decreased e cadherin expression . the increased expression of s100a4 in our study showed a significant correlation with the overall clinical stage of tumor , a finding that has been noted also in colorectal carcinoma and pancreatic cancer . however , no association could be drawn between the elevated s100a4 level and the primary tumor size or histological grading in our analysis . ( 2014 ) in lung cancer cells , in which s100a4 was frequently overexpressed , irrespective of histological subtype . in our study a strong association between s100a4 expression and pattern of invasion could be elicited , with an increased expression noted in type 4c and type 4d pattern of invasion . similar observations have been made in malignant melanomas where increased expression of s100a4 in the nodular variant was noted , linking this association to the pattern of invasion . significantly , our observation of high s100a4 expression with tumor recurrence is a novel finding . s100a4 has been identified as a candidate marker for maintaining the stemness in head and neck cancer initiating cells . s100a4 controls the notch and pten / pi3k / akt signaling pathways , which regulate the self - renewal and tumorigenicity of stem cells or cancer stem cells . s100a4 signaling pathways thus play a major role in the maintenance of cancer initiating cell population and contribute for the tumor recurrence . in summary , the data from our study adds weight to the growing body of evidence that s100a4 can be used to identify the subgroup of patients at high risk of metastasis and recurrence in oscc cases , and that targeting s100a4 signaling might be a potential therapeutic target for increasing the patient survival .	s100a4 , a biomarker of epithelial mesenchymal transition ( emt ) , plays an important role in invasion and metastasis by promoting cancer cell motility . in oral squamous cell carcinoma ( oscc ) , metastasis results in 90% of cancer associated mortality.objectiveto investigate the role of s100a4 expression as an important component of the epithelial mesenchymal transition ( emt ) program in oral squamous cell carcinoma ( oscc).material and methodss100a4 protein expression was assessed semi - quantitatively by immunohistochemistry in 47 histologically confirmed cases of oral squamous cell carcinoma ( oscc ) and 10 normal oral mucosal biopsies . the association between the s100a4 overexpression and the aggressive features of oscc were analyzed by x2 test.resultsmoderate to strong cytoplasmic expression of s100a4 was observed in 30 out of 47 specimens of oscc ( 64% ) . overexpression of s100a4 was significantly associated with the clinical stage , lymph node involvement , metastases , pattern of invasion and recurrence ( p<0.05).conclusions100a4 expression represents an important biomarker of prognostic significance that may be used to identify a subset of patients at high risk of invasion and metast
general remarks . all operations should be performed under an upright dissecting microscope ( leica ) and by using a surgical coagulator . the mice in the experimental and sham groups should be matched in age and weight to ensure comparability of the results . temperature , blood pressure , anesthesia and fluid administration should be stable and monitored during the entire experiment . c57bl/6 mice should be within an age range of 10 to 16 weeks and they should be gender - matched in the respective groups . anesthetize mice with sodium pentobarbital ( 70 mg / kg body weight i.p . ) and maintain anesthesia with approx . place mice on a temperature - controlled heated table ( rt , effenberg , munich , germany ) with a rectal thermometer probe attached to a thermal feedback controller to maintain body temperature at 37 c . place mice in a supine position on the surgery table , with the upper and lower extremities attached to the table with removable tape . in case of survival experiments proper aseptic techniques including clipping of hair , disinfection of skin with iodophors ( e.g. betadine ) , sterile handling of autoclaved instruments , and using masks cover the incision area with mineral oil to prevent inhalation of mouse hair by cutting the skin and thus preventing the risk of mouse hair allergies . perform midline laparotomy and incision of the linea alba up to the upper end so that you can easily expose the liver . if possible perform the incision with a coagulation electrode ( erbe , icc50 , tuebingen , germany ) to prevent bleeding . place the heating table with the mouse in a position that the tail is directed to the surgeon . that position provides you with the best setting to localize the triad and to place the suture . displace the intestine on the right side by using a wet cotton tip swab to expose the portal triad . slightly shift the median and left lobe towards the diaphragm and the right lobe towards the intestine . that will give you a clear view of the portal triad above the bifurcation of right , median / left lobes . once visually identified place the needle with the suture ( 7/0 nylon suture ; ethicon , norderstedt , germany ) via a needle holder under the left portal triad , including the hepatic artery , hepatic vein , and bile duct from the left to the right side of the mouse . a deeper stitch can injure the vena vaca inferior which is located under the triad . therefore hold the right liver lobes down with the forceps to identify the vena cava before placing the stitch . take the tip of the needle on the right side with a forceps and pull it gently out of the liver area without injuring the liver . attach to each end of the suture an eppendorf tube ( approximately 3 g , filled with water ) and place both suture ends over suture holders . place the suture holder distally of the stitch so that the suture does not interfere with the right triad . by applying the weights the triad will be immediately occluded , causing interruption of blood supply to the left and median lobes of the liver . successful occlusion can be easily confirmed by visual inspection of pale blanching of the ischemic lobes . in contrast , the change of color immediately disappears when the hanging weights are released from the poles and the liver is reperfused . this partial hepatic ischemia model avoids mesenteric congestion by preserving blood flow to the right liver lobes . keep the liver and intestine warm with a wet swab soaked with water at 37.0 c . in addition to keep body temperature stable cover the mice with commercially available food wrap after finishing surgery . perform ischemia as required per experimental protocol . after a defined ischemia time release the weights and reperfusion of the median and left liver lobes will start immediately . add approximately 400 l sodium chloride into the abdominal cavity 15 minutes before and directly after reperfusion . continue to substitute fluid every 30 minutes in an amount of 100 l to compensate fluid loss during the reperfusion time . for survival experiments the surgical wound is closed using continuous suture of the muscle wall and skin with a 4/0 suture . the following outcome parameters are recommended to determine liver injury following hepatic ischemia : determine liver enzymes ( e.g. alt , ast ) and liver histology ( h&e staining ) following liver ischemia and reperfusion . model of a hanging - weight system for liver ischemia : a hanging weight system has been established to induce liver ischemia of the left and median lobe . ( a ) therefore the caudate and right lobe were gently separated from the left lobe . the right lobe was then slightly shifted to clearly view the portal triad above the bifurcation of right , median and left lobes . ( b ) at the end of each suture a small weight ( 3 g eppendorf tubes filled with water ) was attached and applied . hepatic ischemia is visible by a color change of the left and median lobe from red to pale . this experimental design allows precise occlusion and reperfusion of the left hepatic triad by applying and releasing the weight load . effect of different liver ischemia times on alanine ( alt ) and aspartate ( ast ) aminotransferase in mice . partial portal triad ischemia was induced as indicated ( 0 - 60 min ischemia ) . after 2 hr of reperfusion , alt ( a ) and ast ( b ) were measured . c57bl/6 mice should be within an age range of 10 to 16 weeks and they should be gender - matched in the respective groups . anesthetize mice with sodium pentobarbital ( 70 mg / kg body weight i.p . ) and maintain anesthesia with approx . overdosing can significantly lower blood pressure and thus can alter the results . place mice on a temperature - controlled heated table ( rt , effenberg , munich , germany ) with a rectal thermometer probe attached to a thermal feedback controller to maintain body temperature at 37 c . place mice in a supine position on the surgery table , with the upper and lower extremities attached to the table with removable tape . in case of survival experiments proper aseptic techniques including clipping of hair , disinfection of skin with iodophors ( e.g. betadine ) , sterile handling of autoclaved instruments , and using masks a sterile gloves for the surgeon are required . cover the incision area with mineral oil to prevent inhalation of mouse hair by cutting the skin and thus preventing the risk of mouse hair allergies . perform midline laparotomy and incision of the linea alba up to the upper end so that you can easily expose the liver . if possible perform the incision with a coagulation electrode ( erbe , icc50 , tuebingen , germany ) to prevent bleeding . place the heating table with the mouse in a position that the tail is directed to the surgeon . that position provides you with the best setting to localize the triad and to place the suture . displace the intestine on the right side by using a wet cotton tip swab to expose the portal triad . slightly shift the median and left lobe towards the diaphragm and the right lobe towards the intestine . that will give you a clear view of the portal triad above the bifurcation of right , median / left lobes . once visually identified place the needle with the suture ( 7/0 nylon suture ; ethicon , norderstedt , germany ) via a needle holder under the left portal triad , including the hepatic artery , hepatic vein , and bile duct from the left to the right side of the mouse . a deeper stitch can injure the vena vaca inferior which is located under the triad . therefore hold the right liver lobes down with the forceps to identify the vena cava before placing the stitch . take the tip of the needle on the right side with a forceps and pull it gently out of the liver area without injuring the liver . attach to each end of the suture an eppendorf tube ( approximately 3 g , filled with water ) and place both suture ends over suture holders . place the suture holder distally of the stitch so that the suture does not interfere with the right triad . by applying the weights the triad will be immediately occluded , causing interruption of blood supply to the left and median lobes of the liver . successful occlusion can be easily confirmed by visual inspection of pale blanching of the ischemic lobes . in contrast , the change of color immediately disappears when the hanging weights are released from the poles and the liver is reperfused . this partial hepatic ischemia model avoids mesenteric congestion by preserving blood flow to the right liver lobes . keep the liver and intestine warm with a wet swab soaked with water at 37.0 c . in addition to keep body temperature stable cover the mice with commercially available food wrap after finishing surgery . perform ischemia as required per experimental protocol . after a defined ischemia time release the weights and reperfusion of the median and left liver lobes will start immediately . add approximately 400 l sodium chloride into the abdominal cavity 15 minutes before and directly after reperfusion . continue to substitute fluid every 30 minutes in an amount of 100 l to compensate fluid loss during the reperfusion time . for survival experiments the surgical wound is closed using continuous suture of the muscle wall and skin with a 4/0 suture . the following outcome parameters are recommended to determine liver injury following hepatic ischemia : determine liver enzymes ( e.g. alt , ast ) and liver histology ( h&e staining ) following liver ischemia and reperfusion . model of a hanging - weight system for liver ischemia : a hanging weight system has been established to induce liver ischemia of the left and median lobe . ( a ) therefore the caudate and right lobe were gently separated from the left lobe . the right lobe was then slightly shifted to clearly view the portal triad above the bifurcation of right , median and left lobes . ( b ) at the end of each suture a small weight ( 3 g eppendorf tubes filled with water ) was attached and applied . hepatic ischemia is visible by a color change of the left and median lobe from red to pale . this experimental design allows precise occlusion and reperfusion of the left hepatic triad by applying and releasing the weight load . effect of different liver ischemia times on alanine ( alt ) and aspartate ( ast ) aminotransferase in mice . partial portal triad ischemia was induced as indicated ( 0 - 60 min ischemia ) . after 2 hr of reperfusion , alt ( a ) and ast ( b ) were measured . the present study describes a technique of performing ischemia in a murine model by using a hanging - weight system for occlusion of the left hepatic portal triad . although it is an easy method to learn the following pitfalls should be taken into account . a very common mistake in the beginning is a deep stitch which includes the portal triad and the vena cava . the breathing and heart rate will slow down within 15 minutes due to the increased resistance and the decrease in returning blood to the heart resulting in an early death of the mice . the best way to prevent the including of the vena cava is to localize the vena cava before the stitch by holding the right lobes down with a forceps . another important issue is that the weights are not to heavy which can result in too much stress on the left and right portal trial which also stops blood flow to the right lobes . therefore the amount of water in the eppendorf vials should be adjusted to the mouse weight and the suture holders should be placed distally from the stitch location . on the opposite both mistakes are easily detectable by an additional color change to pale of the right lobe or insufficient color change of the median and left lobes , respectively . therefore the hepatic color change following applying the weights should be controlled every 10 minutes . therefore the right lobes should be held down with a bland forceps so that the tip of the needle can be easily seen and taken with a needle holder . if these pitfalls in the beginning are taken into account the hanging - weight system for hepatic ischemia provides a highly reproducible injury due to ischemia by minimizing the variability of liver damage associated with clamping of the portal triad . by using a hanging - weight system , the portal triad is only manipulated once throughout the entire surgical procedure , causing significantly less damage to the hepatic lobes . more tissue trauma occurs by removing and replacing vessel clamps , especially with reapplication of the clamp during multiple ischemia cycles like during ischemic preconditioning . the use of hanging weights that are in a remote location from the liver tissue , according to our observations , provides the advantage of reliable occlusion while preventing tissue trauma due to manipulation of the liver lobes by reapplication of a clamp . taken together , the present study provides feasibility of the hanging - weight system for portal triad occlusion during ischemia , minimizing the variability and limitations associated with clamping . thus this technique might be of interest for other investigators who consider studying hepatic ischemia .	acute liver injury due to ischemia can occur during several clinical procedures e.g. liver transplantation , hepatic tumor resection or trauma repair and can result in liver failure which has a high mortality rate1 - 2 . therefore murine studies of hepatic ischemia have become an important field of research by providing the opportunity to utilize pharmacological and genetic studies3 - 9 . specifically , conditional mice with tissue specific deletion of a gene ( cre , flox system ) provide insights into the role of proteins in particular tissues10 - 13 . because of the technical difficulty associated with manually clamping the portal triad in mice , we performed a systematic evaluation using a hanging - weight system for portal triad occlusion which has been previously described3 . by using a hanging - weight system we place a suture around the left branch of the portal triad without causing any damage to the hepatic lobes , since also the finest clamps available can cause hepatic tissue damage because of the close location of liver tissue to the vessels . furthermore , the right branch of the hepatic triad is still perfused thus no intestinal congestion occurs with this technique as blood flow to the right hepatic lobes is preserved . furthermore , the portal triad is only manipulated once throughout the entire surgical procedure . as a result , procedures like pre - conditioning , with short times of ischemia and reperfusion , can be easily performed . systematic evaluation of this model by performing different ischemia and reperfusion times revealed a close correlation of hepatic ischemia time with liver damage as measured by alanine ( alt ) and aspartate ( ast ) aminotransferase serum levels3,9 . taken together , these studies confirm highly reproducible liver injury when using the hanging - weight system for hepatic ischemia and intermittent reperfusion . thus , this technique might be useful for other investigators interested in liver ischemia studies in mice . therefore the video clip provides a detailed step - by - step description of this technique .
hepatitis c virus ( hcv ) is a small , enveloped positive sense , single stranded rna virus that belongs to the genus hepacivirus and the family flaviviridae . it is estimated that 3% of the world s population ( 170 million people ) are infected with hcv ( 1 ) and its prevalence ranges from 0.2% to 40% in different countries ( 2 , 3 ) . hcv is a major cause of chronic viral hepatitis , liver cirrhosis and hepatocellular carcinoma . therefore it has important clinical , epidemiological and economic outcomes throughout the world especially in developing countries ( 4 , 5 ) . genotyping of hepatitis c virus is important because different genotypes have different infectivity and influencing the rate of progression of hcv infection to cirrhosis and hepatocellular carcinoma . besides various hcv genotypes for example , genotypes 1 and 4 are more resistant to interferon based therapies than genotypes 2 and 3 ( 68 ) . currently , hcv is classified into eleven genotypes ( 111 ) with sequence differences range from 30% to 50% that six of them are the major genotypes ( 1 to 6 ) ( 6 , 9 ) . within hcv genotype , several subtypes ( named as a , b , c , etc . ) can be defined by sequence differences range from 15% to 30% ( 10 , 11 ) . types 1a and 1b are the most common , accounting for about 60% of global infections . they predominate in northern europe and north america , and in southern and eastern europe and japan , respectively . genotype 4 is principally detected in the middle east , egypt , north and central africa . type 5 is mostly found in southern africa and genotypes 611 are distributed in asia ( 1216 ) . the determination of the hcv genotypes is important for the prediction of response to antiviral treatment . the distribution of hcv genotypes was reported from various regions of iran ( 1720 ) ; however , such studies were not conducted in arak city . therefore , this investigation was performed to determine the distribution of hcv genotypes in arak , central province of iran . in this retrospective study , 174 cases with chronic hcv infection [ positive result of hepatitis c antibody ( anti - hcv ) more than 6 months and positive hcv - rna ] referred to infectious center of valiasr hospital , arak ; central province of iran were enrolled . hcv infection was confirmed by positive hcv antibody ( anti - hcv ) and hcv - rna tests . all the cases were negative for hepatitis b surface antigen ( hbsag ) and human immunodeficiency virus ( hiv ) antibodies . a questionnaire was used to collect data such as age , sex and possible route of hcv transmission . a peripheral blood sample from each patient was collected in an edta containing sterile tube . anti - hcv and hbsag were tested by enzyme - linked immunosorbent assay ( elisa ) . the commercial enzyme immunoassay kits used were as follows : hbsag ( hepanosticka biomerieux , boxtel , the netherlands ) and anti - hcv ( biorad , segrate , italy ) . recombinant immunoblot assay ( riba innogenetics , ghent , belgium ) was employed to confirm anti - hcv reactivity . anti - hiv was determined by elisa ( mp biomedicals , illkirch , france ) ; with positive tests confirmed by western blot assay ( diaplus , san francisco , usa ) . viral rna was extracted from plasma sample using high pure viral nucleic acid kit ( roche diagnostics gmbh , mannheim , germany ) , according to the manufacturer s instructions . hcv genotyping was carried out using a commercial kit ( sacace , italy ) according to manufacturer s instruction . this kit is designed for the detection of genotypes 1a , 1b , 2 , 3a ( which are most prevalent hcv genotypes in iran ) by generating different size pcr products . the specificity and sensitivity of the kit are 100% and 1000 viral particles / ml respectively . for each patient two sets of pcr amplifications were performed in two separate tubes containing primers for either genotypes 1a+1b or genotypes 2 + 3a . 5 l of cdna was subjected to pcr amplifications using two sets of mixed primers included in the kit . the pcr profile was an initial denaturation at 95c for 5 min , followed by 42 cycles of 95c for 1 min , 68c for 1 min , 72c for 1 min and a final extension at 72c for 10 min . genotype 1a generates a 338 bp pcr product , genotype 1b , 2 and 3a generate 395 , 286 and 227 bp pcr products respectively . the pcr products were electrophoresed in a 2% agarose gel and stained with ethidium bromide . after determination of hcv genotypes , patients with genotypes 2 or 3 , received 800 mg ribavirin ( rbv ) daily in two divided doses for 24 weeks . patients with genotype 1 , received 1000 mg rbv daily if they weighed less than 75 kg and 1200 mg if over 75 kg for 48 weeks . sustained virologic response ( svr ) which is defined as undetectable hcv - rna using a highly sensitive assay ( real time polymerase chain reaction ) was determined in patients after 24 weeks of the end of treatment . the chi - square was used with the spss 16 package program for statistical analysis ( chicago , il , usa ) . data are presented as mean sd or , when indicated , as an absolute number and percentage . in this retrospective study , 174 cases with chronic hcv infection [ positive result of hepatitis c antibody ( anti - hcv ) more than 6 months and positive hcv - rna ] referred to infectious center of valiasr hospital , arak ; central province of iran were enrolled . hcv infection was confirmed by positive hcv antibody ( anti - hcv ) and hcv - rna tests . all the cases were negative for hepatitis b surface antigen ( hbsag ) and human immunodeficiency virus ( hiv ) antibodies . a questionnaire was used to collect data such as age , sex and possible route of hcv transmission . a peripheral blood sample from each patient was collected in an edta containing sterile tube . anti - hcv and hbsag were tested by enzyme - linked immunosorbent assay ( elisa ) . the commercial enzyme immunoassay kits used were as follows : hbsag ( hepanosticka biomerieux , boxtel , the netherlands ) and anti - hcv ( biorad , segrate , italy ) . recombinant immunoblot assay ( riba innogenetics , ghent , belgium ) was employed to confirm anti - hcv reactivity . anti - hiv was determined by elisa ( mp biomedicals , illkirch , france ) ; with positive tests confirmed by western blot assay ( diaplus , san francisco , usa ) . viral rna was extracted from plasma sample using high pure viral nucleic acid kit ( roche diagnostics gmbh , mannheim , germany ) , according to the manufacturer s instructions . hcv genotyping was carried out using a commercial kit ( sacace , italy ) according to manufacturer s instruction . this kit is designed for the detection of genotypes 1a , 1b , 2 , 3a ( which are most prevalent hcv genotypes in iran ) by generating different size pcr products . the specificity and sensitivity of the kit are 100% and 1000 viral particles / ml respectively . for each patient two sets of pcr amplifications were performed in two separate tubes containing primers for either genotypes 1a+1b or genotypes 2 + 3a . 5 l of cdna was subjected to pcr amplifications using two sets of mixed primers included in the kit . the pcr profile was an initial denaturation at 95c for 5 min , followed by 42 cycles of 95c for 1 min , 68c for 1 min , 72c for 1 min and a final extension at 72c for 10 min . genotype 1a generates a 338 bp pcr product , genotype 1b , 2 and 3a generate 395 , 286 and 227 bp pcr products respectively . the pcr products were electrophoresed in a 2% agarose gel and stained with ethidium bromide . after determination of hcv genotypes , patients with genotypes 2 or 3 , received 800 mg ribavirin ( rbv ) daily in two divided doses for 24 weeks . patients with genotype 1 , received 1000 mg rbv daily if they weighed less than 75 kg and 1200 mg if over 75 kg for 48 weeks . sustained virologic response ( svr ) which is defined as undetectable hcv - rna using a highly sensitive assay ( real time polymerase chain reaction ) was determined in patients after 24 weeks of the end of treatment . the chi - square was used with the spss 16 package program for statistical analysis ( chicago , il , usa ) . data are presented as mean sd or , when indicated , as an absolute number and percentage . a total of 174 patients with chronic hcv infection with mean age 37.510.24 ( range : 1876 years ) were enrolled in the study . 170 ( 97.7% ) of cases were male and 4 ( 2.3% ) were female . the main route of hcv transmission was injection drug use ( idu ) which was observed in 104 ( 59.8% ) of cases . other presumed routes of hcv transmission were needle stick in 12 ( 6.9% ) , blood and blood products transfusion in 5 ( 2.9% ) , incarceration in 4 ( 2.3% ) , heterosexual contact in 1 ( 0.6% ) , tattooing in 1 ( 0.6% ) , idu and tattooing in 7 ( 4% ) , idu and blood transfusion in 7 ( 4% ) , idu and incarceration in 6 ( 3.4% ) , idu and heterosexual contact in 3 ( 1.7% ) , tattooing and heterosexual contact in 2 ( 1.2% ) , idu , tattooing and incarceration in 2 ( 1.2% ) , idu , tattooing and heterosexual contact in 3 ( 1.6% ) and in 17 ( 9.8% ) the route of hcv acquisition was not identified . genotyping results demonstrated that subtype 3a 92 ( 52.9% ) was the most prevalent hcv type in arak , followed by subtype 1a 40 ( 22.9% ) and subtype 1ab 31 ( 17.8% ) . hcv genotypes distribution was not significantly associated with age and gender ( p=0.07 and 0.06 respectively ) . distribution of hcv genotypes and subtypes in arak city sustained virologic response was observed in 78 ( 83.8% ) of cases . svr was detected in 42/4 ( 91.3% ) of patients infected with genotype 3a versus 17/22 ( 77.3% ) of those infected with genotype 1a and 9/13 ( 69.2% ) of cases infected with genotype 1ab . epidemiological studies in different region of the world show the wide variation and regional differences in the distribution of hcv genotypes ( 21 ) . hcv genotyping is an important factor in clinical hcv treatment and investigation of hcv genotypes distribution is essential as prognostic factor in chronic hcv infection ( 11 , 22 ) . hcv genotypes are studied in different parts of iran ( 1720 ) . in this survey which is the first study of hcv genotypes distribution in arak city , central province of iran , we showed that subtype 3a ( 52.9% ) was the most prevalent hcv type , followed by subtype 1a ( 22.9% ) and subtype 1ab ( 17.8% ) . ( 23 ) on specimens from 16 provinces of iran revealed that type 3a was the most frequent hcv type ( 46.6% ) , followed by type 1 with rate of 43.2% ( including 1a and 1b with 25.73% and 17.47% for each respectively ) . reviewed the prevalence of hcv genotypes in iranian patients and reported that hcv subtype 1a was maximum in east azarbayjan and guilan respectively but minimum in west azarbayjan . prevalence of subtype 1b was 25% , 25% and 23% in kermanshah , zahedan and hormozgan respectively . prevalence of genotype 2 was 0.7% in tehran and 17.2% in kermanshah ( 24 ) . in a study by esmaeilzadeh et al . ( 17 ) in zanjan ( the northwest of iran ) , subtype 3a was the most current subtype in this city . in another study by omrani et al . ( 25 ) hcv genotype 3a with the prevalence rate of 48.1% was the most prevalent hcv type in west azerbaijan , northwest of iran . a study in shiraz , a city in southern part of iran revealed that the highest level of hcv infection belonging to type 3a followed by 1a ( 26 ) . additionally genotype 3 was the most common genotype in plasma and peripheral blood mononuclear cells ( pbmcs ) of hcv infected patients in jahrom city ( another city in southern part of iran ) ( 27 ) . besides , genotype 3a is the most frequent hcv genotype in isfahan province , iran . hcv subtypes 3a and 1a were determined in the hemodialysis patients living in tehran as the prevalent subtypes ( 28 ) . on the other hand genotype 1a was the more frequent hcv genotype in khorasan razavi province ( northeast of iran ) ( 29 ) . in another survey in southeast of iran , genotype 1 was the more prevalent hcv type followed by genotype 3 , 4 and 2 ( 30 ) . ( 41.7% ) followed by 3a , 2 and 4 were reported as most frequent hcv genotypes in khuzestan province , southwestern iran ( 18 ) . jahanbakhsh sefidi et al.(31 ) showed that in 2003 , genotype 1a was the most common hcv genotype in iran ( 47.8% ) but it decreased over time and hcv genotype 3a was increased in this period of time ( 30.1% in 2003 which was increased up to 39.6% in 2011 . ( 32 ) also reported that while hcv subtype 1a is predominant among hcv infected iranian subjects , subtype 3a is predominant among iranian injecting drug users . our results are in accordance with a recent study from yazd city , another town in central province of iran which showed that hcv genotype 3a was the predominant genotype followed by the subtypes 1a and 1b ( 33 ) and it was also compatible with the results reported from another parts of iran ( 17 , 2528 ) . on the other hand , genotype 3a is significantly associated with hcv transmission through injection drug use ( 34 ) , which is the main route of hcv transmission in our participants that was observed in 59.8% of cases . in our study , svr was more observed in patients infected with genotype 3a versus those infected with genotype 1a and 1ab , which was in accordance to this issue that genotype 3a revealed a great svr among interferon - treated patients , compared to genotype 1(68 ) . this study had limitations like small sample size and lack of the correlation between hcv genotypes and the severity of the disease among the studied population . this study showed that hcv subtype 3a was the most prevalent hcv type , followed by subtype 1a and subtype 1ab in arak , central province of iran . determination of hcv genotype distribution could affect the hcv therapeutic methods and duration and could increase the chance of successful treatment . investigation of hcv genotypes in different parts of the country is needed to facilitate treatment options and preventive strategies .	background and objectives : hepatitis c virus ( hcv ) infection is a worldwide concern and it is the major cause of liver disease . several genotypes of the hcv have been reported from different regions of the world . the determination of the hcv genotypes is important for the prediction of response to antiviral treatment and clinical outcomes . so , hcv genotyping in each region is of great importance . this investigation was performed to determine the distribution of hcv genotypes in arak city , central province of iran.patients & methods : in this cross sectional study , 174 cases with chronic hcv infection from arak city were enrolled . hcv infection was confirmed by positive results in hcv antibody ( anti - hcv ) and hcv - rna tests . hcv genotypes were determined using a pcr based genotyping kit.results:a total of 174 hcv infected patients with mean age of 37.510.24 years were enrolled . 97.7% of cases were male and 2.3% were female . the main route of hcv transmission was injection drug use ( idu ) which was observed in 59.8% of cases . genotyping results demonstrated that subtype 3a ( 52.9% ) was the most prevalent hcv type in arak , followed by subtype 1a ( 22.9% ) and subtype 1ab ( 17.8%).conclusion : this study showed that hcv subtype 3a was the most prevalent hcv type , followed by subtype 1a and subtype 1ab in arak , central province of iran . investigation of hcv genotypes in different parts of the country is needed to facilitate treatment options and preventive strategies .
small , autonomously folding peptides that display -sheet secondary structure in the absence of a specific tertiary context are useful tools for exploring intrinsic relationships between sequence and -sheet stability and for establishing characteristic spectroscopic signatures of this motif . in addition , autonomously folding systems have been valuable for probing noncovalent interactions involving post - translationally introduced units . engineered -sheets have been used to explore preferred modes of intermolecular -strand associations , phenomena that underlie amyloid formation ; such -sheet designs may ultimately lead to diagnostic or therapeutic tools for amyloid diseases . design of -sheet - forming peptides is challenging because the natural residues that promote this secondary structure are hydrophobic ( ile , val , phe , trp , tyr ) or only moderately polar ( thr ) . therefore , sequences intended to adopt -sheet conformations but not self - associate in aqueous solution must be rich both in -promoting residues and in solubility - promoting residues , the latter usually bearing charged side chains . the necessity of using distinct sets of residues for conformational propensity and solubility represents a significant design constraint . here we explore the hypothesis that -sheet propensity and peripheral charge can be combined in a single -amino acid residue . side chain branching adjacent to the backbone ( -branching ) , as in ile , val and thr , is correlated with high -sheet propensity ; therefore , we have synthesized and evaluated new amino acids that contain both a -branch point and an ionizable group in the side chain ( figure 1 ) . our initial design hypothesis focused on derivatives of threonine in which the side chain hydroxyl is used to form an ether linkage to a unit bearing an ionizable group . scheme 1 shows the synthesis of protected amino acid 3 from aziridine 1 , which was generated from l - threonine via well - precedented methods . have shown that nucleophiles can open closely related thr - derived aziridines in the presence of bf3 with high stereoselectivity , and this approach was useful in our case . the residue derived from 3 , which we designate to , was readily incorporated into synthetic peptides via fmoc - based solid - phase synthesis . to can be viewed as an analogue of lys that contains a -branch point in the side chain . we used 12-mer peptide i as a benchmark for evaluating the -sheet propensity of to and other new residues described below ( figure 1 ) . the conformational behavior of i was previously characterized via nmr . in aqueous solution this peptide adopts an antiparallel two - stranded -sheet conformation ( -hairpin ) with a reverse turn at the dpro - gly segment . d - proline was employed to promote a type ii reverse turn , which favors -sheet interactions between flanking strands composed of l - residues . nmr data indicate 68% population of the -hairpin conformation at 4 c in aqueous buffer ( ph 3.8 ) . peptide ii is the analogue of i in which lys-9 has been replaced with to . 2d nmr analysis suggested significant population of the -hairpin conformation at 4 c in aqueous buffer ( ph 3.8 ) . the noes observed for ii are consistent with adoption of the expected -hairpin conformation . for additional insight , we compared chemical shifts for protons ( ch ) of residues within i and ii . ch values are sensitive to secondary structure , with residues that participate in -helix displaying upfield shifts and residues that participate in -sheet showing downfield shifts relative to random coil values . figure 2a shows ch data , the deviation from the random coil chemical shift at each residue , for peptides i and ii . the lpro-6 diastereomers of i and ii were used to provide the random coil ch values because we have previously shown that replacing dpro with lpro abolishes -hairpin folding in i and comparable designs . for both i and ii , ch 0.2 ppm for the segments tyr-2 to val-5 and orn-8 to ile-10 , which is consistent with the expected locations of the two -strand segments . the consistent negative ch values for leu-11 presumably arise from the proximity of the tyr side chain to leu-11 ch in the -hairpin conformation . the near - zero values at dpro-6 and gly-7 are consistent with the expected turn , and the near - zero values at arg-1 and gln-12 are consistent with fraying at the termini . comparison of ch values for the ( a ) parent , ts , and to peptides and ( b ) parent and ts peptides . the ch values at or adjacent to the substitution can not be directly compared as the substitution changes the dynamic range . ch values at hydrogen bonding positions in the core of the peptide have been shown to most accurately reflect the population of the -hairpin . the ch comparison between i and ii suggests that the new to residue may have a slightly lower -sheet propensity than does lys . this surprising conclusion emerges because the absolute ch values for ii are smaller than those for i at most strand residues common to the two peptides . the unexpected behavior of to led us to re - evaluate our design hypothesis , which focused exclusively on -branching in the side chain . the side chain oxygen of thr can form an h - bond with a nearby backbone n h , which leads to thr backbone torsion angles ( and ) more compatible with the -helical than -sheet secondary structure . if the h - bond acceptor ability of the ether oxygen in to works against -sheet propensity , then we hypothesized that the desired conformational properties should be achieved by replacing this oxygen atom with sulfur , to generate ts . a protected -amino acid that could be used to incorporate ts residues was prepared as shown in scheme 1 ; the key step was use of trityl thiol to open the thr - derived aziridine . protected amino acid 5a allowed solid - phase synthesis of peptide iii , which contains ts in place of lys-9 of i. 2d nmr analysis of iii revealed interstrand noes consistent with significant population of the expected -hairpin conformation in aqueous buffer at 4 c . resonance overlap hindered identification of noes involving the tyr-2 side chain at this temperature , but at 10 c such noes could be detected ( figure 3 ) . cross - strand noes observed for peptide iii at 10 c in ph 3.8 aqueous buffer . ch data for iii ( figure 2a ) suggest that the extent of -hairpin formation is higher for this peptide than for ii , which contains the to residue , because the absolute value of ch is larger at each strand residue ( positions 25 and 811 ) for iii than for ii . moreover , the ch values are larger at most strand positions for iii relative to parent peptide i , which suggests that the ts residue stabilizes the -hairpin conformation relative to lys . we explored the scope of this design strategy by evaluating the residue ts , an analogue of ts that bears an acidic rather than a basic side chain along with a thioether - based -branch point . peptide iv , prepared using 5b , is an analogue of i in which glu-4 is replaced by ts . the noes observed at 4 c are consistent with the formation of the expected hairpin . ch data for iv ( figure 2b ) suggest that this peptide forms a more stable -hairpin than that formed by i. circular dichroism spectra of peptides i , iii , and iv ( phosphate buffer , ph = 7.0 ) are consistent with the formation of a hairpin and the stability trends observed by nmr . ch - based population analysis was undertaken in order to estimate the thermodynamic impact of replacing lys-9 with either to or ts or replacing glu-4 with ts . residues 3 , 5 , 8 , and 10 occupy hydrogen bonded positions within the -hairpin conformation adopted by i such peptides are presumed to equilibrate rapidly between -hairpin and unfolded states on the nmr time scale . the ch value measured at each indicator residue represents a populated - weighted average of the contributions from the fully unfolded state and the fully folded ( i.e. , -hairpin ) state . for each sequence , we use the lpro-6 diastereomer to estimate ch for each indicator residue in the fully unfolded state , and we use a macrocyclized analogue with dpro gly turns at both ends to estimate ch in the fully folded state . table 1 shows the -hairpin population deduced in this way at each of the four indicator positions in peptides i iv . the variation among the four values for each molecule is a measure of the intrinsic uncertainty associated with this quantification strategy , which involves independent analysis at four distinct sites within each peptide . despite this uncertainty , the data support the qualitative conclusion that replacing lys-9 of i with to ( ii ) has little effect on -hairpin population , while replacing lys-9 with ts ( iii ) or glu-4 with ts ( iv ) leads to enhanced -hairpin population . the g was calculated for each reporter position in the hairpin and then averaged . iv relative to parent i , and for iii relative to ii , based on the residue - specific population . this thermodynamic analysis shows that replacing lys with to , the basic thr derivative with an ether linkage at the branch point , fails to enhance -hairpin stability , which invalidates our original design hypothesis . in contrast , the analogous basic residue containing a thioether linkage at the branch point , ts , stabilizes the -hairpin conformation . although the gfold values for ii vs i and for iii vs i are not cleanly distinguished given the uncertainties in table 1 , direct comparison of iii vs ii shows unambiguously that ts enhances -hairpin stability relative to to . the magnitude of the -sheet stabilization provided by ts or ts relative to proteinogenic residues with unbranched side chains , lys and glu , respectively , is significant given that the natively folded state of a typical globular protein , containing a few hundred residues , is only 5 kcal / mol more stable than the unfolded state . it should be noted that differences between ch values ( bar heights ) from figure 2a and % folded values from table 1 are not directly comparable . each ch value in figure 2a is determined by the difference between the chemical shift ( ch ) for a given residue in one of three peptides ( i , ii or iii ) and the analogous chemical shift from a reference peptide that has l - proline in place of d - proline . the % folded values in table 1 are generated from an algebraic expression based on three chemical shifts , the two mentioned above and the ch value for the analogous cyclic peptide , which represents the fully folded state . since this last value can vary among macrocyclic peptides with different sequences , quantitative correlations between ch values ( figure 2a ) and % folded values ( table 1 ) may not be evident at specific residues , particularly at or adjacent to substitution positions . in addition , another factor may prevent such quantitative correlations : the relationship between changes in ch and changes in % folding is not linear . for these reasons , and to account for local factors that can influence ch values at particular residues , we draw conclusions based on measurements at multiple sequence positions . circular dichroism ( cd ) provides information on peptide folding that is intrinsically of lower structural resolution than the insights available from 2d nmr ; however , cd can be very useful for qualitative comparisons . cd data in the far - uv region ( 190250 nm ) arise largely from the backbone amide groups and therefore report on secondary structure . the far - uv signatures of peptides i , iii , and iv obtained in the buffer used for nmr studies , 100 mm acetate buffer , ph 3.8 , all manifest a minimum at 215 nm ( figure 5 ) , which is characteristic of -sheet secondary structure and therefore consistent with nmr data for these peptides . cd data for peptides ( a ) i , ( b ) iii , and ( c ) iv in phosphate buffer ( ph 7.0 ) or acetate buffer ( ph 3.8 ) . cd data for peptides ( d ) i , ( e ) iii , and ( f ) iv in phosphate buffer at 4 , 20 , and 37 c . for each peptide , the far - uv cd spectrum obtained in the nmr buffer is not significantly different from the spectrum obtained in 100 mm phosphate buffer , ph 7.0 ( figure 5a c ) , which indicates that -hairpin folding is not ph - dependent over this range . this observation is important because the glu or ts side chain carboxyl group is likely to be largely protonated at ph 3.8 , but fully deprotonated at ph 7.0 . varying temperature from 4 to 20 to 37 c does not significantly alter the far uv cd of i , iii or iv ( figure 5d f ) , which suggests that conclusions drawn from nmr analysis at low temperature are relevant to room or physiological temperatures . we turned to a different -hairpin system to explore the generality of the behavior observed in derivatives of peptide i for new residues to , ts and ts ( figure 6 ) . the design of peptide v was based on the sequence of a -hairpin that occurs at the n - terminus of ubiquitin ; a central dpro previously reported nmr data suggest that the expected -hairpin conformation is significantly populated in aqueous solution . we prepared analogues of v in which lys-15 is replaced with to ( vi ) or ts ( vii ) . the ch data indicate that replacing lys-15 with to does not lead to a significant change in -hairpin population , because the ch values for vi are within error of those for v at strand positions gln-2 to ser-7 ( figure 7 ) . in contrast , replacing lys-15 with ts results in consistently higher ch values among the n - terminal strand residues ( figure 7 ) . these conclusions are similar to those derived from comparisons among i - iii , which also differ at a single residue ( lys vs to vs ts ) . to evaluate ts in this -hairpin system , we evaluated viii , the derivative of v in which ile-3 has been replaced by glu . ch analysis indicates that this change causes a significant decline in -hairpin population , which may reflect two factors , the loss of -branching and the loss of a hydrophobic side chain . replacing glu-3 of viii with ts , to generate ix , leads to an increase in -hairpin population , as judged by ch data ; however , these data indicate that the ts residue in ix is not as effective as the ile residue of v in terms of stabilizing the -hairpin conformation . despite the apparently diminished -sheet propensity of the ts residue relative to the more hydrophobic ile residue , ts represents a useful design tool because one can match the charge provided by glu while enhancing the folding tendency . comparison of ch values for the parent , ts , and to peptides . the ch values at or adjacent to the substitution can not be directly compared as the substitution changes the dynamic range . the ch values at hydrogen bonding positions in the core of the peptide have been shown to most accurately reflect the population of the -hairpin . our design strategy is based on the assumption that new amino acid residues with a side chain that contains both a -branch point and an ionizable group will be more hydrophilic than the proteinogenic -branched residues ( thr , ile and val ) . to test this hypothesis , we evaluated the hydrophilicities of ts and ts along with selected proteinogenic residues , lys , glu , gly , thr and ile , using a previously described system . distribution coefficients are determined ( at equilibrium ) between equal volumes of octanol and aqueous buffer ( 100 mm phosphate , ph 7.0 ) . the parameter of comparison , , is normalized : the logarithm of the distribution coefficient of glycine , log(dglycine ) , is subtracted from the logarithm of distribution coefficient of the amino acid under consideration , log(damino acid ) to calculate for that amino acid . table 2 shows values measured for ts and ts and the selected proteinogenic residue . as we predicted , ts and ts cluster with lys and glu , all significantly preferring aqueous buffer relative to octanol . it is interesting to note that ts and glu are very similar on this scale , while ts is somewhat less hydrophilic than lys . in contrast , ile significantly prefers octanol to aqueous buffer , while thr is similar to gly . the data clearly show that ts and ts are significantly more hydrophilic that thr or ile . we have identified a new family of unnatural -amino acid residues featuring two properties , intrinsic conformational propensity and side chain charge , that have proven to be valuable for design of peptides that fold autonomously to -sheet secondary structure in aqueous solution . these two properties are not paired in any proteinogenic amino acid residue , which has made it challenging to design small peptides that adopt -sheet conformations but do not aggregate . high -sheet propensity is generally associated with -branching in a residue s side chain . our initial attempt to build charge and side chain branching into a new residue , involving an ether linkage at the branch point , was not successful . thioether - based branch points , on the other hand , lead to the desired properties . we have illustrated this approach with two new residues , ts and ts , which feature basic and acidic side chains , respectively . the versatility of the synthetic route will enable preparation of many related thioether - containing -amino acids .	proteinogenic amino acid residues that promote -sheet secondary structure are hydrophobic ( e.g. , ile or val ) or only moderately polar ( e.g. , thr ) . the design of peptides intended to display -sheet secondary structure in water typically requires one set of residues to ensure conformational stability and an orthogonal set , with charged side chains , to ensure aqueous solubility and discourage self - association . here we describe new amino acids that manifest substantial -sheet propensity , by virtue of -branching , and also bear an ionizable group in the side chain .
glucose meters have been used in the hospital setting for decades . traditionally glucose meters were used in the hospital to dose subcutaneous insulin for patients with diabetes when they were hospitalized . as even well - controlled diabetic patients will have their insulin needs , diet and caloric requirements change during periods of acute illness ; glucose must be measured frequently ( four or more times per day ) before meals and/or insulin dosing in the hospital . although most hospital laboratories offer a measurement of serum or plasma glucose , hospitals and healthcare systems find it both convenient and efficient to measure capillary whole blood glucose at the bedside in order to expedite insulin dosing . this can help insure that glucose values are taken before ( rather than after ) meals are consumed , as it is the pre - prandial blood sugar value that is most often used to dose insulin . in 2001 van den berghe and colleagues changed the landscape of glucose control in the hospital by studying the impact of tight glycemic control ( maintaining blood glucose between 80 - 110 mg / dl ) among critically ill patients ( both diabetic and non - diabetic ) after cardiovascular surgery . van den berghe s original study sought to determine whether closely controlling glucose levels in patients in a surgical intensive care unit ( icu ) would improve patient outcome . in the study 1500 patients were divided into two groups : one control group that received what was conventional treatment of hyperglycemia in the icu at that time ( subcutaneous or intravenous insulin to keep glucose levels less than 200 mg / dl ) , and an experimental group that received intravenous insulin to keep blood glucose at relatively normal levels of 80 - 110 mg / dl the experimental group that received intravenous insulin to keep blood glucose relatively normal had much better health outcomes than the control group ( mortality decreased 34% , renal failure 41% , bloodstream infections 46% ) . the outcomes were startling to critical care experts , and almost overnight changed the standard of care in critical care medicine from a relaxed attitude towards hyperglycemia in the icu to vigilant glucose monitoring and insulin treatment to maintain normal or near - normal blood glucose levels . subsequent studies found that depending upon the patient population ( medical vs. surgical icu ) , icu nutrition practices , and protocols to dose insulin and monitor glucose ; intensive glycemic control was of either benefit in only some icu patients or not beneficial at all . finally , in 2011 a multi - center trial called nice - sugar was performed to determine what level of glycemic control was optimal in the icu setting . unlike the preliminary studies done by dr . van den berghe , nice - sugar did not compare conventional treatment to more rigorous management of glycemic control ; as by that time some active management of glucose levels in the icu was standard of care . rather , nice - sugar compared two different glucose management strategies one aimed at controlling glucose levels among critically ill patients to near - normal levels ( similar to the van den berghe strategy ) and one that aimed for slightly higher ( 140 - 180 mg / dl ) glucose levels . nice - sugar , performed in over 40 medical centers , found that patients assigned to the higher ( < 180 mg / dl ) glucose target had significantly better health outcomes than those whose glucose target was near - normal ( 81 - 108 mg / dl ) . among the reasons why more moderate glucose targets may be beneficial to critically ill patients , all studies of intensive glucose control in the icu , including the original studies by dr . van den berghe , found that rates of hypoglycemia are higher among patients whose glucose levels are controlled actively with intravenous insulin . in fact , studies have shown that intravenous insulin therapy increases the rate of hypoglycemia among icu patients on average 5-fold . this is significant because even a single episode of hypoglycemia in the icu may increase the odds of death in the hospital up to two - fold . thus the need to control glucose levels in the icu must be balanced against the risk of hypoglycemia . while the original study ( showing the most positive outcomes ) by dr . van den berghe and colleagues used more accurate blood gas analyzers for all glucose measurements ; the subsequent studies often used less accurate glucose meters for measurement of blood glucose . this has fueled considerable controversy over whether glucose meters , originally intended for use in diabetic patients to monitor glucose and dose subcutaneous insulin , are accurate enough to manage intravenous insulin in critically ill hospitalized patients . traditionally , accuracy requirements for glucose meters were developed based upon the level of accuracy needed for safe and effective subcutaneous insulin dosing in the routine care of diabetes . these specifications are often visually displayed in an error grid , a tool developed by collecting the opinions of endocrinologists and other healthcare providers about the implications of various amounts of glucose measurement error on the safety and efficacy of subcutaneous insulin dosing . these error grid observations were codified in a set of guidelines issued by the international organization for standardization ( iso ) and clinical and laboratory standards institute ( clsi ) some years ago , and until recently used by some regulatory agencies as the measure of required glucose meter accuracy . one such commonly cited guideline , iso 15197 , required that 95% of glucose meter values fall within 15 mg / dl of the true or reference glucose value for serum glucose values < 75 mg / dl ; and 20% of the reference value for serum glucose values 75 mg / dl . because glucose meter use in the hospital has changed as glycemic control strategies have changed , most experts now feel that the original iso guideline is not appropriate as an accuracy guideline for hospital use glucose meters . to address these concerns , more stringent criteria for glucose meter accuracy have been proposed by both national academy of clinical biochemistry ( nacb ) and clsi . the guidelines are similar , and require 95% of glucose meter results to be within either 15 mg / dl ( nacb ) or 12 mg / dl ( clsi ) of reference glucose for glucose values < 100 mg / dl , and within 15% ( nacb ) or 12.5% ( clsi ) for glucose values 100 mg / dl ) several studies have documented that some glucose meters have limited accuracy when used on critically ill patients such as those on intravenous insulin in the icu . the degree to which glucose meters correlate with laboratory glucose measurement varies between glucose meter technologies ; and correlation in the hypoglycemic and hyperglycemic ranges is poor for some meters currently available . in addition , patients in the icu are on multiple medications , and often have abnormal hematocrit and/or oxygen tension , all of which may affect the performance of some glucose meters . finally , target glucose concentrations are narrower for this patient population than they are for patients using handheld meters to dose subcutaneous insulin , logically suggesting that improved accuracy of glucose measurement might be required . a number of studies have examined glucose meter accuracy and its impact on insulin dosing in the context of glycemic control , and concluded that glucose meters could not be safely and effectively used to manage critically ill patients on intravenous insulin in the icu . because studies examining glucose meter accuracy in the icu have been relatively small studies using different meters and reference methods , the larger question of the impact of glucose meter error on patient outcomes during glycemic control remains difficult to address . the primary manner this has been overcome is by utilizing simulation studies to model the effects of various levels of glucose meter error on insulin dosing decisions and glycemic control . boyd and bruns first established the use of simulation modeling as a tool to examine the relationship between glucose meter performance ( bias and precision ) and insulin dosing errors . the initial study was based upon glucose values and insulin doses used for conventional subcutaneous insulin dosing for diabetic patients . the authors used monte carlo simulation to relate glucose meter bias and imprecision to insulin dosing errors during conventional subcutaneous insulin dosing . they found that glucose meters available at that time had sufficient accuracy and precision to avoid large insulin dosing errors in the context of traditional subcutaneous insulin dosing regimens . another study , designed to specifically model glucose meter use during glycemic control in the icu , was based upon 29,920 observed glucose values among patients on intravenous insulin therapy in 2 icu units within one healthcare institution . as expected , most of the values were in a narrow range of glucose value ( 102 - 135 mg / dl ) , such that insulin dose would change with every 20 mg / dl glucose increment according to the insulin dosing protocol in use . the authors found that allowing 20% total error in glucose meter measurements ( previous iso 15197 criteria ) allowed for rare large ( 3 or more insulin dosing categories ) insulin dosing errors ; those that are most likely to produce hypoglycemia . decreasing allowable error to 15% eliminated large insulin dosing errors ; but still allowed for 2 - 5% of insulin dosing decisions to be in error by 2 insulin dosing categories . reducing error tolerance to 10% the authors concluded that 20% glucose measurement error was not safe and effective for intravenous insulin dosing protocols that sought to maintain glucose values at normal or near - normal concentrations ( tight glycemic control ) . after the publication of the nice - sugar study , many institutions changed the glucose target values for icu patients on intravenous insulin therapy to more moderate glucose values . to investigate whether glucose meter accuracy requirements for more moderate glycemic protocols differed from those suggested for tight glycemic control , the authors repeated the simulation studies using 25,948 observed glucose values in 1503 icu patients on a moderate glycemic control protocol ( 110 - 150 mg / dl target value ) . although the median glucose value was significantly higher among patients on moderate ( 134 mg / dl ) compared to tight ( 116 mg / dl ) glycemic control , most glucose values among patients on the moderate glycemic control protocol still fell into insulin dosing categories where insulin dose changed with every 20 mg / dl increment in glucose value . rates of insulin dosing errors as a function of meter bias and precision were nearly identical to those predicted for the population of patients on tight glycemic control . this suggests that the observed relationship between glucose meter and insulin dosing errors can be generalize to insulin infusion protocols where insulin dose changes with every 20 mg / dl change in glucose value . simulation models suggest that 20% error is too much for glucose meters used to manage patients on intravenous insulin therapy . because some studies of glucose meter accuracy in the icu observed that glucose meter error exceeded 20% when used on critically ill patients the simulation models have been used as evidence that glucose meters do not have the level of accuracy required for safe and effective management of critically ill patients placed on intravenous insulin ( glycemic control ) . only a small number of simulation studies have gone beyond relating glucose meter accuracy to insulin dosing errors ; and attempted to relate meter error to the short - term patient outcomes such as rates of hypoglycemia , rates of hyperglycemia , or glycemic variability ( rate and extent of change in glucose levels over time ) . one simulation model used a complex algorithm to predict the impact of glucose meter error over many days on rates of hypoglycemia , hyperglycemia and glycemic variability when glucose meter results were used to dose subcutaneous insulin in the context of diabetes self - management . the authors found that there was a threshold between 10 - 15% meter error that was predicted to result in increased incidences of hypoglycemia , hyperglycemia and increased glycemic variability . one additional study used simulation modeling to assess the impact of both glucose measurement frequency and precision on predicted rates of hypoglycemia in the context of glycemic control in the hospital . using hourly glucose monitoring to adjust insulin dose , the simulation model predicted that increasing imprecision above 10% cv would result in progressively increased rates of hypoglycemia ( glucose < 60 mg / dl ) . the same simulation models suggested that using hourly glucose monitoring rates of hyperglycemia ( > 160 mg / dl ) , time within intended target glucose range , and glycemic variability were all detrimentally affected when precision increased beyond 5 - 10% cv . these studies differed in the type of insulin dosing modeled ( subcutaneous vs. intravenous ) , glucose target ranges assumed , and frequency of glucose monitoring . however both raise concerns about the use of glucose meters to manage patients on intravenous insulin in the icu . both studies suggest a threshold effect of either glucose meter total erroror imprecision ; with a suggested minimum total error of 10 - 15% and imprecision of < 5% . because a number of previous studies demonstrated total error greater than 10 - 15% when glucose meters are used on icu patients , this has fueled concern about their use in this context . while studies of glucose meter use among critically ill patients have demonstrated both systematic differences ( generally positive bias ) and variability between glucose meter and laboratory glucose values , a few studies have concluded that the use of glucose meters during glycemic control may be appropriate . one study used parke s error grid analysis to assess the clinical impact of glucose meter errors when arterial , venous or capillary samples were used to dose glucose meters . these authors concluded that glucose meters may be appropriate for use in glycemic control protocols when arterial or venous ( but not capillary ) samples are used . however it is not clear whether use of the parke s error grid is appropriate for assessing the clinical impact of glucose meter errors in the context of intravenous insulin therapy during icu glycemic control protocols . another study also examined differences between glucose meter and laboratory glucose when either arterial , venous or capillary samples from critically ill patients were used . this study examined the number and magnitude of insulin dosing errors when glucose meter ( compared to laboratory glucose ) results were used to make insulin dosing decisions using the institutional glycemic control protocol ( target glucose 80 - 110 mg / dl ) . this study found that errors in the measurement of both venous catheter and capillary glucose resulted in more frequent large ( 2 or more insulin dosing categories ) dosing errors ; whereas use of arterial catheter whole blood on the glucose meter resulted in predominantly one category dosing errors . finally one study used consensus error grid and bland altman analysis to study whole blood glucose accuracy using several different devices ; and found that by limiting sample type to arterial blood that some glucose meters were accurate enough to be used during glycemic control in assessing the appropriateness of glucose meter use in the icu , choice of sample type is an essential consideration . a number of studies have demonstrated that capillary glucose can be highly inaccurate in patients in shock , or patients with edema or poor tissue perfusion several studies have also demonstrated systematic overestimation of glucose values when venous catheters are used to obtain venous whole blood for analysis on some glucose meter technologies . arterial whole blood is very likely the best sample choice for monitoring whole blood glucose in critically ill patients . in considering the evidence for and against use of glucose meters in the icu , one should pay special attention to sample source as a potential cause for poor glucose meter performance . other investigators have studied whether other factors may be more important than glucose monitor accuracy in determining the effectiveness of a glycemic control protocol . one study compared use of a standardized insulin infusion protocol to physician - directed intravenous insulin dosing in a mixed medical / surgical icu . use of the standardized infusion protocol reduced the rate of hypoglycemia from 16% to 4% , and also reduced the frequency of dextrose rescue . patients using the standardized protocol reached target glucose faster and maintained blood glucose in the target range ( 81 - 110 mg / dl ) longer . glucose in this study was monitored using capillary samples on a glucose meter , perhaps the least desirable sample for critically ill patients . even with this limitation , the study demonstrated that execution of a standardized infusion protocol can improve at least short - term outcomes ( hypoglycemia , time in therapeutic range ) . another study demonstrated that by using an insulin infusion protocol that focused on velocity of glucose change ( rather than absolute glucose levels ) , glucose meters could be used to maintain blood glucose in the range of 100 - 139 mg / dl with very little ( 0.3% of all glucose values < 60 mg / dl ) hypoglycemia . another investigator has described a collaborative approach to establishing both glucose target ranges and insulin infusion algorithms based upon practice and nursing leader opinions about what could be safely accomplished . using this approach they implemented an initial glycemic control protocol to keep glucose levels among critically ill patients below 140 mg / dl they used hourly capillary glucose meter and/or laboratory serum / plasma glucose for all patients on intravenous insulin and observed a rate of severe hypoglycemia ( glucose < 40 mg / dl ) of 0.38% . when staff in the icu was comfortable with the under 140 protocol , the target glucose range was decreased to 80 - 125 mg / dl with only a modest increase in severe hypoglycemia ( 0.92% ) . the authors concluded that by taking an incremental approach to glycemic control , starting with a higher target range and lowering the range only after staff demonstrated they could reliably execute the protocol , safe and effective glycemic control was possible using glucose meters for some monitoring . a more common approach to improving outcomes during glycemic control is to use information technology solutions to computerize insulin doses based upon trended ( rather than individual ) glucose values . this approach mitigates the risk of hypoglycemia from a single aberrant glucose meter value . using this approach one study demonstrated that rates of severe hypoglycemia were 4.25% when mostly capillary whole blood glucose meter values were used to dose insulin among 4588 critically ill patients managed on a glycemic control protocol with an 81 - 110 mg / dl target range . these authors went on to investigate causes of hypoglycemia among all incidents where glucose fell below 40 mg / dl the authors found that ~ 70% of hypoglycemic episodes could be attributed to delay in obtaining glucose measurement ; suggesting that human error ( rather than measurement error ) is responsible for the most insulin - induced hypoglycemia during traditional tight glycemic control protocols . the same authors compared the computerized infusion protocol to a paper - based protocol and found that using a computerized protocol improved the time in therapeutic range , mean blood glucose level , and percent of blood glucose measurements below 70 mg / dl .. finally a study over a one month period in three intensive care units at one institution found that using arterial whole blood to dose glucose meters , and relying upon consistent hourly glucose measurements performed by laboratory ( rather than nursing ) staff , rates of severe hypoglycemia were 1.4% despite a relatively low glucose target range of 80 - 130 mg / dl in addition , 86% of severe hypoglycemic episodes observed were due to protocol violations ( missed hourly glucose measurements or failure to change insulin infusion rate according to protocol instructions ) . when the glucose target range was changed to 110 - 150 mg / dl ( with no change in glucose meter used or measurement frequency ) , no episodes of hypoglycemia were observed in 211 patients over one month . a larger study ( three months , 1503 patients ) within the same icu units found a rate of severe hypoglycemia of 0.25 % . collectively these studies highlight several key points that must be considered before determining the appropriateness of glucose meters for managing glycemic control in the icu . the choice of sample type ( arterial whole blood preferred ) may be as or more important than the type of glucose monitor used for whole blood glucose measurement . glucose meters have been used in effective glycemic control protocols demonstrating both low rates of severe hypoglycemia and reliable glycemic control in the icu . elements of effective protocols are computerized ( rather than paper - based ) insulin dosing algorithms , collaboration and teamwork to determine the appropriate glucose target for a given hospital or icu population , and use of frequent ( often hourly ) arterial whole blood sampling for all patients on intravenous insulin . while many studies demonstrating poor performance of glucose meters in critically ill patients used older glucose meter technologies , newer technologies with improved accuracy have recently become available . some recent studies have demonstrated that newer glucose meter technologies can meet even the more stringent clsi poct12-a3 accuracy guidelines ( 12.5% for values above 100 mg / dl ) when used in the intensive care unit . meters that meet more stringent accuracy guidelines such as poct12-a3 would be performing within the 10 - 15% total error allowance predicted to minimize large insulin dosing errors in the context of icu glycemic control . with the improved performance of newer glucose meters , one might think that the issue of glucose meter accuracy in the icu was close to resolution . to add fuel to the ongoing controversy about glucose meter use in the icu , the food and drug administration ( fda ) released draft guidelines suggesting that improved accuracy was necessary for any future glucose monitors intended for hospital use . while the guidelines are still in draft form at the time of this review , fda draft guidance criteria suggested that 99% of glucose meter values should be within 10% of the reference or true glucose value . there is concern among some that tightening accuracy criteria to this level could impede the development of new meters and monitors , without improving the quality of care delivered in the icu during glycemic control . amidst this cloud of confusion and controversy surrounding glucose meter use in the icu , first and foremost , consider the entire glycemic control protocol in use within your institution , and the role that glucose meters play in the overall scheme of glycemic control . eliminating the use of glucose meters in support of intravenous insulin protocols , without first considering alternatives and implications , would almost certainly have an adverse effect on patient care . understand the effectiveness of the glycemic control protocol ( rates of hypo and hyperglycemia , time within intended glucose range ) as implemented , and the systematic issues that may be leading to adverse outcomes such as hypoglycemia . if the major issues are remembering to obtain glucose values in a timely manner to facilitate insulin dosing decisions , or communicating glucose results to providers in a timely manner , then changing glucose measurement devices ( especially away from the bedside ) would not be expected to improve outcome . if spurious glucose results have been observed in some icu patients , determine whether common interferences ( low hematocrit , some medications ) in the icu environment may be affecting the glucose meter technology in use . if user errors such as incorrect strip codes or under - dosing of strips are suspected ; consider switching to a glucose meter technology that reduces the likelihood of these errors and examining training and competency systems . hospitals and point of care programs should also consider the sample type ( capillary , arterial or venous whole blood ) routinely used for bedside glucose measurements , before making a decision to switch technologies or glucose measurement devices . if capillary sampling is being used as the predominant sample type , switching to arterial whole blood may improve measurement accuracy without requiring large changes in workflow or testing processes . finally , consider evaluating the accuracy of the device being used by comparing whole blood glucose meter values to laboratory serum or plasma glucose obtained from icu patients . if the vast majority of glucose meter values are not within 15% of lab glucose values , then it is likely that more accurate glucose measurements are both possible and desirable . several studies have documented that some glucose meters have limited accuracy when used on critically ill patients such as those on intravenous insulin in the icu . the degree to which glucose meters correlate with laboratory glucose measurement varies between glucose meter technologies ; and correlation in the hypoglycemic and hyperglycemic ranges is poor for some meters currently available . in addition , patients in the icu are on multiple medications , and often have abnormal hematocrit and/or oxygen tension , all of which may affect the performance of some glucose meters . finally , target glucose concentrations are narrower for this patient population than they are for patients using handheld meters to dose subcutaneous insulin , logically suggesting that improved accuracy of glucose measurement might be required . a number of studies have examined glucose meter accuracy and its impact on insulin dosing in the context of glycemic control , and concluded that glucose meters could not be safely and effectively used to manage critically ill patients on intravenous insulin in the icu . because studies examining glucose meter accuracy in the icu have been relatively small studies using different meters and reference methods , the larger question of the impact of glucose meter error on patient outcomes during glycemic control remains difficult to address . the primary manner this has been overcome is by utilizing simulation studies to model the effects of various levels of glucose meter error on insulin dosing decisions and glycemic control . boyd and bruns first established the use of simulation modeling as a tool to examine the relationship between glucose meter performance ( bias and precision ) and insulin dosing errors . the initial study was based upon glucose values and insulin doses used for conventional subcutaneous insulin dosing for diabetic patients . the authors used monte carlo simulation to relate glucose meter bias and imprecision to insulin dosing errors during conventional subcutaneous insulin dosing . they found that glucose meters available at that time had sufficient accuracy and precision to avoid large insulin dosing errors in the context of traditional subcutaneous insulin dosing regimens . another study , designed to specifically model glucose meter use during glycemic control in the icu , was based upon 29,920 observed glucose values among patients on intravenous insulin therapy in 2 icu units within one healthcare institution . as expected , most of the values were in a narrow range of glucose value ( 102 - 135 mg / dl ) , such that insulin dose would change with every 20 mg / dl glucose increment according to the insulin dosing protocol in use . the authors found that allowing 20% total error in glucose meter measurements ( previous iso 15197 criteria ) allowed for rare large ( 3 or more insulin dosing categories ) insulin dosing errors ; those that are most likely to produce hypoglycemia . decreasing allowable error to 15% eliminated large insulin dosing errors ; but still allowed for 2 - 5% of insulin dosing decisions to be in error by 2 insulin dosing categories . reducing error tolerance to 10% further reduced the rate of 2 category insulin dosing errors to less than 0.2% . the authors concluded that 20% glucose measurement error was not safe and effective for intravenous insulin dosing protocols that sought to maintain glucose values at normal or near - normal concentrations ( tight glycemic control ) . after the publication of the nice - sugar study , many institutions changed the glucose target values for icu patients on intravenous insulin therapy to more moderate glucose values . to investigate whether glucose meter accuracy requirements for more moderate glycemic protocols differed from those suggested for tight glycemic control , the authors repeated the simulation studies using 25,948 observed glucose values in 1503 icu patients on a moderate glycemic control protocol ( 110 - 150 mg / dl target value ) . although the median glucose value was significantly higher among patients on moderate ( 134 mg / dl ) compared to tight ( 116 mg / dl ) glycemic control , most glucose values among patients on the moderate glycemic control protocol still fell into insulin dosing categories where insulin dose changed with every 20 mg / dl increment in glucose value . rates of insulin dosing errors as a function of meter bias and precision were nearly identical to those predicted for the population of patients on tight glycemic control . this suggests that the observed relationship between glucose meter and insulin dosing errors can be generalize to insulin infusion protocols where insulin dose changes with every 20 mg / dl change in glucose value . simulation models suggest that 20% error is too much for glucose meters used to manage patients on intravenous insulin therapy . because some studies of glucose meter accuracy in the icu observed that glucose meter error exceeded 20% when used on critically ill patients the simulation models have been used as evidence that glucose meters do not have the level of accuracy required for safe and effective management of critically ill patients placed on intravenous insulin ( glycemic control ) . only a small number of simulation studies have gone beyond relating glucose meter accuracy to insulin dosing errors ; and attempted to relate meter error to the short - term patient outcomes such as rates of hypoglycemia , rates of hyperglycemia , or glycemic variability ( rate and extent of change in glucose levels over time ) . one simulation model used a complex algorithm to predict the impact of glucose meter error over many days on rates of hypoglycemia , hyperglycemia and glycemic variability when glucose meter results were used to dose subcutaneous insulin in the context of diabetes self - management . the authors found that there was a threshold between 10 - 15% meter error that was predicted to result in increased incidences of hypoglycemia , hyperglycemia and increased glycemic variability . one additional study used simulation modeling to assess the impact of both glucose measurement frequency and precision on predicted rates of hypoglycemia in the context of glycemic control in the hospital . using hourly glucose monitoring to adjust insulin dose , the simulation model predicted that increasing imprecision above 10% cv would result in progressively increased rates of hypoglycemia ( glucose < 60 mg / dl ) . the same simulation models suggested that using hourly glucose monitoring rates of hyperglycemia ( > 160 mg / dl ) , time within intended target glucose range , and glycemic variability were all detrimentally affected when precision increased beyond 5 - 10% cv . these studies differed in the type of insulin dosing modeled ( subcutaneous vs. intravenous ) , glucose target ranges assumed , and frequency of glucose monitoring . however both raise concerns about the use of glucose meters to manage patients on intravenous insulin in the icu . both studies suggest a threshold effect of either glucose meter total erroror imprecision ; with a suggested minimum total error of 10 - 15% and imprecision of < 5% . because a number of previous studies demonstrated total error greater than 10 - 15% when glucose meters are used on icu patients , this has fueled concern about their use in this context . while studies of glucose meter use among critically ill patients have demonstrated both systematic differences ( generally positive bias ) and variability between glucose meter and laboratory glucose values , a few studies have concluded that the use of glucose meters during glycemic control may be appropriate . one study used parke s error grid analysis to assess the clinical impact of glucose meter errors when arterial , venous or capillary samples were used to dose glucose meters . these authors concluded that glucose meters may be appropriate for use in glycemic control protocols when arterial or venous ( but not capillary ) samples are used . however it is not clear whether use of the parke s error grid is appropriate for assessing the clinical impact of glucose meter errors in the context of intravenous insulin therapy during icu glycemic control protocols . another study also examined differences between glucose meter and laboratory glucose when either arterial , venous or capillary samples from critically ill patients were used . this study examined the number and magnitude of insulin dosing errors when glucose meter ( compared to laboratory glucose ) results were used to make insulin dosing decisions using the institutional glycemic control protocol ( target glucose 80 - 110 mg / dl ) . this study found that errors in the measurement of both venous catheter and capillary glucose resulted in more frequent large ( 2 or more insulin dosing categories ) dosing errors ; whereas use of arterial catheter whole blood on the glucose meter resulted in predominantly one category dosing errors . finally one study used consensus error grid and bland altman analysis to study whole blood glucose accuracy using several different devices ; and found that by limiting sample type to arterial blood that some glucose meters were accurate enough to be used during glycemic control in assessing the appropriateness of glucose meter use in the icu , choice of sample type is an essential consideration . a number of studies have demonstrated that capillary glucose can be highly inaccurate in patients in shock , or patients with edema or poor tissue perfusion several studies have also demonstrated systematic overestimation of glucose values when venous catheters are used to obtain venous whole blood for analysis on some glucose meter technologies . arterial whole blood is very likely the best sample choice for monitoring whole blood glucose in critically ill patients . in considering the evidence for and against use of glucose meters in the icu , one should pay special attention to sample source as a potential cause for poor glucose meter performance . other investigators have studied whether other factors may be more important than glucose monitor accuracy in determining the effectiveness of a glycemic control protocol . one study compared use of a standardized insulin infusion protocol to physician - directed intravenous insulin dosing in a mixed medical / surgical icu . use of the standardized infusion protocol reduced the rate of hypoglycemia from 16% to 4% , and also reduced the frequency of dextrose rescue . patients using the standardized protocol reached target glucose faster and maintained blood glucose in the target range ( 81 - 110 mg / dl ) longer . glucose in this study was monitored using capillary samples on a glucose meter , perhaps the least desirable sample for critically ill patients . even with this limitation , the study demonstrated that execution of a standardized infusion protocol can improve at least short - term outcomes ( hypoglycemia , time in therapeutic range ) . another study demonstrated that by using an insulin infusion protocol that focused on velocity of glucose change ( rather than absolute glucose levels ) , glucose meters could be used to maintain blood glucose in the range of 100 - 139 mg / dl with very little ( 0.3% of all glucose values < 60 mg / dl ) hypoglycemia . another investigator has described a collaborative approach to establishing both glucose target ranges and insulin infusion algorithms based upon practice and nursing leader opinions about what could be safely accomplished . using this approach they implemented an initial glycemic control protocol to keep glucose levels among critically ill patients below 140 mg / dl they used hourly capillary glucose meter and/or laboratory serum / plasma glucose for all patients on intravenous insulin and observed a rate of severe hypoglycemia ( glucose < 40 mg / dl ) of 0.38% . when staff in the icu was comfortable with the under 140 protocol , the target glucose range was decreased to 80 - 125 mg / dl with only a modest increase in severe hypoglycemia ( 0.92% ) . the authors concluded that by taking an incremental approach to glycemic control , starting with a higher target range and lowering the range only after staff demonstrated they could reliably execute the protocol , safe and effective glycemic control was possible using glucose meters for some monitoring . a more common approach to improving outcomes during glycemic control is to use information technology solutions to computerize insulin doses based upon trended ( rather than individual ) glucose values . this approach mitigates the risk of hypoglycemia from a single aberrant glucose meter value . using this approach one study demonstrated that rates of severe hypoglycemia were 4.25% when mostly capillary whole blood glucose meter values were used to dose insulin among 4588 critically ill patients managed on a glycemic control protocol with an 81 - 110 mg / dl target range . these authors went on to investigate causes of hypoglycemia among all incidents where glucose fell below 40 mg / dl the authors found that ~ 70% of hypoglycemic episodes could be attributed to delay in obtaining glucose measurement ; suggesting that human error ( rather than measurement error ) is responsible for the most insulin - induced hypoglycemia during traditional tight glycemic control protocols . the same authors compared the computerized infusion protocol to a paper - based protocol and found that using a computerized protocol improved the time in therapeutic range , mean blood glucose level , and percent of blood glucose measurements below 70 mg / dl .. finally a study over a one month period in three intensive care units at one institution found that using arterial whole blood to dose glucose meters , and relying upon consistent hourly glucose measurements performed by laboratory ( rather than nursing ) staff , rates of severe hypoglycemia were 1.4% despite a relatively low glucose target range of 80 - 130 mg / dl in addition , 86% of severe hypoglycemic episodes observed were due to protocol violations ( missed hourly glucose measurements or failure to change insulin infusion rate according to protocol instructions ) . when the glucose target range was changed to 110 - 150 mg / dl ( with no change in glucose meter used or measurement frequency ) , no episodes of hypoglycemia were observed in 211 patients over one month . a larger study ( three months , 1503 patients ) within the same icu units found a rate of severe hypoglycemia of 0.25 % . collectively these studies highlight several key points that must be considered before determining the appropriateness of glucose meters for managing glycemic control in the icu . the choice of sample type ( arterial whole blood preferred ) may be as or more important than the type of glucose monitor used for whole blood glucose measurement . glucose meters have been used in effective glycemic control protocols demonstrating both low rates of severe hypoglycemia and reliable glycemic control in the icu . elements of effective protocols are computerized ( rather than paper - based ) insulin dosing algorithms , collaboration and teamwork to determine the appropriate glucose target for a given hospital or icu population , and use of frequent ( often hourly ) arterial whole blood sampling for all patients on intravenous insulin . while many studies demonstrating poor performance of glucose meters in critically ill patients used older glucose meter technologies , newer technologies with improved accuracy have recently become available . some recent studies have demonstrated that newer glucose meter technologies can meet even the more stringent clsi poct12-a3 accuracy guidelines ( 12.5% for values above 100 mg / dl ) when used in the intensive care unit . meters that meet more stringent accuracy guidelines such as poct12-a3 would be performing within the 10 - 15% total error allowance predicted to minimize large insulin dosing errors in the context of icu glycemic control . with the improved performance of newer glucose meters , one might think that the issue of glucose meter accuracy in the icu was close to resolution . to add fuel to the ongoing controversy about glucose meter use in the icu , the food and drug administration ( fda ) released draft guidelines suggesting that improved accuracy was necessary for any future glucose monitors intended for hospital use . while the guidelines are still in draft form at the time of this review , fda draft guidance criteria suggested that 99% of glucose meter values should be within 10% of the reference or true glucose value . there is concern among some that tightening accuracy criteria to this level could impede the development of new meters and monitors , without improving the quality of care delivered in the icu during glycemic control . amidst this cloud of confusion and controversy surrounding glucose meter use in the icu , what is the point of care program to do ? first and foremost , consider the entire glycemic control protocol in use within your institution , and the role that glucose meters play in the overall scheme of glycemic control . eliminating the use of glucose meters in support of intravenous insulin protocols , without first considering alternatives and implications , would almost certainly have an adverse effect on patient care . understand the effectiveness of the glycemic control protocol ( rates of hypo and hyperglycemia , time within intended glucose range ) as implemented , and the systematic issues that may be leading to adverse outcomes such as hypoglycemia . if the major issues are remembering to obtain glucose values in a timely manner to facilitate insulin dosing decisions , or communicating glucose results to providers in a timely manner , then changing glucose measurement devices ( especially away from the bedside ) would not be expected to improve outcome . if spurious glucose results have been observed in some icu patients , determine whether common interferences ( low hematocrit , some medications ) in the icu environment may be affecting the glucose meter technology in use . if user errors such as incorrect strip codes or under - dosing of strips are suspected ; consider switching to a glucose meter technology that reduces the likelihood of these errors and examining training and competency systems . hospitals and point of care programs should also consider the sample type ( capillary , arterial or venous whole blood ) routinely used for bedside glucose measurements , before making a decision to switch technologies or glucose measurement devices . if capillary sampling is being used as the predominant sample type , switching to arterial whole blood may improve measurement accuracy without requiring large changes in workflow or testing processes . finally , consider evaluating the accuracy of the device being used by comparing whole blood glucose meter values to laboratory serum or plasma glucose obtained from icu patients . if the vast majority of glucose meter values are not within 15% of lab glucose values , then it is likely that more accurate glucose measurements are both possible and desirable . simulation models have provided the best evidence available to relate glucose meter accuracy to insulin dosing errors during glycemic control in the icu . however they do not provide a way to measure the impact of glucose meter error on patient outcome . studies directly relating glucose monitor accuracy to glycemic control outcome ( mortality , infections , transfusions , etc ) or effectiveness ( hypoglycemia , hyperglycemia , time in therapeutic range ) are needed to understand the level of glucose meter accuracy required for management of critically ill patients on intravenous insulin therapy .	glucose meters are a fast and convenient way to measure circulating blood glucose . like many technologies in healthcare , the use of glucose meters within the hospital has evolved significantly over the last few decades . this change has been driven predominantly by changes in the approach to glycemic control for critically ill patients . both glycemic control in the intensive care unit ( icu ) , and use of glucose meters to manage insulin dosing during glycemic control , are likely to remain controversial topics in the years to come . this review will elaborate on the evidence for and against use of glucose meters in the icu to monitor glucose concentrations during glycemic control , and provide some tips for point of care programs on how to evaluate glucose monitors for this purpose .
some of the substances used for these dyes are either allergenic or have other side effects in some persons . 6 ) , the disodium salt of 3-hydroxy-4-[(4-methyl-2-sulfophenyl ) azo]-2-naphthalenecarboxylic acid , which is an anionic azo compound ( ci : 15850 ) , is one such substance . lithol rubine b is mutagenic because of intermediate reactions or by - products found in commercial samples . there are several methods for determining trace concentrations of lithol rubine b in cosmetic products . some use high - performance liquid chromatography ( hplc ) with ultraviolet visible ( uv / vis ) or mass spectrometry ( ms ) detection [ 26 ] , raman spectra , and voltammetry [ 8 , 9 ] . most methods for the quantitative detection and characterization of azo dyes use hplc analysis [ 1014 ] . uv visible spectrometry is a useful tool , but the azo dyes are largely affected by solvent influence , and require a photodiode array detector . detection using mass spectrometry is a more sensitive , but more expensive , approach and is not yet available for every laboratory . lc / ms has been recommended for analyzing disperse and low - molecular ( < 300 ) azo dyes , but it is not applicable to sulfonated azo dyes . when the mixture sample contains more than two azo dyes , the voltammetric waves of these dyes are seriously overlapped , which makes their quantification difficult without any preanalysis separation and purification . recent research on the electrodeposition of antimony ( sb ) focuses on ( a ) sb / sb2o3 particles for lithium - ion battery anodes ; ( b ) binary and ternary alloys for use in thermoelectric elements ; ( c ) the electrodeposition of sb on tin oxide ( sno2 ) electrodes , gold ( au ) electrodes , glassy carbon electrodes ( gces ) [ 19 , 20 ] , and mercury ( hg ) electrodes ; ( d ) one ternary film , ( antimony / tellurium ) ( sb2te3 ) , on a silver ( ag ) electrode . ag and metal - sb alloys [ 24 , 25 ] cause the reduction of electrocatalytic activity and oxidation in organic compounds . however , there are no published studies on using an electrocatalytic sensor with liquid chromatography - electrochemical detection ( lc - ec ) and flow - injection analysis ( fia ) determination of lithol rubine b in cosmetic products . directive 76/768/eec allows lithol rubine b to be used in cosmetic products as a coloring agent at a level of 0.22.0% . in the present study , we developed a more sensitive and low - cost method for detecting lithol rubine b in cosmetic products . we also report the development of a flow - through voltammetric sensor to catalyze the electroduction of lithol rubine b. the hplc system consisted of a hitachi model l-7110 pump with a rheodyne 7125 injection valve with a 20 l sample loop and was coupled with an eg&g parc 400 controlled potentiostat . the flow - through electrolysis cell was designed with the following electrodes : an ag / agcl/0.1 m kcl reference electrode ( bas ) , a platinum auxiliary electrode , and modified silver electrodes as working electrodes for detecting lithol rubine b azo dye . all solvents and the analyte were filtered through 0.45 m cellulose acetate and polyvinylidene fluoride ( pvdf ) syringe membrane filters , respectively . a chromatogram of lithol rubine b was acquired , and the peak height was calculated using an sisc chromatogram data integrator . lithol rubine b ( d&c red 6 ) pure dye content ( > 90% ) was purchased from unipure lc sensient cosmetic technologies / lcw ( milwaukee , wi , usa ) . a thin - film antimony electrode was produced using the following method . before the analysis , the silver wire electrode ( 4 cm long , 3 mm in diameter ) was mirror - polished sequentially with aqueous suspensions of 1.0 , 0.5 , and 0.05 m alumina . the electrode was then rinsed with deionized water and electrolytically plated with antimony ions ( 1.0 10 to 4.0 10 m ) from 10 ml of acetate buffer ( ph 4.5 ) . plating time was 8 min according to a potential scan of between 1.0 and 0 v ( versus ag / agcl ; at 10 mv / s ) . the antimony - modified silver electrode ( 0.3 mm diameter ) was constructed from a length ( ~8 cm ) of teflon tubing ( 1/32 in . , i.d . ; 1/16 in . , o.d . ) , inserted into one end of the teflon tube , and then sealed with acrylic resin ( struers ) . a small copper wire was placed at the other end of the teflon tube to allow an electrical connection to the antimony - modified silver wire electrode . the platinum wire , which served as a counter , and the ag / agcl wire , a reference electrode , the voltammetric detector , that is , the eluate , was fed to the antimony - modified silver wire electrode , which had been placed in an overflow vessel containing counter - electrodes and reference electrodes . the cosmetics ( rouge , lipstick , and nail polish ) contained oils ( mineral oil , petrolatum , and lanolin ) , ester ( isopropyl myristate ) , waxes ( paraffin and carnauba ) , and a powder base ( zinc oxide , titanium dioxide , inorganic pigments , and talc ) as the primary constituents of the base . the base was bound with the required color . raw samples received for analysis were converted into a form suitable for making useful measurements by pretreatment , separation , and preconcentration . taking into account the content of lithol rubine b azo dye in each sample , about 1.0 g of each sample was accurately weighed in a 50 ml beaker , diluted to about 10 ml with methanol , dissolved , and then stirred for 2 h. the mixture solution was concentrated , 9 ml of acetate buffer was added , and then the mixture was centrifuged at 6000 g for 30 min . the supernatant was transferred into a 10 ml calibrated flask ; acetate buffer was added to ensure that the flask contained 10 ml of liquid . the sample ( 1 ml ) was loaded onto a c18 cartridge and washed with 1 ml of ethyl acetate , and then the eluent was discarded . the resulting solution was extracted twice with methanol , and the organic layer was evaporated at 40c under nitrogen . the dried extract was reconstituted with 1 ml of methanol and filtered through 0.45 m membrane filters before hplc analysis . a stock standard sample solution was prepared by dissolving 10 mg of lithol rubine b in 10 ml of water , because lithol rubine b is a soluble sulfonated salt . working standard solutions in the range 0.051.2 mg / l were prepared from the stock standard solution . reversed - phase- ( rp- ) hplc was done on a phenomenex hyperclone c18 5 m ( 250 mm 4.6 mm ) column eluted with methanol - water ( 30 : 70 , v / v ) and acetonitrile - water ( 30 : 70 , v / v ) , respectively , containing 0.1 mm of k2hpo4 ( ph 4.08 ) as the mobile phase , at flow rates of 0.7 , 0.5 , and 0.3 ml / min . after the azo dye components in the cosmetics sample had been separated on the hyperclone c18 column , they were examined using an ultraviolet detector set at 235 nm . the electrochemical detector was operated at 0.6 v. a chromatograph was obtained with 20 l of the prepared sample solution and a standard solution under the operating conditions described above . azo linkages are reducible ; a typical two - electron , two - proton reduction was found for azobenzene . azobenzene undergoes a fast ecec reduction to hydrazobenzene which is then oxidized at approximately the same potential as azobenzene is reduced . azobenzene was reduced to the corresponding anilines using controlled potential coulometry since the initially formed intermediate hydrazobenzene decomposes to aniline . the proposed pathway for the electrochemical reduction of lithol rubine b is shown in scheme 2 . for both the gce and the sb / ag electrodes , good linearity was observed between the peak height ( current ) and the square root of the scan rate ( figure 1 ) . two different eluents , methanol - water ( 30 : 70 , v / v ) and acetonitrile - water ( 30 : 70 , v / v ) containing 0.1 mm of k2hpo4 ( ph 4.08 ) as the mobile phases of both solvent systems , were investigated at various flow rates . the retention times of acetonitrile - water were 9.8 , 5.3 , and 4.6 min for flow rates of 0.3 , 0.5 , and 0.7 ml / min , respectively . using methanol - water was inappropriate for determining lithol rubine b under our analytical conditions because the acetonitrile content in the mobile phase affects capacity factors and sensitivity . in most reversed - phase - hplc separations of highly unipolar colorants , broad , tailing peaks are obtained . a flow rate of 0.5 ml / min and acetonitrile - water were , therefore , used to determine lithol rubine b. the linear slope ( 5.3641 ) of the sb / ag electrode was higher than that ( 1.3568 ) of the gce ( figure 1 ) . lc - ecd chromatograms of lithol rubine b azo dye at various working electrodes ( a gce , a bare ag electrode , and an sb / ag electrode ) . figure 2 shows that the height of the reduction peak ( 0.3 g / ml ) of the sb / ag electrode was higher than those of the other electrodes ( gce : 1.0 g / ml ; bare ag : 0.3 g / ml ) . sb / ag film gave a better performance than the others did ; therefore , we used the sb / ag electrode to determine lithol rubine b levels in cosmetic products . to determine the optimum applied potential for electrochemical detection , after the lc analysis , hydrodynamic voltammograms were constructed for lithol rubine b ( figure 3 ) . the voltammetric detector was operated at 0.6 v. using the injection valve , 20 l of the prepared sample solution and of the standard solution were chromatographed under the operating conditions described above . the calibration curve showed good linearity over the range 0.01881.203 g / ml ; the regression equation was y = 430x + 150 , and the correlation coefficient was r = 0.9997 . the detection limit of our method was approximately 2.0 ng / ml . quantitation was based on the peak area of the sample . the operational stability of the bare ag and sb / ag sensors was tested and compared by continuously exposing them to the flow stream , and by monitoring the amperometric response ( at 0.6 v versus ag / agcl ) of acetonitrile - water ( 30 : 70 , v / v ) containing 0.1 mm k2hpo4 ( ph 3.97 ) over several hours of repetitive injections . scanning electron microscope ( sem ; at 15 kv ) images of 4 mm antimony ( sb ) particles distributed ( figure 4(a ) ) at the bare ag working electrode surface ( 0.3 mm i.d . ) as the working electrode in the flow cell after ( b ) 0 h and ( c ) 6 h. figure 4(c ) illustrates the stability of the sensor after 6 h of repetitive injections . the calibration plots obtained by plotting the peak area against the concentration of lithol rubine b show good linearity over the range 1080 mg / l . the regression equations were y = 1.17x + 0.387 ( correlation coefficient r = 0.9999 ) ; the range 0.301.2 mg / l and y = 202x + 10.0 ( correlation coefficient r = 0.9999 ) for lc - uv and lc - ed , respectively ( figures 5(a ) and 5(b ) ) . subsequently , we developed a simple and sensitive green electrochemical procedure for determining lithol rubine b in real samples . commercial lipstick was spiked with 0.1 , 0.2 , or 0.3 mg / l and then analyzed . the calibration plot ( figure 6 ) shows good linearity . because it is less expensive than lc - uv analysis , hplc should be done using a conventional variable wavelength detector to achieve some selectivity and close to the optimum sensitivity of all the colorants . thus , our proposed analytical method offers a valid and economical alternative to lc - uv detection of lithol rubine b. our proposed lc - ec method was used to determine lithol rubine b in cosmetic products . chromatograms of a comparison of lc - uv ( figure 7(a ) ) and lc - ec ( figure 7(b ) ) for lithol rubine b in cosmetics show that the sensitivity for the lithol rubine b investigated was about two orders of a magnitude higher with lc - ec than with lc - uv detection ; however , there were no significant differences in the values obtained using both types of analysis ( table 1 ) . we constructed an antimony - modified silver electrode to use as an electrocatalytic sensor liquid chromatography - electrochemical ( lc - ec ) detection and flow - injection analysis ( fia ) determination of lithol rubine b in cosmetics . the electrode not only exhibited catalytic activity toward this analyte , but also provided a stable , quantitatively reproducible performance in the chromatographic stream . thus , the proposed analytical method offers a valid , and economical , alternative to uv detection and mass spectrometry detection of lithol rubine b.	lithol rubine b ( lrb ; the disodium salt of 3-hydroxy-4-[(4-methyl-2-sulfophenyl ) azo]-2-naphthalenecarboxylic acid ) was detected using high - performance liquid chromatography with an electrochemical ( antimony film on silver ) detector ( hplc - ecd ) . for direct current ( dc ) mode , with the current at a constant potential , and measurements with suitable experimental parameters , a linear concentration from 0.125 to 1.80 g / ml was found . the detection limit of our method was approximately 2.0 ng / ml . an antimony - modified silver detector was used to demonstrate that lrb is electrochemically reduced in acidic media and to analyze commercial cosmetics to determine their lrb content . findings using hplc - ecd and hplc with an ultraviolet detector were comparable .
primitive neuroectodermal tumors ( pnets ) are highly malignant embryonal neoplasms of bone and soft tissues accounting for 4%17% of all pediatric soft tissue tumors . metastasis at presentation is quite common , with central pnets metastasizing to cranium and leptomeninges while peripheral pnets ( ppnets ) disseminating to lungs , bone , liver , and lymph nodes . primary pulmonary pnet is known to metastasize to pancreas , adrenal gland and ovaries while it metastasizing to brain is exceedingly rare . a 29-year - old female presented with a cough and right - sided chest pain of 1-month duration . computed tomography scan of chest [ figure 1 ] showed an 11.3 cm 11 cm 10 cm soft tissue mass lesion in right perihilar region with the loss of fat planes with esophagus , aorta , and diaphragm without chest wall or pleural involvement . biopsy showed atypical round to oval cells with scanty cytoplasm intermixed with respiratory epithelium [ figure 2 ] . immunohistochemistry ( ihc ) was positive for neuron - specific enolase ( nse ) [ figure 3 ] , synaptophysin , chromogranin , cd 99 , and vimentin . smooth muscle actin ( sma ) , epithelial membrane antigen ( ema ) , cytokeratin ( ck ) , leukocyte common antigen ( lca ) , cd 20 , cd 45 , thyroid transcription factor ( ttf-1 ) and desmin were negative , confirming the diagnosis of pnet lung . computed tomography scan ( axial section ) of thorax showing a large heterogeneously enhancing soft tissue mass lesion in the right perihilar region involving right lower lobe extending into the mediastinum with loss of fat planes with esophagus , aorta , and right crus of the diaphragm . there is no chest wall or pleural involvement biopsy from lung lesion showing small round cells with scanty cytoplasm with condensed chromatin and inconspicuous nucleoli ( h and e , 200 ) immunohistochemistry picture from lung lesion showing tumor cells positive for neuron specific enolase ( 200 ) magnetic resonance imaging ( mri ) brain and positron emission tomography scan showed localized disease . received chemotherapy vincristine , adriamycin , and cyclophosphamide alternating with ifosfamide plus etoposide ( vac / ie ) resulting in partial response ( pr ) to therapy . conformal radiotherapy ( rt ) was given to postchemotherapy residual volume to a dose of 60 ( gray ) gy in 30 fractions . post - rt patient experienced significant symptomatic relief with improved performance status though with a static disease . the patient was on follow up for 9 months when she presented with a headache , vomiting , seizures and left - sided hemiparesis . mri brain [ figure 4 ] showed an 8.6 cm 7.5 cm solitary lesion with areas of altered signal intensity at right periventricular and periatrial parietal lobe with significant perilesional edema suggestive of hemorrhagic metastatic deposit . she underwent right fronto - temporo - parietal craniotomy with excision of tumor and evacuation of hematoma . ihc was positive for synaptophysin [ figure 5a ] , chromogranin [ figure 5b ] , vimentin [ figure 5c ] , and cd 99 [ figure 5d ] with ki67 of 90% while negative for ck , ttf-1 , and melan - a which suggested metastasis from primary pnet lung . we treated her with whole brain rt ( wbrt ) to a dose of 30 gy in 10 fractions followed by oral pazopanib 800 mg once a day as a palliative intent . the patient is under follow up for over 1 year with a karnofsky performance status of 70% . magnetic resonance imaging brain ( a = coronal section and b = axial section ) showing a solitary lesion with areas of altered signal intensity at right periventricular and periatrial parietal lobe with significant perilesional edema suggestive of hemorrhagic metastatic deposit immunohistochemistry picture from brain metastasis showing tumor cells positive for ( a ) synaptophysin ( 100 ) , ( b ) chromogranin ( 200 ) , ( c ) vimentin ( 100 ) , ( d ) cd 99 ( 200 ) thoracic pnets are the most aggressive form of ppnets arising from chest wall or underlying lung pleura invading bone , lung or mediastinum in children and young adults . they are extremely aggressive with a dismal prognosis as patients may present with an upfront metastatic disease to contralateral lung , bone , bone marrow , lymph nodes , liver , pancreas , adrenals , and ovaries . primary lung pnet can often be misdiagnosed with a metastatic lung lesion , mucinous adenocarcinoma or squamous cell lung carcinoma . since pnets and other small round cell tumors share the same histological picture , ihc plays a vital role in their differentiation . cd 99 , vimentin and s100 are nonspecific while nse , synaptophysin and chromogranin confirm the diagnosis of pnet . ttf-1 is found in pulmonary adenocarcinomas and thyroid malignancies , ck for carcinoma , desmin for rhabdomyosarcoma , sma and ema for soft tissue sarcoma while lca , bcl-2 , cd 20 , cd 45 are for acute lymphoblastic lymphoma and leukemia . however , to confirm the diagnosis , amplification of ewing sarcoma breakpoint region-1 by situ hybridization techniques is required . according to javery et al . and previous other series on extraskeletal ewing sarcoma ( ees ) , lung is the most common site of metastasis followed by bone comprising 80% and 40% cases , respectively . huh et al . reported lymph nodes as the most frequent metastatic site ( 75.9% ) followed by bone , lung , peritoneum , pleura while least being brain ( 3.4% ) . the most prevalent primary sites were extremities , abdomen , pelvis , thorax , paravertebral space followed by head and neck current treatment recommendations for lung pnet and ppnets / ewing 's are same with vac / ie . rt concurrent with chemotherapy is preferred for the limited stage while sequential rt is beneficial for extensive lesions . we have treated two more cases of adult primary lung pnet although with a radiological pr but a significant symptomatic response . both patients are on follow - up for more than a year now without any evidence of disease progression or metastasis . for the management of brain metastasis from pnet lung , at present wbrt to a dose of 30 gy was exhibited as it is the standard of care in patients with brain metastasis which has shown superiority in preventing neurologic morbidity and mortality . pazopanib is an oral multikinase inhibitor of angiogenesis indicated mainly for progressive or chemotherapy refractory soft tissue sarcomas . although pazopanib is not the standard therapeutic option in ewing sarcoma , it has demonstrated its efficacy in refractory and ees metastatic cases . since ewing 's tumors and ppnets share similar genetic characteristics with reciprocal translocations of chromosomes 11 and 22 at ewsr1 , differing only in degree of neural differentiation with the former being more differentiated than latter , we , therefore , exhibited pazopanib to our patient with a palliative intent as she had progressed after standard chemotherapeutic regimen . the effectiveness of pazopanib by children 's oncology group ( cog ) nct01956669 phase-2 study and another multi - kinase inhibitor regorafenib by nct02048371 and nct02389244 phase-2 studies for refractory ewing sarcoma are underway , and the results are awaited . newer prognostic markers should be developed to identify primary pnet lung at risk of developing brain or distant metastases . a better understanding and interpretation of the molecular and biological mechanisms of this entity we recommend craniotomy and excision followed by wbrt for brain metastasis and oral pazopanib to check further dissemination as an incisive treatment guideline , though more prospective data favoring this recommendation is required .	primitive neuroectodermal tumors ( pnets ) are highly malignant neoplasms of embryonal origin manifesting in children and adolescents , rarely seen in adults . carcinoma lung with hemorrhagic metastasis to the brain is very common , but primary lung pnet with hemorrhagic brain metastasis is extremely uncommon . we hereby report a 29-year - old female diagnosed as pnet lung was treated with vincristine , adriamycin , and cyclophosphamide alternating with ifosfamide plus etoposide followed by radiotherapy ( rt ) . after 9 months , she developed hemorrhagic brain metastasis from pnet lung confirmed from tissue immunohistology postcraniotomy . received palliative whole brain rt followed by oral pazopanib resulting in significant improvement in performance status . a thorough review of literature reveals that our case may be the second case of primary lung pnet with hemorrhagic brain metastasis and also the first to be exhibited oral pazopanib resulting in a significant therapeutic effect to be reported in world literature till date .
pu - erh tea is a special tea species in china and has become one of the most popular beverages in southwestern china and southeast asian , owing to its special flavour properties and potential healthy benefits . it is originated from yunnan province ( china ) through a special post - fermentative process , using crude green tea prepared from the leaves of c. sinensis var . assamica as original materials . because pu - erh tea has a malty flavour and low - stimulation taste in tea infusions which may fit female appetite , recent years , interest in the flavor and the healthy properties and the related scientific investigations were increasing [ 3 , 4 ] . up to now , a little information on the relationship of the chemical composition to the flavor is available . volatile flavor component is one of the most important factors to influence the flavor , taste , and quality of pu - erh tea , in which the contents are different from the green and black tea because of different processing procedure and variety of species and cultivar . investigation on the components in green and black tea are reported elsewhere , yet few on those in pu - erh teas . in order to explore the influence of those components to flavor , taste , and quality , quantitative analysis of main volatile flavor components in pu - erh tea for analysis of volatile components or essential oils , several extraction techniques , including soxhlet extraction , liquid - liquid extraction ( lle ) , simutaneous distillation - solvent extraction ( sde ) , solid phase microextraction ( spme ) , and headspace microextraction ( hsme ) and so forth , had been used to diferent matices . soxhlet extraction is a classical method for decades in extraction of organic compounds from solid sample , and this apparatus has been developed to several types for special use . thorough extraction method because the organic phase cooled from condensation tube continuously passes through the target solid sample for hours . however , poor recovery commonly occurred for extraction of high - volatile or heat - labile compounds . lle is a conventional method for isolation of all boiling range volatile compounds , based on the compatibility of compounds with organic phase selected . the main disadvantage is solvent - consuming , tedious and , low - recovery for some target compounds . sde , proposed by godefroot and so forth , has been widely applied to analysis of volatile components in tea samples . although low recovery has been found for extracting the most volatile or heat - labile components , this technique has achieved higher recoveries and greater repeatability of volatile or semi - volatile and heat - stable components than other isolation technique such as spme or hsme when low water temperature in the circulating system was used . spme and hsme are relatively new techniques that are able to address the need for concentrating the components in the headspace [ 10 , 11 ] . both of them use a small piece of fused silica , on which a liquid or solid phase , similar to a gc stationary phase , has been coated to absorb the desired components and concentrate them on the fibre . thus the two techniques are more sensitive for the isolation of high volatile components than sde , while less sensitive and lower repeatability for the low volatiles than sde . conventionally , pu - erh tea is condensed to a pie - like tea - biscuit in the final processing procedure . extraction of the volatile flavor components will be different from the green and black tea . in order to select the best extraction technique for studying the volatile flavor components of pu - erh tea , a modified sde was evaluated for quantitative determination of the analytes using ethyl decylate as internal standard , and the two classical techniques , steam distillation - liquid / liquid extraction and soxhlet extraction , pu - erh tea samples were obtained from dayi limited incorporation ( menghai , yunnan , china ) . the sample was dried at 40c in electric oven for 6 h , ground to 3060 mesh , and sealed for use . the reference volatile chemicals were purchased from sigma ( st . louis , mo , usa ) . the stock solution was prepared by dissolving single solid / liquid standard in dichloromethane to an appropriate concentration depending on the content in pu - erh tea . ultra - pure water was obtained from a milli - q water purification system ( pall co , il , usa ) . the other solvents used in the test were all of analytical - grade and disdillated before use . dichloromethane and ethyl decylate were employed as solvent and internal standards , respectively . for each extraction , 15 g of tea sample , 10 g sodium sulphate , 100 l internal standard solution and 300 ml ultra - pure water were placed in a 1 l flask , 50 ml dichloromethane was in a 100 ml flask , and temperature of the circulating water system was operated at 8c . stream distillation was stopped after 2 h , while the solvent extraction was continued for a further 15 min . the extract was concentrated to 1 ml at 10c by a nitrogen - purge apparatus ( shanghai anpel scientific instrument co. ltd ) . the concentated solution was dehydrated with anhydrous sodium sulphate for at least 12 h , of which 2 l was injected to gc or gc - ms system for analysis . for sd - lle , 15 g of tea sample , 10 g sodium sulphate , 100 l internal standard solution and 500 ml ultra - pure water were placed in a 1 l distillation flask , respectively . the liquid was transferred to a 500 ml of separation funnel and then extracted three times ( 30 ml 3 ) using dichloromethane . the extracted organic phase was combined and concentrated to 1 ml at 30c by a nitrogen - purge apparatus after internal standard was added . 15 g of tea sample containing 100 l internal standard solution were placed in soxhlet 's apparatus and 50 ml of dichloromethane in an 150 ml distillation flask . at both ends of the sample in soxhlet 's apparatus , there are 2 cm - height of celite to help fix the sample . extraction was carried out at 50c for 2 hours , and extraction was concentrated to 1 ml at 30c by a nitrogen - purge apparatus after internal standard was added . the concentrated extracts were chromtographed by an hp 6890 series gc system ( agilent , usa ) . a 30 m 0.25 mm db-5 quartz capillary column ( supelco , bellefonte , pa , usa ) with 0.25 m film thickness was used to resolve the volatiles . temperature programming was as follows : initial oven temperature was set at 60c and kept for 3 min , then raised to 200c at a ramp of 4c / min and kept for 2 min ; to 210c at of 1c / min and kept for 2 min ; and finally , it was raised to 270c and kept for 7 min . nitrogen was used as carrier gas with column head pressure at 12.26 kpa in constant pressure mode . programming split / splitless injection temperature was set at 260c with split ratio of 10 : 1 and fid detector at 280c . auto system shimaszu qp 2010 gc - ms was employed for qualitative analysis to confirm the target components . the oven temperature was set at 50c and kept for 2 min , then raised to 60c at a ramp of 1c / min and kept for 2 min , to 200c at of 4c / min and kept for 2 min , and finally , to 270c at 10c / min and kept for 5 min . the mass spectrometry was operated at 200c in the electron impact mode ( 70 ev ) , scanning from m / z 40 to 600 in 0.3 s with an 0.2 s interval time of the scan ; the temperature of the gc - ms interface was 280c ; the voltage of the photoelectric multiplier tube ( pmt ) was 200 v. the mass spectral identifications of the target components were carried out by comparing to the nist 107 ( national institute of standards and technology , gaithersburg , usa ) mass spectral library as well as to wiley 6.0 ( wiley , new york , ny , usa ) mass spectral library . for gc separation of volatile components in tea samples , bp-20 sge column ( polar column , 30 m 0.25 mm i.d . film thickness 0.25 m ) was commonly used for quantitative analysis . in this work , film thickness 0.25 m ) was employed to separate the target components with temperature programming described above , and the chromatograms of standard compounds by gc - fid and gc - ms was shown in figures 1(a ) and 2(a ) . from the chromatograms , it can be seen each peak of the target compounds was baseline separated , and separating degree between two vicinity peaks was beyond 2 , confirming weak - polar capillary column can be used to separate the target components if gc separation condition was well optimized . according to gc - fid chromatogram , we calculated the relative factor of each of the standard compounds , it was shown in table 1 , and the relative factor was used to determination of the corresponding components in real pu - erh teas . figures 1(b ) and 2(b ) show the chromatograms of pu - erh raw tea that was obtained from gc - fid and gc - ms determination , in which the target peaks of real sample could be easily discerned and accurately quantified . the mass spectra of each target peaks in figure 2(b ) was checked by nist or wiley mass spectral library respectively , and campared with those in figure 2(a ) , confirming that each mass spectra of the components was the same as those of the corresponding standard compound , and no interference was appeared in the target peaks . comparing figures 1(b ) and 2(b ) , we found that both gc - fid and gc - ms separation methods can be applied to quantitative analysis of volatile flavor components in pu - erh teas . nevertheless , quantitative determination of the target components using gc - fid was more cost - saving than doing gc - ms , so the rest determinations for all of the samples were carried out in gc - fid . during sde operation , dichloromethane and diethylether are the most suitable extraction solvents for extraction of volatile components because of their weakly polar characteristics [ 15 , 17 ] . in this experimental , we select dichloromethane as extraction solvent for sde , due to its property of low combustion , and higher boiling point ( 40c ) than diethylether ( 35c ) thus liable to storage . zhu et al . had applied sde to extract volatile constitutes in green tea , confirming of 2 h was adequate to extract all the target compounds . initially , we operated sde apparatus as zhu et al . done , and found that there are about 6% of target components dwelling on the tea residue . this is because pu - erh tea was condensed to a pie - like shape , and the components were more difficult to release from tea matrix although the teas had been ground to 3060 mesh . then we modified this method by adding 10 g of sodium sulphate to the 1 l of steam flask containing 300 ml of water , and then operating sde device . after 2 h of sde and a further 15 min of the solvent extraction , the tea residue was re - extracted by sde , we found the area of the target peaks were less than 2% of the total . obviously , boiling point of water in steam flask was enhanced by adding sodium sulphate , accelerating the releasing velocity of target compounds . to confirm the repeatability , parallel the relative standard deviations ( rsd ) were in range of 1.44%12.6% , which were shown in table 3 . to check accuracy of sde for volatile flavor components , a known amount of standard solutions were added to aliquot of pu - erh tea . the adding level and corresponding recoveries were listed in table 3 . for quantitative extraction of volatile flavor components from complex matrices , common technique used is sde . because both liquid / liquid extraction and soxhlet extraction are classical techniques [ 8 , 10 ] , in this test , the modified classical techniques were compared with sde in extraction of the volatile flavor components from pu - erh tea in order to select the best one for sample preparation . the results were shown in table 2 . generally , sde was the best one among them , because the amounts of the components extracted were greater than those by employing sd - llc or soxhlet for the high - volatile components such as benzyl alcohol , linalool oxide , and linalool . this may attributes to the fact that sde was a closed and continuous extraction system , in which the target components can be furthermore , the temperature of the circulating water in sde was set at 8c , reducing the lose of the high - volatile components . as for extraction of the low - volatile components in pu - erh tea , such as 1,2,4-trimethoxylbenzene , sde is less poor compared with soxhlet extraction , probably due to the characteristics of high - boiling point and incompatibility with water steam . although sd - lle shares a same extraction principle with sde , the limitation of the technique is clear in table 2 . this is due to its open steam distillation system and following step - liquid - liquid extraction . during this procedure , not only high - volatile components such as benzyl alcohol , linalool oxide , and linalool and so forth had a chance to escape from the distillation system , but the analytes hardly transferred to organic phase . we could not confirm whether isomerization reaction was happened between the two compounds during extraction , and further investigation was beyond the work . table 2 shows that it can almost exhaustively extract all the 1,2,3- and 1,2,4-trimethoxylbenzene from pu - erh tea compared with sde , nevertheless it is very poor for benzyl alcohol , linalool oxide , linalool , phenethyl alcohol , and geraniol . these findings indicate soxhlet was suitable for high - boiling point ( low - volatile ) compounds such as essential oil . as for the trace high - volatile component , , we analyzed several tea samples , including four pu - erh ripe tea , four pu - erh raw tea , one green tea ( huangshan , anhui ) and one black tea ( qimen , anhui ) , and the contents of the target volatile components were shown in table 4 . it can be seen that the content of high volatile components , such as benzyl alcohol , linalool oxide , linalool in raw teas are higher than those in ripe teas , these findings are mainly due to the high temperature ( 4555c ) during the pile - fermentation process of ripe pu - erh teas , leading to the lose of these components . however , in raw teas , the content of 1,2,3- and 1,2,4-trimethoxylbenzene are much lower than those in ripe teas , this is because microbes play an important role in the synthesis of the two compounds during the pile - fermentation process of ripe pu - erh teas , which promote reaction procedure of methylation . generally , the contents of the volatile components in the green and the black teas are higher than those in pu - erh teas , except for 1,2,3- and 1,2,4-trimethoxylbenzene , which are not found in both of teas , probably due to lack of post - pile - fermentation process in manufacture , and linalool , lower than those in pu - erh raw teas due to different cultivar , variety of species and processing procedure . sde combined gc method was constructed for determination of volatile flavor components in pu - erh tea samples . 10 of volatile flavor components were quantitatively determined , and the recoveries and rsds were in the range of 66.4%109% and 1.44%12.6% , respectively . the method was compared with sd - lle and soxhlet extraction , comfirming sde was suitable for pu - erh teas among them . pu - erh raw tea and ripe tea samples were analyzed by the method , indicating the high - volatile components , such as benzyl alcohol , linalool oxide , and linalool , were rich in pu - erh raw teas , while the contents of 1,2,3- and 1,2,4-trimethoxylbenzene were much high in pu - erh ripe teas .	a simutaneous distillation extraction ( sde ) combined gc method was constructed for determination of volatile flavor components in pu - erh tea samples . dichloromethane and ethyl decylate was employed as organic phase in sde and internal standard in determination , respectively . weakly polar db-5 column was used to separate the volatile flavor components in gc , 10 of the components were quantitatively analyzed , and further confirmed by gc - ms . the recovery covered from 66.4%109% , and repeatability expressed as rsd was in range of 1.44%12.6% . sde was most suitable for the extraction of the anlytes by comparing with steam distillation - liquid / liquid extraction and soxhlet extraction . commercially available pu - erh tea samples , including pu - erh raw tea and ripe tea , were analyzed by the constructed method . the high - volatile components , such as benzyl alcohol , linalool oxide , and linalool , were greatly rich in pu - erh raw teas , while the contents of 1,2,3-trimethoxylbenzene and 1,2,4-trimethoxylbenzene were much high in pu - erh ripe teas .
we used data collected in the capital of chad , ndjamna , which in 2001 had a human population of 776,000 and a dog population of 23,600 ( 4 ) . dog rabies is endemic to chad ; before the vaccination campaigns , the prevalence of dog rabies was 1.41.7 cases/1,000 unvaccinated dogs ( 5,6 ) . we obtained direct observations of the association between compliance ( i.e. , percentage of dogs vaccinated ) and amount charged to owners from 2 pilot dog vaccination campaigns held in ndjamna in 2002 and 2006 ( 7 ; unpub . each campaign covered the same 3 city quarters , which had high - density dog populations ( 4 ) . only owned animals were vaccinated , but owned dogs comprise 90%99% of all dogs in ndjamna ( 7 ) . owners brought their animals ( dogs , cats , monkeys ) to 1 of 10 vaccination sites . in the 2002 campaign , vaccinations were free to owners ( 7 ) ; in the 2006 campaign , owners were charged 2,000 cfa francs ( us $ 3.88)/animal vaccinated ( unpub . ( exchange rate us $ 1 = 515.71 cfa francs as of february 2007 ; www.oanda.com/convert/classic ) for each campaign , the percentage of dogs vaccinated was estimated by using a capture recapture method ( 7 ; unpub . data ) . for the 2002 free - to - owners campaign , 71%87% ( 95% confidence interval [ ci ] 64%89% ; mean 79% ) of all dogs ( owned and unowned ) were vaccinated in 2 of the zones ( 1 zone per quarter ) included in the campaign ( 7 ) . for the 2006 campaign , in which owners were charged , the mean vaccination coverage among all dogs was estimated at 24% ( 95% ci 0.13%24.82% ) ( unpub . vaccination rates for owned dogs averaged only 78% and 25% in the 2002 and 2006 campaigns , respectively ( 7 ; unpub . , we used the latter estimates because we were interested in measuring owner compliance to charges for dog vaccination . additional observational data were obtained from a household survey conducted in 2001 ( 4 ) , which recorded that 19% of owned dogs were vaccinated against rabies . such vaccinations the charge for such vaccinations at the urban government - run veterinary clinic and the 3 private veterinary practices of ndjamna was 3,0005,000 cfa francs ( us $ 5.82$9.69 ) . we did not inflate the 2001 charges because we encountered problems identifying an appropriate conversion factor that considered veterinary medical services . during the 2006 campaign we surveyed dog owners by using a short questionnaire ( technical appendix ) . the survey was conducted in the vaccination zones ; households ( containing at least 1 animal ) were chosen randomly . the questions ( written in french ) were translated , when needed , into local languages by 4 interviewers . one question asked owners how much they were willing to pay for the vaccination of their animals . we graphed the 3 observational data points ( assuming a straight - line interpolation between points ) and the reverse cumulative probability of the owner - stated amounts that they would be willing to pay for their animal to be vaccinated against rabies ( figure ) . an initial statistical ( regression ) analysis of the relationship between the amounts that owners said they would pay and the variables collected during the survey ( table ) provided an adjusted r value of 0.07 ( data not shown ) . average probability of having a dog vaccinated against rabies by charge for vaccination : observed versus owner - stated values for vaccination . the observed values of charges to vaccinate an owned dog against rabies and probability of vaccination came from 3 sources . points a and b ( recording vaccination coverage for all owned dogs vs. costs charged ) come from 2 vaccination campaigns held in ndjamna in 2002 and 2006 , respectively . point c represents the midpoint of the range of recorded 2001 clinic charges in ndjamna for vaccinating a dog against rabies ( costs not adjusted for any potential inflation ) . the owner - stated amounts that they would be willing to pay for their dogs to be vaccinated against rabies came from a survey of 356 households , conducted in 2006 . interviewed households provided 356 questionnaires from which we estimated owner - stated willingness - to - pay for pet vaccination and calculated the resultant reverse cumulative probability of having their animal vaccinated . when asked how much they would be willing to pay , 5 ( 1% ) owners stated that they were against vaccination . we interpreted that response to indicate that such owners would , essentially , have to be paid to have their animals vaccinated . when the proposed cost of vaccination was < 1,500 cfa francs / animal vaccinated , owners were more likely to state that they would pay to have their pet vaccinated than they were to actually do it . the stated values and observed values were closest at 2,000 cfa francs ( 25% probability of animal being vaccinated ) ( figure ) . this finding was probably because the questionnaire was administered immediately after the campaign in which owners were charged 2,000 cfa francs / animal vaccinated . for > 2,000 cfa francs , the observed values indicated that compliance would be greater than that stated by owners responses to the willingness - to - pay question . the figure shows that to achieve a minimum of 70% of owned animals vaccinated , the maximum amount that could be charged would be 400 cfa francs ( us $ 0.78 ) ( observed values ) to 700 cfa francs ( us $ 1.36 ) ( owner - stated values ) . because the data shown in the figure reflect owned animals only , to get vaccination coverage up to 70% of all animals ( owned and stray ) , vaccination rates among owned animals would have to be > 70% . to attain these higher rates few studies have compared what members of the general public state they are willing to pay for a public health intervention with their actual observed behavior ( 8) . direct comparison between stated and observed behavior , as influenced by charges to owner , provides public health officials with an understanding of the reliability of owner surveys . furthermore , to maintain dog vaccination rates at the who - recommended rate of 70% , dog vaccination campaigns would have to be held every 16 years , which could reduce compliance . the survey was , by design , short , but a longer questionnaire may have allowed us to better identify why 75% of respondents did not wish to pay > 500 cfa francs ( us $ 0.97 ) . to achieve the who - recommended goal , public health officials can not charge owners more than the equivalent of 400 cfa francs ( us $ 0.78 ) . full - cost recovery concepts will not ensure that enough dogs are vaccinated in chad ( or , most likely , other african countries ) to interrupt rabies transmission in dogs in urban areas . clearly , to have > 70% of all dogs vaccinated , public health officials and policy makers must consider methods to substantially subsidize dog rabies vaccinations .	we estimated the association between amount charged and probability that dog owners in ndjamna , chad , would have their dogs vaccinated against rabies . owners would pay 400700 cfa francs ( us $ 0.78$1.36)/animal . to vaccinate > 70% of dogs , and thus interrupt rabies transmission , health officials should substantially subsidize these vaccinations .
( r)-n - benzyl 2-acetamido-3-methoxypropionamide ( lacosamide , ( r)-1 ) is a first - in - class antiseizure drug ( asd ) that is marketed worldwide for adjunctive treatment of partial - onset seizures . whole animal pharmacological studies showed that ( r)-1 displayed potent anticonvulsant activity in the maximal electroshock ( mes ) , 6 hz psychomotor , and hippocampal kindled seizure models . whole - cell , patch - clamp electrophysiology studies indicated that ( r)-1 reduced neuronal hyperexcitability by a mechanism that was consistent with its enhancing the transition of voltage - gated na channels ( vgscs ) to the slow - inactivated state . recent structure activity relationship studies demonstrated that modifying either the n - benzyl or the o - methoxy sites in ( r)-1 provided analogues that retained excellent anticonvulsant activities . importantly , we found that the two n-(biphenyl-4-yl)methyl analogues , ( r)-2 and ( r)-3 , when given orally to rats in the mes model , exhibited activities that exceeded those of ( r)-1 ( figure 1 ) . we further showed that the ic50 value for na channel slow inactivation ( the concentration at which half of the na channels transitioned to the slow - inactivated state ) in catecholamine a differentiated ( cad ) cells was 29-fold lower ( more potent ) for ( r)-3 than for ( r)-1 ( figure 1 ) . while these findings showed that introducing 4-aryl substituents to ( r)-1 provided pharmacologically potent compounds , they also showed that the 4-aryl substituent affected animal behavioral neurotoxicity . compound ( r)-2 was significantly more neurotoxic than ( r)-1 , but ( r)-3 was not ( figure 1 ) . key pharmacological properties of ( r)-1 , ( r)-2 , and ( r)-3 . in this study , we report the synthesis and pharmacological activity of 13 substituted n-(biphenyl-4-yl)methyl ( r)-2-acetamido-3-methoxypropionamides ( compound class ( r)-a ) where the r - substituent in the terminal aryl unit was systematically varied . the ( r)-a s r - substituent significantly impacted the compound s whole animal and cellular pharmacological activity . numerous compounds with a polar , aprotic r - substituent exhibited excellent anticonvulsant and potent na channel activities while producing minimal behavioral neurotoxicities . thirteen ( r)-a derivatives , ( r)-4(r)-16 , containing different r - substituents were synthesized and evaluated in animal and cellular pharmacological models . one set , ( r)-4(r)-12 , contained polar , aprotic r - substituents ( i.e. , f , cl , br , i , cn , cf3 , ocf3 ) , and the second set , ( r)-13(r)-16 , contained polar , protic r - substituents ( i.e. , n(h)c(o)ch3 , ch2oh , co2h , nh3cl ) . we expected that the co2h substituent in ( r)-15 and the nh3cl substituent in ( r)-16 were likely ionized at the physiological - like ph values used in the evaluations . when the r - substituent was br , i , cn , cf3 , n(h)c(o)ch3 , ch2oh , co2h , or nh3cl , we installed the group at the 3-position of the terminal aryl ring ( ( r)-7 , ( r)-8 , ( r)-9 , ( r)-10 , ( r)-13 , ( r)-14 , ( r)-15 , ( r)-16 ) ; when the r - substituent was f , cl , or ocf3 , we prepared both the 3 ( ( r)-3 , ( r)-5 , ( r)-11 ) and the 4 ( ( r)-4 , ( r)-6 , ( r)-12 ) regioisomers . we expected that the r - substituents would affect the compound s biodistribution , anticonvulsant activities , and ability to modulate na channel function . compounds ( r)-4(r)-16 were prepared by similar routes ( schemes 16 ) using the mixed anhydride coupling ( mac ) method and employing ( r)-n - tert - butoxycarbonylserine ( ( r)-45 ) , ( r)-2-acetamido-3-hydroxypropionic acid ( ( r)-69 ) , or ( r)-2-acetamido-3-methoxypropionic acid ( ( r)-70 ) as the carboxylic acid component . the choice of the carboxylic acid was dictated by the ease of purification of the intermediate products and the need to avoid competing side reactions in the latter stages of the synthesis . for compounds ( r)-14(r)-16 , we removed the protecting group in the final step to facilitate the isolation of the desired polar products . to prepare compounds ( r)-4(r)-13 , ( r)-n - tert - butoxycarbonylserine ( ( r)-45 ) was coupled with the requisite ( biphenyl-4-yl)methylamine ( 2939 , 43 , 44 ) using the mac reagents , isobutyl chloroformate ( ibcf ) and n - methylmorpholine ( nmm ) , to give the amides ( r)-46(r)-55 ( scheme 3 ) . the crude ( biphenyl-4-yl)methylamines were prepared by suzuki coupling using tetrakis(triphenylphoshine)palladium(0 ) . for 2939 , 4-bromobenzylamine ( 17 ) was reacted with the commercially available arylboronic acids ( 1828 ) ( scheme 1 ) , and for 43 and 44 , p - aminomethylboronic acid ( 40 ) was coupled with the appropriate 1,3-dihalobenzene ( 41 , 42 ) . the serine hydroxyl group in ( r)-46(r)-55 was methylated ( ch3i , ag2o ) to provide ethers ( r)-56(r)-65 , respectively . deprotection of the tert - butoxycarbonyl group in ( r)-56(r)-65 with acid ( hcl / dioxane ) followed by acetylation ( accl , et3n ) gave the desired products ( r)-4(r)-13 , respectively . for ( r)-14 and ( r)-15 , we coupled ( r)-69 with ( biphenyl-4-yl)methylamines 37 and 38 ( scheme 5 ) , and for ( r)-16 , we combined ( r)-70 with ( 3-n-(tert - butoxycarbonyl)aminobiphenyl-4-yl)methylamine ( 39 ) ( scheme 6 ) . to facilitate the preparation of ( r)-14(r)-16 , we first developed convenient synthetic procedures for ( r)-69 and ( r)-70 ( scheme 4 ) . commercially available d - serine benzyl ester hydrochloride ( ( r)-66 ) was selectively converted to ( r)-2-n - acetamido-3-hydroxypropionate benzyl ester ( ( r)-67 ) using 0.9 equiv of accl at 20 c . compound ( r)-67 was methylated ( ch3i , ag2o ) at room temperature to give ( r)-68 , without racemization . catalytic hydrogenation of ( r)-67 and ( r)-68 using 10% pd / c in methanol provided the desired acids ( r)-69 and ( r)-70 , respectively , which were used directly in the mac reaction without purification . the enantiomeric purity of ( r)-4(r)-13 was assessed by detecting a single acetyl methyl peak and a single o - methyl peak in the h nmr spectrum ( cdcl3 ) for each compound when a saturated solution of ( r)-()-mandelic acid was added . in the cases of ( r)-14(r)-16 , their poor solubility in cdcl3 thus , we demonstrated with h nmr the enantiomeric purity of their immediate precursors ( r)-73(r)-75 , respectively . in the experimental section , we report the details ( synthetic procedure , characterization ) of the final step for all compounds evaluated in the anticonvulsant and cellular electrophysiology studies . in supporting information , we provide the experimental procedures and physical and spectroscopic properties for all synthetic compounds prepared in this study . compounds ( r)-4(r)-16 were tested for anticonvulsant activity at the anticonvulsant screening program ( asp ) of the national institute of neurological disorders and stroke ( ninds ) , u.s . screening was performed using the established protocols and procedures at the asp described by stables and kupferberg . we relied principally on two rodent models to assess anticonvulsant activity , the mes and the 6 hz psychomotor seizure tests . the mes test is a model for generalized tonic clonic seizure and is thought to provide an indication of a compound s ability to prevent seizure spread when all neuronal circuits in the brain are maximally active . correspondingly , the 6 hz psychomotor test ( 32 ma ) has been used as a model for therapy - resistant limbic seizures . previous studies have shown that some compounds with minimal activity in the mes test are effective in the 6 hz psychomotor model . the anticonvulsant data from the mes model ( mice , ip ; rat , po ) and psychomotor 6 hz ( 32 ma ) seizure test for therapy - resistant limbic seizures ( mice , ip ) are summarized in table 1 along with similar results obtained for ( r)-1 , ( r)-2 , ( r)-3 , and the asds phenytoin , phenobarbital , and valproate . for compounds that showed significant activity , we also include the median doses for 50% neurological impairment ( td50 ) in mice , using the rotorod test , and the behavioral toxicity effects observed in rats . the protective index ( pi = td50/ed50 ) for the test compounds , where possible , is also listed . compounds ( r)-2(r)-6 and ( r)-11 and ( r)-12 were evaluated in the subcutaneous metrazol ( scmet ) seizure model . no activity was observed below 100 mg / kg ( data not shown ) . similarly , we observed no activity in the scmet model for ( r)-1 and structurally related compounds . numbers in parentheses are 95% confidence intervals . a dose response curve was generated for all compounds that displayed sufficient activity . effect for these compounds was obtained at the time of peak effect ( indicated in hours in the brackets ) . numbers in parentheses are 95% confidence intervals . a dose response curve was generated for all compounds that displayed sufficient activity . effect for these compounds was obtained at the time of peak effect ( indicated in hours in the brackets ) . ic50 , concentration at which half of the na channels have transitioned to a slow inactivated state . the r - substituent in ( r)-4(r)-16 markedly affected the observed anticonvulsant activity in the mes and 6 hz seizure tests ( table 1 ) . we identified compounds with similar potency compared with the parent compound in this series , the unsubstituted n-(biphenyl-4-yl)methyl ( r)-2-acetamido-3-methoxypropionamide ( ( r)-2 ) , and the asd ( r)-1 , while other compounds showed little or no activity . the most potent anticonvulsant compounds in the mes test contained a polar , aprotic r - substituent ( i.e. , f ( ( r)-3 , ( r)-4 ) ; cl ( ( r)-5 , ( r)-6 ) ; br ( ( r)-7 ) ; cf3 ( ( r)-10 ) ; ocf3 ( ( r)-11 , ( r)-12 ) . the mes ed50 values in mice ( ip ) ranged from 4.7 to 12 mg / kg and in the rat ( po ) from 2.4 to < 30 mg / kg . the corresponding ed50 values for ( r)-2 in mice ( ip ) and rats ( po ) were 8.0 and 2.0 mg / kg , and for ( r)-1 they were 4.5 and 3.9 mg / kg . we observed little differences in the anticonvulsant activities in the mes test in mice ( ip ) for the 3-substituted f , cl , and ocf3 compounds ( ( r)-3 , ( r)-5 , ( r)-11 ) compared with their 4-substituted regioisomers ( ( r)-4 , ( r)-6 , ( r)-12 ) ( mes ed50 ( mice , ip , mg / kg ) : ( r)-3 , 12 ; ( r)-4 , 6.1 ; ( r)-5 , 9.8 ; ( r)-6 , 12 ; ( r)-11 , 4.7 ; ( r)-12 , 6.5 ) . the 3-cn substituted compound ( ( r)-9 ) exhibited only moderate activity in the mes seizure model ( mice , ip , mg / kg ) : ( r)-9 , 30100 ) . a pronounced decrease in activity was seen for compounds ( r)-13(r)-16 containing a polar , protic r - substituent ( i.e. , n(h)c(o)ch3 ( ( r)-13 ) ; ch2oh ( ( r)-14 ) ; co2h ( ( r)-15 ) ; nh3cl ( ( r)-16 ) . most of these compounds showed little activity in the mes seizure model in mice ( ip ) , with only ( r)-14 exhibiting modest activity ( ed50 , mice , ip , mg / kg : ( r)-13 , > 300 ; ( r)-14 , 30100 ; ( r)-15 , 100300 ; ( r)-16 , 100 ) . we found that the neurotoxicity of ( r)-a in the rotorod ( mice , ip ) and behavioral ( rat , po ) tests varied with the nature of the r - substituent and its position in the terminal aryl ring . many of the compounds ( ( r)-3 , ( r)-5(r)-11 , ( r)-13(r)-16 ) were significantly less neurotoxic in mice than the parent , unsubstituted compound ( r)-2 ( td50 = 11 mg / kg ) and exhibited comparable or less neurotoxicity than the asd ( r)-1 ( td50 = 27 mg / kg ) . thus , the excellent activities in the mes test ( mice , ip ) of ( r)-3 , ( r)-5 , ( r)-6 , and ( r)-10 ( ed50 ( mg / kg ) , ( r)-3 , 12 ; ( r)-5 , 9.8 ; ( r)-6 , 12 ; ( r)-7 , 12 ; ( r)-10 , 8.3 ) coupled with their low neurotoxicities ( td50 ( mg / kg ) , ( r)-3 , 50 ; ( r)-5 , 74 ; ( r)-6 , 82 ; ( r)-7 , 86 ; ( r)-10 , 50 ) provided compounds with pis ( 4.17.6 ) that were notably higher than that of the parent , unsubstituted compound ( r)-2 ( pi = 1.4 ) and that were similar to that of ( r)-1 ( pi = 6.0 ) . when r was either f or ocf3 , we found that the 3-derivative was less neurotoxic than the 4-substituted isomer ( td50 ( mg / kg ) , ( r)-3 ( 3-f ) , 50 ; ( r)-4 ( 4-f ) , 11 ; ( r)-11 ( 3-ocf3 ) , 23 ; ( r)-12 ( 4-ocf3 ) , 13 ) and provided higher pi values ( pi , ( r)-3 ( 3-f ) , 4.1 ; ( r)-4 ( 4-f ) , 1.8 ; ( r)-11 ( 3-ocf3 ) , 4.9 ; ( r)-12 ( 4-ocf3 ) , 2.0 ) ; however , when r was cl , there was little difference in the neurotoxicity ( td50 ( mg / kg ) , ( r)-5 ( 3-cl ) , 74 ; ( r)-6 ( 4-cl ) , 82 ) and in the pi values ( pi , ( r)-5 ( 3-cl ) , 7.6 ; ( r)-6 ( 4-cl ) , 6.8 ) for the regioisomers . a drop in neurotoxicity was observed for ( r)-13(r)-16 , the four compounds with polar , protic r - substituents ( td50 of 100 to > 300 mg / kg ) . these compounds showed moderate or little protection in the seizure models . taken together , these findings demonstrated the importance of the r - substituent in ( r)-a in eliciting the optimal responses in the animal models . we found that specific substituents and their placement in the terminal aryl ring afforded compounds ( ( r)-5 ( 3-cl ) , ( r)-6 ( 4-cl ) , ( r)-7 ( 3-br ) , ( r)-10 ( 3-cf3 ) , ( r)-11 ( 3-ocf3 ) ) with excellent anticonvulsant activities and improved pi values compared with the parent , unsubstituted compound , ( r)-2 , in the mouse ( ip ) . similar results were observed in the rat ( po ) ( pi : ( r)-2 , 25 ; ( r)-3 , > 210 ; ( r)-5 , > 91 ; ( r)-6 , 78 ; ( r)-11 , 34 ) . three of the more active ( r)-a compounds were tested in the rat hippocampal seizure model ( ip ) . this is a test of partial complex or temporal lobe seizures , the most common and most drug - resistant type of adult focal epilepsy . we obtained ed50 values ( mg / kg ) for ( r)-3 , ( r)-5 , and ( r)-11 of 13 , 36 , and 1.7 , respectively . the ed50 value for ( r)-11 was 8-fold lower than that for ( r)-1 ( 14 mg / kg ) . compounds of interest were characterized for their abilities to interact with vgscs by whole - cell patch - clamp electrophysiological studies . in neurons , these channels undergo inactivation in response to activity over two distinct time courses and mechanisms : fast inactivation occurs over a few milliseconds , whereas slow inactivation occurs over several hundred milliseconds . inactivation is reversible , but for a time , these channels are unable to produce current in response to stimulus . the function of several asds , such as carbamazepine and ( r)-1 , has been attributed to their ability to inactivate vgscs and reduce synaptic activity . we assessed the ability of the ( r)-a compounds to alter na channel kinetics of fast inactivation and slow inactivation and also for frequency ( use ) dependence . these studies were carried out in several cell models : cad cells allow for rapid characterization of compounds ; rat cortical neurons allow for characterization in cells related to those that generate epileptic phenotypes in humans ; and hek293 cells allow for characterization of activity on single vgsc isoforms . we previously showed that ( r)-3 inhibited several parameters of vgsc activities in neuronal model cad cells . here initially , we focused on ( r)-a compounds with polar , aprotic r - substituents ( i.e. , r = f , cl , br , i , cf3 , ocf3 ) at either the 3 or the 4 position of the terminal aryl ring , since these compounds exhibited the greatest anticonvulsant activities ( table 1 ) . these cells express endogenous tetrodotoxin - sensitive na currents that display rapid activation and inactivation kinetics upon membrane depolarization and likely comprise a majority of nav1.7 channels , with minor contributions by nav1.1 , nav1.3 , and nav1.9 channels . importantly , we earlier demonstrated that the na channel properties of ( r)-1 in cad cells were similar to those reported in cultured neurons and in mouse n1e-115 first , we examined ( r)-3 , ( r)-5 , ( r)-7 , ( r)-8 , ( r)-10 , and ( r)-11 , compounds with a 3-polar , aprotic r - substituent ( r = f , cl , br , i , cf3 , ocf3 ) , for their ability to transition na channels to the slow - inactivated state . cad cells were held at 80 mv and conditioned to potentials ranging from 120 to + 20 mv ( in + 10 mv increments ) for 5 s. then fast - inactivated channels were allowed to recover for 150 ms at a hyperpolarized pulse to 120 mv , and then the fraction of channels available was tested by a single depolarizing pulse , to 0 mv , for 15 ms ( figure 2a , left ) . brief hyperpolarization to 120 mv allows channel recovery from fast inactivation while limiting recovery from slow inactivation such that the property of slow inactivation can be isolated . representative slow inactivation traces , at 120 and 50 mv , from cad cells treated with 0.1% dmso ( control ) or 10 m ( r)-11 are shown in figure 2a ( right ) . at 50 mv , a majority of the channels undergo steady - state inactivation that involves contributions from slow- and fast - inactivating pathways . this voltage is also near the resting membrane potential ( rmp ) and approaches the action potential firing threshold for cns neurons , where slow inactivation appears to be physiologically relevant during sustained subthreshold depolarizations . finally , changes in the na channel availability near 50 mv can affect the overlap of na current activating and inactivating under steady - state conditions . for these reasons , we utilized 50 mv conditioning pulses to compare pharmacological effects on slow inactivation and to develop concentration response curves and ic50 values for each tested compound ( figure 2b g ) . the slow inactivation ic50 values are shown in figure 2h and summarized in table 1 . compared with the reported ic50 calculated value of 85 m for slow inactivation induced by ( r)-1 , the ic50 values for these 3-substituted ( r)-a compounds were considerably lower . for example , the ic50 values for ( r)-3 and ( r)-8 were 29- and 133-fold lower than ( r)-1 . collectively , these data indicate that these ( r)-a derivatives more effectively induced na channel transition to a slow - inactivated state than did ( r)-1 . effects of ( r)-a compounds with a polar , aprotic substituent at the 3-position on the steady - state , slow inactivation state of na currents in cad cells . currents were evoked by 5 s prepulses between 120 and + 20 mv , and then fast - inactivated channels were allowed to recover for 150 ms at a hyperpolarized pulse to 120 mv . representative current traces from cad cells were taken in the absence ( control , 0.1% dmso ) or presence of 10 m ( r)-11 . the black and red traces represent the currents evoked at 120 and 50 mv , respectively ( also highlighted in the voltage protocol ) . ( b g ) summary of steady - state , slow activation curves for cad cells treated with 0.1% dmso ( control ) or with various concentrations of the indicated compounds . ( h ) the concentrations at which half of the channels have transitioned to the slow - inactivated state ( see text for details ) , the si ic50 , are indicated . a similar series of experiments were undertaken to investigate the effects of ( r)-a compounds with polar , aprotic substituents ( r = f , cl , ocf3 ) at the 4-aryl site on na current slow inactivation . we observed pronounced , concentration - dependent slow inactivation for ( r)-4 , ( r)-6 , and ( r)-12 ( supporting information , figure s1b d ) . as for the 3-substituted compounds , the ic50 values of slow inactivation were calculated by fitting the concentration responses of slow inactivation against the v1/2 of slow inactivation ( table 1 ; supporting information , figure s1e ) . the ic50 values for ( r)-4 , ( r)-6 , and ( r)-12 were 101- , 708- , and 129-fold lower than for ( r)-1 . we next asked if the polar , aprotic ( r)-a derivatives could enhance steady - state fast inactivation in cad cells . for these studies we used a protocol ( figure 3a ) designed to induce a fast - inactivated state , similar to cells were held at 80 mv , stepped to inactivating prepulse potentials ranging from 120 to 10 mv ( in 10 mv increments ) for 500 ms . then the cells were stepped to 0 mv for 20 ms to measure the available current . a 500 ms conditioning pulse was used because it allowed all of the endogenous channels to transition to a fast - inactivated state at all potentials assayed . in figure 3 , we provide the steady - state , fast inactivation curves of na currents ( representative family of current traces shown in figure 3a from control ( 0.1% dmso ) and figure 3b ( r)-3- , ( r)-5- , ( r)-7- , ( r)-8- , ( r)-10- , and ( r)-11-treated cad neurons were well fitted with a single boltzmann function ( r > 0.969 for all conditions ) . the v1/2 inactivation value for 0.1% dmso - treated cells was 68.3 3.1 mv ( n = 5 ) , which was significantly different from that of ( r)-8 ( 10 m ) treated cells ( 76.5 1.3 mv ; n = 5 ; p < 0.05 ; student s t - test ; see figure 3e ) . compared with the 8.2 mv shift in v1/2 of fast inactivation in the hyperpolarizing direction observed in the presence of ( r)-8 , the shifts caused by the other five compounds were not significantly different from control ( figure 3b d , f , g ) ( p < 0.05 vs 0.1% dmso ( control ) ; student s t - test ) . effects of ( r)-a compounds with a polar , aprotic substituent at the 3-position on fast inactivation and steady - state activation state of na currents in cad cells . ( a ) voltage protocol for fast inactivation ( left ) and activation ( right ) . ( b g ) representative boltzmann fits for steady - state fast inactivation and activation for cad cells treated with 0.1% dmso ( control ) and various concentrations of the indicated compounds are shown . values for v1/2 , the voltage of half - maximal inactivation and activation and the slope factors ( k ) were derived from boltzmann distribution fits to the individual recordings and were averaged to determine the mean ( sem ) voltage dependence of steady - state inactivation and activation , respectively . the v1/2 and k of steady - state fast inactivation or activation other than ( r)-8 were not different among any of the conditions tested ( p > 0.05 , one - way anova ) . the effects of ( r)-a derivatives with 4-polar , aprotic substituents ( f , cl , ocf3 ) on na current fast inactivation was also investigated ( supporting information , figure s2 ) . steady - state , fast inactivation curves of na currents from 0.1% dmso- ( control ) and ( r)-4- , ( r)-6- , and ( r)-12-treated cad cells were well fitted with a single boltzmann function ( r > 0.989 for all conditions ) . the v1/2 inactivation value for 0.1% dmso - treated cells was not significantly different from any of the conditions ( p > 0.05 ; student s t - test ; supporting information figure s2b d ) . because changes in current amplitudes can result from changes in channel gating , we tested whether the nine 3- and 4-polar , aprotic ( r)-a derivatives ( ( r)-4(r)-12 ) could alter voltage - dependent activation properties of na currents in cad cells . activation changes for the ( r)-a derivative treated cad cells were measured by whole - cell ionic conductances by comparing their midpoints ( v1/2 ) and slope factors ( k ) in response to changes in command voltage ( figure 3a , top right ) . representative traces in response to the voltage protocol are shown in figure 3a ( bottom right ) . boltzmann fits for 0.1% dmso ( control ) and various concentrations of ( r)-3 , ( r)-5 , ( r)-7 , ( r)-8 , ( r)-10 , and ( r)-11 are shown in figure 3b g and those for ( r)-4 , ( r)-6 , and ( r)-12 in supporting information , figure s2b d . an analysis of the six 3-substituted compounds ( ( r)-3 , ( r)-5 , ( r)-7 , ( r)-8 , ( r)-10 , ( r)-11 ) v1/2 and k values showed that , with the exception of two ( r)-8 concentrations , there were no changes in the steady - state activation properties of na currents between cad cells treated with 0.1% dmso ( control ) or with any of the tested compounds ( figure 3c g ) . the v1/2 steady - state activation value for 0.1% dmso - treated ( control ) cells was 12.8 0.5 mv ( n = 4 ) , which was significantly different from that of ( r)-8-treated cells at 100 nm ( 24.6 3.9 mv , n = 5 ) ( p < 0.05 ; student s t - test ; see figure 3e ) . similarly , we found that the v1/2 or k values for steady state activation for 0.1% dmso - treated ( control ) cells were not significantly different from those of the 4-substituted ( r)-a compound ( 10 m ) treated cad cells ( ( r)-4 , ( r)-6 , and ( r)-12 ; p > 0.05 ; student s t - test ; supporting information figure s2b d ) . these data indicate that , except for ( r)-8 , polar , aprotic ( r)-a derivatives did not affect a closed channel s transition to an open conformation . finally , we tested whether ( r)-4(r)-12 could elicit a frequency ( use ) dependent block of na currents in cad cells . use dependency represents an ability to bind more efficiently to a channel undergoing repeated transitions between closed , open , and inactivated conformations . the ability to block na currents in an activity- or use - dependent manner is a useful property for asds , since it allows for preferential decreases in na channel availability during high - frequency ( seizure - like ) but not low - frequency firing . several theories have been proposed to account for the complexities of use - dependent inhibition , including the modulated receptor hypothesis , which postulates that the receptor on the na channels can exist in multiple configurations and the affinity and binding rates of drugs depend on channel s state , which in turn can depend on voltage . the response to voltage changes can be viewed as reflecting voltage - sensitive shifts in equilibrium between conducting , unblocked channels and nonconducting , blocked channels . the modulated - receptor hypothesis postulates shifts in equilibrium as the result of a variable - affinity receptor and modified inactivation gate kinetics in drug - complexed channels . we tested compounds ( r)-5 , ( r)-7 , ( r)-8 , ( r)-10 , and ( r)-11 at a range of concentrations from below to above the determined slow inactivation ic50 values . a train of 30 test pulses ( 20 ms to 10 mv ) was delivered from a holding potential of 80 mv at 10 hz ( figure 4a ) . the available current in control cells ( 0.1% dmso ) and cells in the presence of the ( r)-a compounds was calculated by dividing the peak current at any given pulse ( pulsen ) by the peak current in response to the initial pulse ( pulse1 ) . representative currents elicited by the voltage protocol are illustrated for control and 5 m ( r)-5-treated cells ( figure 4a ) . compounds ( r)-5 , ( r)-10 , and ( r)-11 exhibited a statistically significant concentration - dependent frequency ( use ) inhibition of na currents ( figure 4b , e , f ) by the last pulse , compared with control ( 0.1% dmso ) . the peak current was 50% lower in the presence of 5 m ( r)-5 , 18% lower in the presence of 10 m ( r)-10 , and 65% lower in the presence of 6 m ( r)-11 . these test concentrations were 7.17.6 times higher than their slow inactivation ic50 values . by comparison , as we previously reported , there was a 45% diminution of na currents in the presence of 15 m ( r)-3 ( figure 4b , purple circles ) , a concentration that is 5.2 times higher than the slow inactivation ic50 value . similarly , the traces for ( r)-7 and ( r)-8 ( figure 4c , d ) suggested frequency ( use ) inhibition of the na currents at a 10 m concentration , but the values did not reach statistical significance . effect of ( r)-a compounds with a polar , aprotic substituent at the 3-position on frequency ( use ) dependent block of na currents in cad cells . ( a ) the frequency dependence of the block was examined by holding cells at the hyperpolarized potential of 80 mv and evoking currents at 10 hz by 20 ms test pulses to 10 mv ( inset middle ) . representative overlaid traces are illustrated by pulses 1 and 30 for control ( predrug ) and in the presence of ( r)-5 ( 5 m ) . ( b f ) summary of average frequency ( use ) dependent decrease in current amplitude ( sem ) produced by control ( 0.1% dmso ) or by the presence of various concentrations of the indicated compounds ( p > 0.05 , one - way anova with dunnett s post hoc test ) . data are from three to six cells per condition . as a comparison , a use - dependent block imposed by ( r)-3 is also shown . compounds ( r)-3 , ( r)-5 , ( r)-10 , and ( r)-11 caused a significant decrease ( indicated by ) in current amplitude compared with 0.1% dmso - treated cells ( control ) ( p < 0.05 , one - way anova with dunnett s post hoc test ) . note the rapid frequency - dependent facilitation of the block by ( r)-3 and ( r)-5 , which was observed beginning as early as pulse 4 . we also examined whether the three polar , aprotic 4-substituted derivatives ( r)-4 , ( r)-6 , and ( r)-12 affected frequency ( use ) dependent block of na currents using a single concentration of 1 m , a concentration at which near maximal effects on slow inactivation were observed ( ic50 ( m ) : ( r)-4 , 0.82 ; ( r)-6 , 0.12 ; ( r)-12 , 0.66 ; supporting information figure s1 ) . only ( r)-4 and ( r)-6 exhibited small but statistically significant reduction in frequency ( use ) dependent bock of na currents ; by the last pulse , compared with control , the peak current was 15% lower in the presence of 1 m ( r)-4 and 16% lower in the presence of 1 m ( r)-6 ( supporting information , figure s3b ) . r - substituents ( r = f , cl , br , i , cf3 , ocf3 ) in rat embryonic cortical neurons . these cells express channel subtypes ( nav1.1 , nav1.2 , nav1.3 , nav1.6 ) that participate in seizure propagation , and we assumed contributions from all four cns nav isoforms to the observed effects . slow inactivation , fast inactivation , steady - state activation , and frequency ( use ) dependence of na currents in rat cortical neurons grown for 710 days in vitro were examined using protocols described earlier . because of the reduced availability of cortical neurons and their viability under the patch - clamp electrophysiology conditions , we used only 10 m ( r)-a compounds . furthermore , because the cad cell patch - clamp electrophysiology data for the 3- and 4-substituted isomers were similar , we examined only the 3-substituted derivatives . using cortical neurons , we also evaluated several ( r)-a compounds with 3-polar , protic r - substituents ( r = n(h)c(o)ch3 , co2h , nh3cl ) to try to correlate their activities on vgscs with their diminished anticonvulsant activities . first , we determined the ability of the ( r)-a derivatives with a 3-polar , aprotic r - substituent ( ( r)-3 , ( r)-5 , ( r)-7 , ( r)-8 , ( r)-10 , ( r)-11 ) to modulate the vgsc transition to a slow - inactivated state . cortical cells were held at 70 mv and conditioned to potentials ranging from 100 to + 20 mv ( in + 10 mv increments ) for 5 s. then fast - inactivated channels were allowed to recover for 1000 ms at a hyperpolarized pulse to 70 mv , and the fraction of channels available was tested by a single depolarizing pulse , to 10 mv , for 20 ms ( figure 5a , left ) . the hyperpolarization pulse allows channel recovery from fast inactivation while limiting recovery from slow inactivation . representative traces that illustrate the extent of slow inactivation observed at 50 mv are compared with the prepulse at 100 mv in the absence or presence of ( r)-5 in figure 5a , with complete slow inactivation curves ( normalized peak vs prepulse potential ) shown in figure 5b . for the 0.1% dmso ( control ) treated cells ( 50 mv ) , 0.82 0.03 fractional unit ( n = 5 ) of the na current was available , suggesting a small fraction ( 0.18 0.03 , calculated as 1 minus the normalized ina ) of the channels transitioned to a nonconducting ( slow - inactivated ) state ( figure 5b , c ) . compared with control ( 0.1% dmso ) , all of the 3-polar , aprotic substituted ( r)-a compounds at 10 m caused significant increase in the maximal fraction of current unavailable by depolarization , with maximal induction observed in the presence of ( r)-11 : 0.82 0.02 ( n = 5 ; p < 0.01 , mann whitney u test ) . the effectiveness of these ( r)-a compounds to promote slow inactivation in cortical neurons at 10 m approximated the ic50 values observed for slow inactivation in cad cells where the order of potency was ( r)-5 , ( r)-8 > ( r)-11 > ( r)-7 , ( r)-10 > ( r)-3 ( figure 2 , table 1 ) . when we tested the ( r)-a derivatives with a 3-polar , protic r - substituent ( ( r)-13 , ( r)-15 , ( r)-16 ) , only ( r)-15 caused an increase in the extent of slow inactivation ( supporting information , figure s4a , c , e ) while neither ( r)-13 nor ( r)-16 ( supporting information , figure s4b , e ) was different from 0.1% dmso ( control ) treated neurons . effects of ( r)-a compounds with a polar , aprotic substituent at the 3-position on steady - state , slow inactivation state of na currents in embryonic cortical neurons . . currents were evoked by 5 s prepulses between 100 and + 20 mv ( in + 10 mv increments ) , and then fast - inactivated channels were allowed to recover for 1000 ms at a hyperpolarized pulse to 70 mv before testing for the fraction of available channels for 20 ms at 10 mv . the fraction of channels available at 10 mv was analyzed . shown are representative current traces from cortical neurons in the absence ( control , 0.1% dmso ) or presence of 10 m ( r)-5 . the black and gray traces represent the currents evoked at 100 and 50 mv , respectively ( highlighted in the voltage protocol as thick , dashed line ) . ( b ) summary of steady - state , slow - activation curves for neurons treated with 0.1% dmso ( control ) or 10 m ( r)-5 . significant drug - induced slow inactivation was evident at voltages more depolarizing than 80 mv in neurons treated with ( r)-5 . ( c ) summary of the available current fraction at 50 mv for neurons treated with dmso ( control ) or 10 m ( r)-3 , ( r)-5 , ( r)-7 , ( r)-8 , ( r)-10 , and ( r)-11 . asterisks ( ) indicate statistically significant differences in available current fraction between control ( 0.1% dmso ) and the indicated compounds ( p < 0.05 , student s t - test ) . we next asked if the ( r)-a derivatives that contained a 3-polar r - substituent that was either aprotic ( ( r)-3 , ( r)-5 , ( r)-6 , ( r)-7 , ( r)-8 , ( r)-10 , ( r)-11 ) or protic ( ( r)-13 , ( r)-15 , ( r)-16 ) could enhance steady - state fast inactivation . to investigate , we used a protocol ( figure 6a , left ) designed to induce a fast - inactivated state , similar to that previously described . neurons were held at 70 mv , stepped to inactivating prepulse potentials ranging from 120 to 10 mv ( in 10 mv increments ) for 500 ms . then the cells were stepped to 0 mv for 20 ms to measure the available current . a 500 ms conditioning pulse was used because it allowed all of the endogenous channels to transition to a fast - inactivated state at all potentials assayed . steady - state , fast inactivation curves of na currents ( representative family of control current traces shown in figure 6b , left ) from control ( 0.1% dmso ) treated and ( r)-3- , ( r)-5- , ( r)-6- , ( r)-7- , ( r)-8- , ( r)-10- , and ( r)-11-treated cortical neurons were well fitted with a single boltzmann function ( r > 0.983 for all conditions ) . the v1/2 inactivation value for 0.1% dmso - treated cells was 53.2 1.2 mv ( n = 4 ) , which was significantly different from that of ( r)-5 ( 59.9 3.1 mv ; n = 5 ) , ( r)-7 ( 58.3 1.4 mv ; n = 4 ) , and ( r)-11 ( 60.1 1.2 mv ; n = 5 ) treated cells ( p < 0.05 vs control ( 0.1% dmso ) ; student s t - test ; figure 6c , d ) ; none of the other compounds ( ( r)-3 , ( r)-6 , ( r)-8 , ( r)-10 ) were different from controls ( 0.1% dmso ) . the slope values were unchanged between control and any of the conditions tested . by comparison , we did not see evidence to indicate that the ( r)-a compounds with 3-polar , protic r - substituents , ( r)-13 , ( r)-15 , and ( r)-16 , affected v1/2 or k values of steady - state fast inactivation ( figure 6d ) . effects of ( r)-a compounds with a polar , aprotic or a polar , protic substituent at the 3-position on fast inactivation and steady - state activation states of na currents in cortical embryonic neurons . ( a ) voltage protocol for fast inactivation ( left ) and activation ( right ) . ( b ) representative family of currents in response to these protocols is illustrated . ( c , d ) representative boltzmann fits for steady - state fast inactivation and activation for cortical neurons treated with 0.1% dmso ( control ) and various concentrations of the indicated compounds are shown . values for v1/2 , the voltage of half - maximal inactivation and activation , and the slope factors ( k ) were derived from boltzmann distribution fits to the individual recordings and were averaged to determine the mean ( sem ) voltage dependence of steady - state inactivation and activation , respectively . statistically significant differences between control and fast inactivation or activation are indicated by asterisks ( p < 0.05 , one - way anova ) . next , we tested whether ( r)-a compounds that contained polar r - substituents , aprotic or protic , at the 3-position could alter voltage - dependent activation properties of na currents in cortical neurons . compound - treated cortical neuron activation changes were measured by whole - cell ionic conductances by comparing their midpoints ( v1/2 ) and slope factors ( k ) in response to command voltage changes ( figure 6a , right ) . representative traces for the 0.1% dmso - treated neurons ( control ) in response to the voltage protocol are shown in figure 6b ( right ) . boltzmann fits for dmso ( control ) and the ( r)-a compounds with polar , aprotic and polar , protic r - substituents are shown in figure 6c and figure 6d , respectively . the v1/2 value for steady - state activation for 0.1% dmso - treated ( control ) neurons was 27.3 3.3 mv ( n = 5 ) , which was significantly different from those of ( r)-5 ( 37.5 4.9 mv ; n = 5 ) , ( r)-10 ( 35.4 5.4 mv ; n = 5 ) , ( r)-11 ( 33.2 1.9 mv ; n = 4 ) , ( r)-13 ( 39.0 2.6 mv ; n = 4 ) , ( r)-15 ( 36.8 3.0 mv ; n = 5 ) , and ( r)-16 ( 33.4 3.2 mv ; n = 5 ) ( p < 0.05 vs control ; student s t - test ; figure 6c , d ) . the hyperpolarizing shifts induced by these compounds suggest that in the presence of the compounds , nav currents are likely to activate earlier ( i.e. , at less - depolarized potentials ) . finally , we tested whether the ( r)-a derivatives containing polar r - substituents , aprotic or protic , at the 3-position could affect frequency ( use ) dependent block of na currents . unlike the data obtained with cad cells , none of the compounds tested at a 10 m concentration in cortical neurons affected frequency ( use ) dependent block of na currents , compared with controls ( 0.1% dmso ) ( supporting information , figure s5 ) . in order to assess the relative effect of an ( r)-a compound on na channel isoforms , we examined the na currents in hek293 cells that stably express cns ( hnav1.1 and rnav1.3 ) , peripheral nervous system ( hnav1.7 ) , or cardiac ( hnav1.5 ) isoforms . compound ( r)-5 ( 10 m ) was chosen for study because it displayed excellent anticonvulsant activity in mice ( ip ) and rats ( po ) ( table 1 ) , and it modulated na currents in cad cells and rat embryonic cortical neurons . properties of slow inactivation , fast inactivation , steady - state activation , and frequency ( use ) dependent inhibition were examined in the four cell lines using voltage protocols illustrated in figure 7a , k . notably , ( r)-5 exhibited similar , but not identical , effects on these biophysical properties irrespective of na channels , indicating that this compound exhibited little isoform specificity . we observed that ( r)-5 transitioned the four na channel subtypes to the slow - inactivated state and that differences were observed in the degree to which ( r)-5 affected na channel fast inactivation , fast activation , and the frequency ( use ) inhibition of na currents . analysis of ( r)-5 on electrophysiological properties of nav1.1 , nav1.3 , nav1.7 , and nav1.5 currents in hek293 cells . ( a , k ) voltage protocols for examining slow inactivation , fast inactivation , activation , and frequency ( use ) dependent block . nav1.5 uses hyperpolarized protocols because of differences in the hyperpolarized activation of this isoform . ( b , e , h , l ) summary of steady - state slow - activation curves for hek293 cells treated with 0.1% dmso ( control ) or 10 m ( r)-5 . insets illustrate representative current traces from hek293 cells in the absence ( control , 0.1% dmso ) or presence of 10 m ( r)-5 . the black and colored traces represent the currents evoked at 120 and 50 mv , respectively ( highlighted in the voltage protocol ) . ( c , f , i , m ) representative boltzmann fits for steady - state fast inactivation and activation for hek293 cells treated with 0.1% dmso ( control ) or 10 m ( r)-5 . values for v1/2 , the voltage of half - maximal inactivation and activation , and the slope factors ( k ) were derived from boltzmann distribution fits to the individual recordings and were averaged to determine the mean ( sem ) voltage dependence of steady - state inactivation and activation , respectively . ( d , g , j , n ) summary of average frequency ( use ) dependent decrease in current amplitude over time ( sem ) produced by control ( 0.1% dmso ) or 10 m ( r)-5 . asterisks represent statistically significant differences compared to control within each tested group ( p < 0.05 , student s t test ) . the extent of slow inactivation was calculated as 1 minus the normalized peak ina and denotes the fraction of channels that have transitioned to a nonconducting ( slow - inactivated ) state at 50 mv . values for v1/2 , the voltage of half - maximal activation , and slope were derived from boltzmann distribution fits to the individual recordings and averaged to determine the mean and standard error of the mean ( sem ) . fraction of current remaining at the end of the 30-pulse train , normalized to the first pulse . recently , we merged the structures of functionalized amino acids ( faas ) and -aminoamides ( aaas ) , two classes of compounds that have shown excellent anticonvulsant activities in seizure models , to give compounds ( r)-b and reported on their anticonvulsant activities ( figure 8) . the substituted ( r)-a compounds described herein are examples of ( r)-b where r is a methoxymethyl moiety ( r = ch2och3 ) and the x - y - z unit is a single bond ; they are also 4-aryl derivatives of ( r)-1 . in 2010 , we showed that the unsubstituted n-(biphenyl-4-yl)methyl derivative ( r)-2 exhibited excellent anticonvulsant activity but that seizure protection in mice and rats was accompanied by appreciable neurotoxicity ( figure 1 , table 1 ; mes ed50 ( mg / kg ) , 8.0 ( mice , ip ) , 2.0 ( rat , po ) ; td50 ( mg / kg ) , 11 ( mice , ip ) , 49 ( rat , po ) ) . subsequently , we showed that the corresponding 3-f - substituted analogue ( r)-3 retained high anticonvulsant activity but that neurotoxicity was reduced ( figure 1 , table 1 ; mes ed50 ( mg / kg ) , 12 ( mice , ip ) , 2.4 ( rat , po ) ; td50 ( mg / kg ) , 50 ( mice , ip ) , rat > 500 ( rat , po ) ) . while the pi value in mice for ( r)-3 was higher than for ( r)-2 , it was still below that of the asd ( r)-1 ( pi ( mice , ip ) : ( r)-2 , 1.4 ; ( r)-3 , 4.1 ; ( r)-1 , 6.0 ) . in this study , we explored the effect of the terminal aryl r - substituent in ( r)-a on anticonvulsant activity , neurotoxicity , and na channel function . we found that the placement of polar , aprotic r - substituents on the terminal aryl ring of ( r)-a provided compounds with excellent anticonvulsant activities and that these activities were only modestly affected by the site of aryl substitution ( 3 vs 4 ) . in addition , we found that several ( r)-a derivatives with polar , aprotic r - substituents had improved pi values over ( r)-2 ( pi = 1.4 ) but that the pi values varied with the r - substituent and the site of substitution . for the 3-cl and 4-cl derivatives ( ( r)-5 , ( r)-6 ) , high pi values were obtained regardless of the site of substitution ( pi ( mice , ip ) : ( r)-5 ( 3 ) , 7.6 ; ( r)-6 ( 4 ) , 6.8 ) and that the values exceeded that of ( r)-2 ( pi = 1.4 ) and even ( r)-1 ( pi = 6.0 ) . correspondingly , for the 3- and 4-f derivatives ( ( r)-3 , ( r)-4 ) and the 3- and 4-ocf3 compounds ( ( r)-11 , ( r)-12 ) , high pi values were observed for only the 3-substituted isomers ( pi ( mice , ip ) : ( r)-3 ( 3-f ) , 4.1 ; ( r)-4 ( 4-f ) , 1.8 ; ( r)-11 ( 3-ocf3 ) , 4.9 ; ( r)-12 ( 4-ocf3 ) , 2.0 ) . correspondingly , when a polar , protic r - substituent was placed in the terminal aryl ring in ( r)-a to give ( r)-13(r)-16 , we observed minimal or no anticonvulsant activity . accompanying this loss of activity was a reduction in neurotoxicity , suggesting that the activity loss resulted from either reduced levels of these compounds in the cns or a reduced interaction with the receptor(s ) responsible for the seizure protection and toxicity or both . when we tested whether ( r)-13 , ( r)-15 , and ( r)-16 modulated na currents in rat embryonic cortical neurons , only ( r)-15 ( r = 3-co2h ) displayed notable activity ( supporting information , figure s4 ) . this latter finding indicated that the diminished anticonvulsant activities for ( r)-13 and ( r)-16 may have resulted , in part , from their inability to promote na channel slow inactivation during the seizure test . both faas and aaas likely exert their anticonvulsant activities by interacting with na channels . the faa , ( r)-1 , has been shown to preferentially promote the transition of na channels to the slow inactivation state without affecting fast inactivation . we demonstrated that ( r)-3 displayed potent na channel slow inactivation in cad cells that exceeded ( r)-1 ( ic50 ( m ) : ( r)-3 , 2.9 ; ( r)-1 , 85 ) . moreover , ( r)-3 displayed notable frequency ( use ) dependent inhibition of na currents at concentrations ( 15 m ) above its ic50 value ( figure 4b ) . in this study , we determined the na channel properties of the ( r)-a compounds in three different cell types ( cad cells , rat embryonic cortical neurons , hek293-transfected cells ) . overall , we found it difficult to compare the effects of individual ( r)-a compounds across the different cell systems . for several compounds , we observed differences in the three cell types in their ability to affect na channel fast inactivation and fast activation and frequency ( use ) inhibition of na currents . we have tentatively attributed these differences to the different channel compositions , expression levels , auxiliary proteins , and origins ( mouse , rat , human ) in the cad , cortical , and hek293 cell systems and the test ( r)-a concentrations used in the studies . accordingly , the experimental results in any given cell system were used to identify interesting compounds , and direct comparisons were only made within data sets from the same cell type . we found that ( r)-4(r)-8 and ( r)-10(r)-12 , compounds that contained polar , aprotic r - substituents , potently transitioned cad cell na channels to the slow - inactivated state ( ic50 = 0.122.9 m ) and that na channel fast inactivation was not observed ( figures 2 , 3 ; supporting information figures s1 , s2 ) . furthermore , we observed that many compounds ( ( r)-4(r)-6 , ( r)-10 , ( r)-11 ) showed frequency ( use ) dependent inhibition of na currents , provided that concentrations higher than the slow inactivation ic50 value were employed ( figure 4 ; supporting information , figure s3 ) . we and others have not observed notable frequency ( use ) inhibition of na currents for ( r)-1 at 100 m . our finding that frequency ( use ) inhibition for selected ( r)-a compounds was observed only at concentrations 5-fold higher than their slow inactivation ic50 values in cad cells led us to retest ( r)-1 at concentrations above its ic50 value ( 85 m ) . at 500 m ( r)-1 , we observed noticeable frequency ( use ) inhibition of na currents ( figure 9 ) similar to that seen for the ( r)-a compounds ( figure 4 ) . significantly , this concentration is higher than the reported human therapeutic ( r)-1 plasma concentrations ( 2050 m ) , suggesting that frequency ( use ) inhibition of na currents may not be a clinically important pathway for its drug function . in contrast , we found that several substituted ( r)-a derivatives , at low micromolar concentrations , promoted na channel slow inactivation and frequency ( use ) dependent inhibition of na currents . this finding provides a potential comparative pharmacological advantage for these compounds , since both mechanisms are proven pathways to control neuronal hyperexcitability in the epileptic neuron . effect of ( r)-1 in cad cells on frequency ( use ) dependent block . summary of average frequency ( use ) dependent current amplitude decrease ( sem ) produced by control ( 0.1% dmso ) or by the presence of various concentrations of ( r)-1 is shown ( p > 0.05 , one - way anova with dunnett s post hoc test ) . concentrations greater than 500 m ( r)-1 caused a significant inhibition of use - dependence starting at about pulse 8 ( p < 0.05 , one - way anova with dunnett s post hoc test ) . to better approximate cns vgsc activities , we tested many of the ( r)-a compounds with a 3-polar , aprotic r - substituent in rat embryonic cortical neurons that expressed the nav1.1 , nav1.2 , nav1.3 , and nav1.6 channels . we observed that ( r)-3 , ( r)-5 , ( r)-7 , ( r)-8 , ( r)-10 , and ( r)-11 all promoted slow inactivation at 10 m ( figure 5 ) and their relative effectiveness mirrored the slow inactivation ic50 values measured in cad cells ( figure 2 ) . differences were found between the cortical neurons and the cad cells on the ability of these compounds to affect fast inactivation and their ability to inhibit frequency ( use ) dependent inhibition of na currents ( figures 3 , 4 , 6 ; supporting information , figure s5 ) . there are nine na channel isoforms . despite structural similarity , the isoforms distribution and biophysical properties ( e.g. , kinetics of activation and inactivation , voltage , and frequency ( use ) dependency ) are distinct , each having specific functions . thus , we evaluated ( r)-5 in hek293 cells expressing hnav1.1 , rnav1.3 , hnav1.5 , or hnav1.7 channels . the patch - clamp electrophysiology protocols for the cells containing hnav1.5 were different from those used for hnav1.1 , rnav1.3 , and hnav1.7 ( figure 7a , k ) . we found that 10 m ( r)-5 transitioned the na channels to the slow - inactivated state regardless of channel type ( figure 7b , e , h , l ) . correspondingly , differences in the fast inactivation profiles were observed for the four isoforms . compound ( r)-5 did not affect rnav1.3 fast inactivation compared with 0.1% dmso ( control ) , while a depolarizing shift was found for the hnav1.1- , hnav1.5- , and hnav1.7-transfected cells , with the most pronounced shift observed in hnav1.7-expressing cells ( figure 7c , f , i , m ) . finally , we observed notable frequency ( use ) inhibition of na currents for the transfected hnav1.5 cells ( figure 7n ) , lesser amounts for the hnav1.1- and hnav1.7-expressing cells ( figure 7d , j ) , and no inhibition for the rnav1.3-expressing cells ( figure 7 g ) . together these findings showed that ( r)-5 , and perhaps the other polar , aprotic r - substituted ( r)-a derivatives , displayed little na channel isoform specificity . rather , ( r)-5 appears to act as a functionally selective na channel inhibitor that controls hyperexcitability upon excitation by transitioning a greater fraction of na channels to their slow - inactivated state and by inhibiting na currents in a frequency ( use ) dependent manner . the successful merger of faas and aaas ( figure 8) provided substituted n-(biphenyl-4-yl)methyl ( r)-2-acetamido-3-methoxypropionamide derivatives ( ( r)-a ) . select ( r)-a compounds displayed low ed50 values and high pi values that , in rodents , compared favorably with most clinical asds . we found compounds that potently transitioned na channels into the slow - inactivated state and displayed frequency ( use ) inhibition of na currents at low micromolar concentrations . the activities of ( r)-as along with their pharmacokinetic properties are being investigated further . melting points were determined in open capillary tubes using a thomas - hoover melting point apparatus and are uncorrected . optical rotations were obtained on a jasco p-1030 polarimeter at the sodium d line ( 589 nm ) using a 1 dm path length cell . nmr spectra were obtained at 400 mhz ( h ) and 100 mhz ( c ) using tms as an internal standard . chemical shifts ( ) are reported in parts per million ( ppm ) from tetramethylsilane . low - resolution mass spectra were obtained with a biotof - ii - bruker daltonics spectrometer by dr . the high - resolution mass spectrum was performed on a bruker apex - q 12 telsa fticr spectrometer by dr . reactions were monitored by analytical thin - layer chromatography ( tlc ) plates ( aldrich , catalog no . the mixtures were purified by flash column chromatography using silica gel ( dynamic adsorbents inc . 02826 - 25 ) . all chemicals and solvents were reagent grade and used as obtained from commercial sources without further purification . compounds were checked by tlc , h and c nmr , ms , and elemental analyses . the nmr and the analytical data confirmed that the purity of the products was 95% . a ch2cl2 solution ( 0.10.3 m ) of the ( r)-n-(biphenyl-4-yl)methyl 2-n-(tert - butoxycarbonyl)amino-3-methoxypropionamide derivative was treated with 4 m hcl in dioxane ( 34 equiv ) at room temperature ( 26 h ) . the resulting residue was dissolved in ch2cl2 ( 0.10.3 m ) , and then triethylamine ( 23 equiv ) and acetyl chloride ( 1.01.2 equiv ) were carefully added at 0 c . the resulting solution was washed with an aqueous 10% citric acid solution followed by a saturated aqueous nahco3 solution . the residue was purified by column chromatography on sio2 and/or recrystallized with etoac / hexanes . using method 1 , ( r)-56 ( 3.17 g , 7.9 mmol ) and 4 m hcl ( 6.90 ml , 27.6 mmol ) followed by et3n ( 2.42 ml , 17.3 mmol ) and accl ( 0.61 ml , 8.7 mmol ) gave ( r)-4 as a white solid ( 2.50 g , 92% ) : rf = 0.40 ( meoh / ch2cl2 1/20 ) ; mp 189190 c ; [ ]d 17.8 ( c 1.0 , chcl3 ) ; h nmr ( cdcl3 ) 2.04 ( s , 3h ) , 3.40 ( s , 3h ) , 3.443.48 ( m , 1h ) , 3.83 ( dd , j = 3.9 , 9.0 hz , 1h ) , 4.454.59 ( m , 3h ) , 6.435.46 ( br d , 1h ) , 6.816.85 ( m , 1h ) , 7.12 ( t , j = 8.6 hz , 2h ) , 7.32 ( d , j = 7.6 hz , 2h ) , 7.497.54 ( m , 4h ) ; addition of excess ( r)-()-mandelic acid to a cdcl3 solution of ( r)-4 gave only one signal for the acetyl methyl and one signal for the ether methyl protons ; c nmr ( dmso - d6 ) 22.6 , 41.8 , 52.7 , 58.2 , 72.1 , 115.7 ( d , j = 21.3 hz , 2c ) , 126.5 , 127.6 , 128.5 ( d , j = 8.0 hz , 2c ) , 136.4 ( d , j = 2.9 hz ) , 137.6 , 138.6 , 161.8 ( d , j = 242.4 hz ) , 169.5 , 169.8 ; lrms ( esi ) 367.1 [ m + na ] ( calcd for c19h21fn2o3na 367.1 ) ( c19h21fn2o3 ) c , h , f , n. using method 1 , ( r)-57 ( 4.00 g , 9.6 mmol ) and 4 m hcl ( 8.36 ml , 33.4 mmol ) followed by et3n ( 2.93 ml , 21.0 mmol ) and accl ( 0.74 ml , 10.5 mmol ) gave ( r)-5 as a white solid ( 2.85 g , 83% ) : rf = 0.40 ( meoh / ch2cl2 1/20 ) ; mp 172173 c ; [ ]d 15.8 ( c 1.1 , chcl3 ) ; h nmr ( cdcl3 ) 2.00 ( s , 3h ) , 3.37 ( s , 3h ) , 3.49 ( dd , j = 7.0 , 9.0 hz , 1h ) , 3.78 ( dd , j = 4.0 , 9.0 hz , 1h ) , 4.43 ( 1/2hhq , j = 5.8 , 15.0 hz , 1h ) , 4.49 ( 1/2hhq , j = 5.8 , 15.0 hz , 1h ) , 4.624.67 ( m , 1h ) , 6.71 ( d , j = 6.8 hz , 1h ) , 7.157.18 ( m , 1h ) , 7.307.52 ( m , 8h ) ; addition of excess ( r)-()-mandelic acid to a cdcl3 solution of ( r)-5 gave only one signal for the acetyl methyl and one signal for the ether methyl protons ; c nmr ( cdcl3 ) 23.3 , 43.3 , 52.7 , 59.2 , 72.1 , 125.3 , 127.3 , 127.5 , 128.1 , 130.2 , 134.8 , 137.9 , 139.1 , 142.6 , 170.3 , 170.6 ; the remaining aromatic peak was not detected and is believed to overlap with the observed signals ; lrms ( esi ) 383.1 [ m + na ] ( calcd for c19h21cln2o3na 383.1 ) . ( c19h21cln2o3 ) c , h , cl , n. using method 1 , ( r)-58 ( 2.80 g , 6.7 mmol ) , and 4 m hcl ( 6.70 ml , 26.7 mmol ) followed by et3n ( 2.10 ml , 14.7 mmol ) and accl ( 0.52 ml , 7.4 mmol ) gave ( r)-6 as a white solid ( 1.83 g , 76% ) : rf = 0.40 ( meoh / ch2cl2 1/20 ) ; mp 215216 c ; [ ]d 16.9 ( c 1.1 , chcl3 ) ; h nmr ( cdcl3 ) 2.03 ( s , 3h ) , 3.39 ( s , 3h ) , 3.443.48 ( m , 1h ) , 3.82 ( dd , j = 4.0 , 9.2 hz , 1h ) , 4.454.61 ( m , 3h ) , 6.456.49 ( br d , 1h ) , 6.866.91 ( m , 1h ) , 7.32 ( d , j = 8.0 hz , 2h ) , 7.39 ( d , j = 8.4 hz , 2h ) , 7.487.52 ( m , 4h ) ; addition of excess ( r)-()-mandelic acid to a cdcl3 solution of ( r)-6 gave only one signal for the acetyl methyl and one signal for the ether methyl protons ; c nmr ( cdcl3 ) 23.4 , 43.4 , 52.7 , 59.3 , 71.9 , 127.5 , 128.2 , 128.5 , 129.2 , 133.7 , 137.5 , 139.3 , 139.4 , 170.3 , 170.5 ; lrms ( esi ) 361.2 [ m + h ] ( calcd for c19h21cln2o3h 361.2 ) . ( c19h21cln2o3 ) c , h , cl , n. using method 1 , 4 m hcl in dioxane ( 3.5 ml ) , ( r)-59 ( 1.30 g , 2.8 mmol ) , et3n ( 862 mg , 8.5 mmol ) , and accl ( 268 mg , 3.4 mmol ) gave compound ( r)-7 as a white solid ( 620 mg , 54% ) : rf = 0.44 ( 1:20 meoh / ch2cl2 ) ; mp 171173 c ; [ ]d 17.3 ( c 1.0 , chcl3 ) ; h nmr ( cdcl3 ) 2.00 ( s , 3h ) , 3.37 ( s , 3h ) , 3.453.49 ( m , 1h ) , 3.79 ( dd , j = 4.0 , 9.0 hz , 1h ) , 4.404.55 ( m , 2h ) , 4.564.66 ( m , 1h ) , 6.60 ( d , j = 6.4 hz , 1h ) , 6.987.08 ( br s , 1h ) , 7.247.35 ( m , 3h ) , 7.397.56 ( m , 4h ) , 7.68 ( s , 1h ) ; addition of excess ( r)-()-mandelic acid to a cdcl3 solution of ( r)-7 gave only one signal for the acetyl methyl and one signal for the ether methyl protons ; c nmr ( cdcl3 ) 23.1 , 43.1 , 52.5 , 59.1 , 71.8 , 122.9 , 125.6 , 127.3 , 127.9 , 130.0 , 130.2 , 130.3 , 137.6 , 138.8 , 142.7 , 170.1 , 170.3 ; lrms ( esi ) 405.0 [ m + h ] ( 100% ) , 407.0 [ m + 2 + h ] ( 100% ) ; hrms ( esi ) 405.0814 [ m + h ] ( calcd for c19h21brn2o3h 405.0814 ) . anal . ( c19h21brn2o3 ) c , h , br , n. using method 1 , 4 m hcl in dioxane ( 2.0 ml ) , ( r)-60 ( 670 mg , 1.3 mmol ) , et3n ( 515 mg , 3.9 mmol ) , and accl ( 123 mg , 1.6 mmol ) gave compound ( r)-8 as a white solid ( 515 mg , 87% ) : rf = 0.41 ( 1:20 meoh / ch2cl2 ) ; mp 161163 c ; [ ]d 11.0 ( c 1.0 , chcl3 ) ; h nmr ( cdcl3 ) 2.05 ( s , 3h ) , 3.41 ( s , 3h ) , 3.433.50 ( m , 1h ) , 3.84 ( dd , j = 4.3 , 9.3 hz , 1h ) , 4.464.55 ( m , 2h ) , 4.574.60 ( m , 1h ) , 6.45 ( d , j = 6.8 hz , 1h ) , 6.83 ( t , j = 4.8 hz , 1h ) , 7.17 ( t , j = 7.8 hz , 1h ) , 7.34 ( d , j = 8.2 hz , 2h ) , 7.487.55 ( m , 3h ) , 7.68 ( d , j = 7.8 hz , 1h ) , 7.92 ( s , 1h ) ; addition of excess ( r)-()-mandelic acid to a cdcl3 solution of ( r)-8 gave only one signal for the acetyl methyl and one signal for the ether methyl protons ; c nmr ( cdcl3 ) 23.2 , 43.2 , 52.4 , 59.1 , 71.6 , 94.8 , 126.3 , 127.4 , 127.9 , 130.4 , 136.0 , 136.2 , 137.6 , 138.9 , 142.9 , 170.0 , 170.3 ; hrms ( esi ) 453.0675 ( c19h21in2o3 ) c , h , i , n. using method 1 , ( r)-61 ( 3.10 g , 7.6 mmol ) and 4 m hcl ( 9.5 ml ) followed by et3n ( 2.30 g , 22.7 mmol ) and accl ( 0.71 g , 9.1 mmol ) gave the desired product ( r)-9 ( 2.00 g , 75% ) as a white solid : rf = 0.42 ( ch2cl2/ch3oh 19/1 ) ; mp 122123 c ; [ ]d 16.0 ( c 1.0 , chcl3 ) ; h nmr ( cdcl3 ) 2.03 ( s , 3h ) , 3.40 ( s , 3h ) , 3.473.52 ( m , 1h ) , 3.793.84 ( m , 1h ) , 4.484.54 ( m , 2h ) , 4.594.63 ( m , 1h ) , 6.566.59 ( br d , 1h ) , 7.037.07 ( br t , 1h ) , 7.36 ( d , j = 8.4 hz , 2h ) , 7.487.64 ( m , 4h ) , 7.767.82 ( m , 2h ) ; addition of excess ( r)-()-mandelic acid to a cdcl3 solution of ( r)-9 gave only one signal for the acetyl methyl and one signal for the ether methyl protons ; c nmr ( cdcl3 ) 23.4 , 43.3 , 52.7 , 59.3 , 71.9 , 113.2 , 119.0 , 127.5 , 128.3 , 129.8 , 130.8 , 130.9 , 131.6 , 138.2 , 138.5 , 142.9 , 170.4 , 170.6 ; lrms ( esi ) 352.2 [ m + h ] ( calcd for c20h22n3o3 352.2 ) ; hrms ( esi ) 352.1660 [ m + h ] ( calcd for c20h22n3o3 352.1661 ) . ( c20h21n3o3 ) c , h , n. using method 1 , 4 m hcl in dioxane ( 3.0 ml ) , ( r)-62 ( 1.34 g , 3.0 mmol ) , et3n ( 896 mg , 8.9 mmol ) , and accl ( 348 g , 4.4 mmol ) gave compound ( r)-10 as a white solid ( 1.02 g , 88% ) : rf = 0.42 ( 1:20 meoh / ch2cl2 ) ; mp 161162 c ; [ ]d 13.7 ( c 1.0 , chcl3 ) ; h nmr ( cdcl3 ) 2.03 ( s , 3h ) , 3.40 ( s , 3h ) , 3.49 ( dd , j = 4.3 , 9.3 hz , 1h ) , 3.81 ( dd , j = 4.3 , 9.3 hz , 1h ) , 4.454.58 ( m , 2h ) 4.614.64 ( m , 1h ) , 6.58 ( d , j = 7.0 hz , 1h ) , 7.04 ( t , j = 5.3 hz , 1h ) , 7.36 ( d , j = 8.2 hz , 2h ) , 7.517.62 ( m , 4h ) , 7.73 ( d , j = 7.4 hz , 1h ) , 7.80 ( s , 1h ) ; addition of excess ( r)-()-mandelic acid to a cdcl3 solution of ( r)-10 gave only one signal for the acetyl methyl and one signal for the ether methyl protons ; c nmr ( cdcl3 ) 23.1 , 43.1 , 52.5 , 59.1 , 71.7 , 123.7 ( q , j = 4.0 hz ) , 124.0 ( q , j = 4.0 hz ) , 124.1 ( q , j = 272.0 hz ) , 126.0 , 127.4 , 129.2 , 130.2 , 131.2 ( q , j = 32.0 hz ) , 137.9 , 138.9 , 141.4 , 170.1 , 170.4 ; ms ( esi ) 395.2 [ m + h ] ( calcd for c20h22f3n2o3 395.2 ) ; hrms ( esi ) 395.1583 [ m + h ] ( calcd for c20h22f3n2o3 395.1582 ) . anal . ( c20h21f3n2o3 ) c , h , f , n. using method 1 , ( r)-63 ( 2.14 g , 4.6 mmol ) and 4 m hcl ( 4.00 ml , 16.0 mmol ) followed by et3n ( 1.40 ml , 10.1 mmol ) and accl ( 0.36 ml , 5.0 mmol ) gave ( r)-11 as a white solid ( 1.69 g , 90% ) : rf = 0.40 ( meoh / ch2cl2 1/20 ) ; mp 139140 c ; [ ]d 13.5 ( c 1.3 , chcl3 ) ; h nmr ( cdcl3 ) 2.03 ( s , 3h ) , 3.40 ( s , 3h ) , 3.47 ( dd , j = 7.4 , 9.2 hz , 1h ) , 3.82 ( dd , j = 4.0 , 9.2 hz , 1h ) , 4.464.61 ( m , 3h ) , 6.49 ( d , j = 6.4 hz , 1h ) , 6.896.93 ( m , 1h ) , 7.187.21 ( m , 1h ) , 7.337.54 ( m , 7h ) ; addition of excess ( r)-()-mandelic acid to a cdcl3 solution of ( r)-11 gave only one signal for the acetyl methyl and one signal for the ether methyl protons ; c nmr ( dmso - d6 ) 22.5 , 41.7 , 52.7 , 58.2 , 72.1 , 119.1 , 119.6 , 120.1 ( q , j = 254.9 hz ) , 125.7 , 126.7 , 127.7 , 130.8 , 136.8 , 139.5 , 142.3 , 149.0 , 169.4 , 169.8 ; lrms ( esi ) 433.1 [ m + na ] ( calcd for c20h21f3n2o4na 433.1 ) . ( c20h21f3n2o4 ) c , h , f , n. using method 1 , ( r)-64 ( 0.81 g , 1.7 mmol ) and 4 m hcl ( 1.49 ml , 6.0 mmol ) followed by et3n ( 0.53 ml , 3.8 mmol ) and accl ( 0.13 ml , 1.9 mmol ) gave ( r)-12 as a white solid ( 0.59 g , 84% ) : rf = 0.40 ( meoh / ch2cl2 1/20 ) ; mp 194195 c ; [ ]d 14.3 ( c 0.9 , chcl3 ) ; h nmr ( cdcl3 ) 2.02 ( s , 3h ) , 3.39 ( s , 3h ) , 3.48 ( dd , j = 7.4 , 9.2 hz , 1h ) , 3.81 ( dd , j = 4.0 , 9.2 hz , 1h ) , 4.454.55 ( m , 2h ) , 4.584.63 ( m , 1h ) , 6.56 ( d , j = 6.4 hz , 1h ) , 6.987.01 ( m , 1h ) , 7.27 ( d , j = 8.0 hz , 2h ) , 7.33 ( d , j = 8.0 hz , 2h ) , 7.497.58 ( m , 4h ) ; addition of excess ( r)-()-mandelic acid to a cdcl3 solution of ( r)-12 gave only one signal for the acetyl methyl and one signal for the ether methyl protons ; c nmr ( dmso - d6 ) 22.5 , 41.7 , 52.7 , 58.2 , 72.1 , 120.1 ( q , j = 254.9 hz , ocf3 ) , 121.4 , 126.6 , 127.7 , 128.4 , 137.1 , 139.1 , 139.3 , 147.7 , 169.4 , 169.8 ; lrms ( esi ) 433.1 [ m + na ] ( calcd for c20h21f3n2o4na 433.1 ) . anal . ( c20h21f3n2o4 ) c , h , f , n. using method 1 , ( r)-65 ( 1.80 g , 4.1 mmol ) and 4 m hcl ( 3.1 ml , 12.4 mmol ) followed by et3n ( 1.24 g , 12.2 mmol ) and accl ( 0.38 g , 4.9 mmol ) gave the desired product ( r)-13 ( 1.05 g , 71% ) as a white powder : rf = 0.20 ( ch2cl2/ch3oh 19/1 ) ; mp 150151 c ; [ ]d + 11.1 ( c 1.0 , ch3oh ) ; h nmr ( cdcl3/cd3od ) 2.04 ( s , 3h ) , 2.16 ( s , 3h ) , 3.47 ( s , 3h ) , 3.523.57 ( m , 1h ) , 3.683.73 ( m , 1h ) , 4.404.52 ( m , 2h ) , 4.544.60 ( m , 1h ) , 7.297.40 ( m , 4h ) , 7.507.56 ( m , 3h ) , 7.76 ( s , 1h ) ; c nmr ( cdcl3/cd3od ) 22.8 , 23.9 , 43.1 , 53.1 , 59.1 , 72.0 , 118.6 , 119.0 , 122.8 , 127.4 , 127.9 , 129.3 , 137.1 , 138.8 , 140.3 , 141.5 , 170.4 , 171.5 ; one carbonyl resonance was not detected ; lrms ( esi ) 384.2 [ m + h ] ( calcd for c21h26n3o3 384.2 ) ; hrms ( esi ) 384.1924 [ m + h ] ( calcd for c21h26n3o3 384.1923 ) . to a solution of ( r)-73 ( 0.65 g , 1.6 mmol ) in ch2cl2 ( 16 ml ) was added 4 m hcl in dioxane ( 1.5 ml , 6.0 mmol ) , and the mixture was stirred at room temperature ( 4 h ) . the reaction mixture was evaporated in vacuo and recrystallized ( ch3oh / etoac / hexanes ) to give the desired product ( r)-14 ( 0.51 g , 90% ) as a white solid : rf = 0.48 ( ch2cl2/ch3oh 9/1 ) ; mp 152153 c ; [ ]d + 12.7 ( c 1.0 , ch3oh ) ; h nmr ( cdcl3/cd3od ) 2.00 ( s , 3h ) , 3.34 ( s , 3h ) , 3.453.49 ( m , 1h ) , 3.673.72 ( m , 1h ) , 4.424.46 ( m , 2h ) , 4.514.55 ( m , 1h ) , 4.69 ( s , 2h ) , 7.277.31 ( m , 3h ) , 7.357.41 ( m , 1h ) , 7.447.46 ( m , 1h ) , 7.507.54 ( m , 3h ) ; c nmr ( cdcl3/cd3od ) 23.1 , 43.3 , 52.9 , 59.3 , 65.0 , 72.0 , 125.7 , 126.1 , 126.3 , 127.5 , 128.0 , 129.1 , 137.1 , 140.4 , 141.0 , 141.8 , 170.3 , 171.2 ; lrms ( esi ) 357.1 [ m + h ] ( calcd for c20h24n2o4h 357.2 ) . ( c20h24n2o4 ) c , h , n. using the previous procedure , ( r)-74 ( 0.31 g , 0.7 mmol ) and 4 m hcl ( 0.8 ml , 3.2 mmol ) gave the desired product ( r)-15 ( 0.21 g , 82% ) as a white solid : rf = 0.56 ( ch2cl2/ch3oh 9/1 ) ; mp 196197 c ; [ ]d + 9.6 ( c 1.0 , ch3oh ) ; h nmr ( cdcl3/cd3od ) 1.99 ( s , 3h ) , 3.463.50 ( m , 1h ) , 3.633.66 ( m , 1h ) , 4.39 ( dd , j = 5.8 , 14.0 hz , 1h ) , 4.46 ( dd , j = 5.8 , 14.0 hz , 1h ) , 4.504.55 ( br t , 1h ) , 6.786.84 ( m , 1h ) , 7.167.21 ( m , 1h ) , 7.287.56 ( m , 8h ) ; the methoxy proton resonance was not detected and believed to be overlapped with the adjacent peak near 3.40 ; c nmr ( cdcl3/cd3od ) 22.8 , 43.1 , 52.9 , 59.1 , 72.1 , 127.4 , 128.0 , 128.4 , 128.8 , 128.9 , 131.0 , 131.5 , 137.5 , 139.4 , 141.0 , 168.9 , 170.4 , 171.5 ; lrms ( esi ) 371.2 [ m + h ] ( calcd for c20h22n2o5h 371.2 ) ; hrms ( esi ) 371.1607 [ m + h ] ( calcd for c20h22n2o5h 371.1607 ) . anal . ( c20h22n2o50.13h2o ) c , h , n. using the procedure for ( r)-14 , ( r)-75 ( 1.18 g , 3.59 mmol ) and 4 m hcl ( 2.7 ml , 10.8 mmol ) gave the desired product ( r)-16 ( 0.91 g , 90% ) as a yellow solid : rf = 0.00 ( etoac / hexanes 1/1 ) ; mp 99100 c ; [ ]d + 10.4 ( c 1.0 , ch3oh ) ; h nmr ( cdcl3/cd3od ) 1.99 ( s , 3h ) , 3.36 ( s , 3h ) , 3.50 ( dd , j = 4.0 , 9.2 hz , 1h ) , 3.78 ( dd , j = 7.2 , 9.2 hz , 1h ) , 4.43 ( dd , j = 5.8 , 15.0 hz , 1h ) , 4.48 ( dd , j = 5.8 , 15.0 hz , 1h ) , 4.594.64 ( m , 1h ) , 6.636.67 ( m , 2h ) , 6.85 ( m , 1h ) , 6.936.96 ( m , 1h ) , 6.997.05 ( br t , 1h ) , 7.20 ( t , j = 7.8 hz , 1h ) , 7.26 ( d , j = 6.0 hz , 2h ) , 7.49 ( dd , j = 1.6 , 6.4 hz , 2h ) ; c nmr ( cd3od ) 22.6 , 42.8 , 52.8 , 58.3 , 72.1 , 121.2 , 122.0 , 126.0 , 126.6 , 127.8 , 130.4 , 132.8 , 137.2 , 139.5 , 141.4 , 169.6 , 169.9 ; lrms ( esi ) 364.2 [ m + na hcl ] ( calcd for c19h23nan3o3 364.2 ) ; hrms ( esi ) 342.1817 [ m cl ] ( calcd for c19h24n3o3 342.1817 ) . compounds were screened under the auspices of the national institutes of health s asp . experiments were performed in male rodents ( albino carworth farms no . 1 mice ( ip ) , albino sprague dawley rats ( ip , po ) ) . housing , handling , and feeding were in accordance with recommendations contained in the guide for the care and use of laboratory animals . anticonvulsant activity was established using the mes test , 6 hz , and the scmet test , according to previously reported methods . cad cells were grown at 37 c and in 5% co2 ( sarstedt , newton , nc ) in ham s f12/emem ( gibco , grand island , ny ) , supplemented with 8% fetal bovine serum ( sigma , st . louis , mo ) and 1% penicillin / streptomycin ( 100% stocks , 10 000 u / ml penicillin g sodium and 10 000 g / ml streptomycin sulfate ) . rat cortical neuron cultures were prepared from cortices dissected from embryonic day 19 brains exactly as described . the cdna gene encoding nav1.1 was codon - optimized and synthesized using the open reading frame ( accession no . the cdna genes encoding nav1.3 from rat and nav1.7 from human were subcloned into the vector pcdna3.1-mod . the cdna encoding nav1.5 from human was introduced into pcdna3.1(+ ) with the cmv promoter . the constructs were then transfected into hek293 cells using the calcium phosphate precipitation technique . after 48 h , the cells were passaged into 100 mm dishes and treated with g418 ( geneticin , life technologies ) at 800 g / ml to select for neomycin resistant cells . after 2 weeks the colonies were then tested for channel expression with whole - cell patch - clamp technique . nav1.1 , nav1.3 , nav1.5 , and nav1.7 stable cells were grown under standard tissue culture conditions ( 5% co2 at 37 c ) in dulbecco s modified eagle medium supplemented with 10% fetal bovine serum . whole - cell voltage clamp recordings were performed at room temperature on hek293 cells , cad cells , and cortical neurons using an epc 10 amplifier ( heka electronics , lambrecht / pfalz germany ) . electrodes were pulled from thin - walled borosilicate glass capillaries ( warner instruments , hamden , ct ) with a p-97 electrode puller ( sutter instrument , novato , ca ) such that final electrode resistances were 12 m when filled with internal solutions . the internal solution for recording na currents contained the following ( in mm ) : 110 cscl , 5 mgso4 , 10 egta , 4 atp na2-atp , 25 hepes ( ph 7.2 , 290310 mosm / l ) . the external solution contained the following ( in mm ) : 100 nacl , 10 tetraethylammonium chloride ( tea - cl ) , 1 cacl2 , 1 cdcl2 , 1 mgcl2 , 10 d - glucose , 4 4-ap , 0.1 nicl2 , 10 hepes ( ph 7.3 , 310315 mosm / l ) . cells were considered only when the seal resistance was less than 3 m. linear leak currents were digitally subtracted by p/4 . analysis was performed using fitmaster and origin8.1 ( originlab corporation , ma , usa ) . for activation curves , conductance ( g ) through na channels was calculated using the equation g = i/(vm vrev ) , where vrev is the reversal potential , vm is the membrane potential at which the current was recorded , and i is the peak current . activation and inactivation curves were fitted to a single - phase boltzmann function g / gmax = 1/{1 + exp[(v v50)/k ] } , where g is the peak conductance , gmax is the fitted maximal g , v50 is the half activation voltage , and k is the slope factor . additional details of specific pulse protocols are described in the results text or figure legends . differences between mean values were compared by either paired or unpaired two - tailed student s t - tests or an analysis of variance ( anova ) , when comparing multiple groups ( repeated measures whenever possible ) . if a significant difference was determined by anova , then a dunnett s or tukey s post hoc test was performed . data are expressed as the mean sem , with p < 0.05 considered as the level of significance .	we prepared 13 derivatives of n-(biphenyl-4-yl)methyl ( r)-2-acetamido-3-methoxypropionamide that differed in type and placement of a r - substituent in the terminal aryl unit . we demonstrated that the r - substituent impacted the compound s whole animal and cellular pharmacological activities . in rodents , select compounds exhibited excellent anticonvulsant activities and protective indices ( pi = td50/ed50 ) that compared favorably with clinical antiseizure drugs . compounds with a polar , aprotic r - substituent potently promoted na+ channel slow inactivation and displayed frequency ( use ) inhibition of na+ currents at low micromolar concentrations . the possible advantage of affecting these two pathways to decrease neurological hyperexcitability is discussed .
endometrial carcinoma is the second most common gynecological cancer in korea , accounting for approximately 1.8% of all the cancers diagnosed in women total abdominal hysterectomy with bilateral salpingo - oophrectomy and lymph node dissection is essential for staging and treatment of endometrial carcinoma . adjuvant treatment after surgery is indicated in high - risk endometrial carcinoma , and postoperative radiotherapy has shown relatively good outcomes . in a recent retrospective analysis that evaluated 382 patients , adjuvant radiothrapy significantly improved recurrence - free , disease - specific , and overall survival in patients with early - stage endometrial carcinoma . the national cancer comprehensive network guideline recommends various ways of administering adjuvant therapy according to stage and grade of tumor in endometrial carcinoma . no one single therapy has shown superiority over the others , leading to no treatment being established as the gold - standard . several randomized trials have shown that the use of pelvic radiotherapy in stage i endometrial carcinoma provides a highly significant improvement of pelvic control in patients with selected risk factors , as well as progression - free survival [ 4 - 8 ] . for stage i - iii endometrial carcinoma patients with high risk features , the use of adjuvant chemotherapy has been investigated , and no survival benefit has been shown compared to pelvic radiotherapy [ 9 - 12 ] . with the unsatisfactory results of previous trials involving single treatment modality , there are some trials investigating the roles of concurrent chemoradiotherapy compared to chemotherapy or radiotherapy alone . however , the role of chemoradiotherapy after surgery has not been established definitely in stage i - ii endometrial carcinoma . thus , this study was designed to compare the overall and relapse - free survival of adjuvant pelvic radiotherapy and concurrent chemoradiotherapy after surgical treatment of stage i - ii endometrial carcinoma . from march 1999 to july 2013 , a total 83 patients with stage i - ii endometrial carcinoma , who underwent curative surgery and postoperative pelvic radiotherapy with or without concurrent chemotherapy , were reviewed retrospectively . however , two patients were excluded from the study due to both patients having lung metastases during the course of radiotherapy , and one patient proved to have uterine sarcoma on examination of the permanent pathologic specimen . the patients had histologically proven adenocarcinoma arising from the uterine body , with a tumor stage of pt1n0 with one or more risk factors , such as high grade tumor , old age ( > 60 ) , lymphovascular invasion , and outer half of myometrial invasion or t2n0 . low grade represents a well or moderately differentiated histology and high grade represents poorly differentiated , papillary serous or clear - cell histology . all patients underwent open or laparoscopic hysterectomy with adnexectomy and pelvic and paraaortic lymph node dissection . external beam radiation therapy was delivered with the standard whole pelvic four - box field technique 2 to 8 weeks ( median , 3 weeks ) after surgery . two patients with postoperative pelvic abscess received a delayed radiation therapy , 6 and 8 weeks after surgery , respectively . patients were in supine position and a candle - shaped cylinder was inserted into the vagina to indicate a stump . clinical target volume included distal common and iliac lymph nodes , tumor bed , and vaginal stump . the lateral border of the anterior - posterior field was defined to be 1.5 cm from the lateral margin of the bony pelvis . in the lateral field , the anterior border was the anterior aspect of the symphysis pubis , and the posterior border was the s2/s3 interspace . the median external beam radiation therapy dose was 45 gy with a daily dose of 1.8 gy , ranging from 40 gy to 60 gy . all other patients tolerated the treatment well , and the initial planned dose was fully delivered . intracavitary brachytherapy was recommended to stage i - ii endometrial cancer patients , except to those who had toxicity grade 3 or higher during the external beam radiotherapy period . adjuvant vaginal brachytherapy was delivered with the largest fitting vaginal cylinder ( 2.5 - 4.0 cm in diameter ) . a total of 32 patients received six cycles of weekly cisplatin , in which 40 mg / m was delivered concurrently with radiotherapy . basically , radiotherapy alone after surgery was recommended to patients with stage i - ii endometrial cancer . radiotherapy with concurrent cisplatin was considered in patients with high - grade histology or lymphovascular invasion . during radiotherapy , patients were to have a weekly physical examination and complete blood count for the evaluation of acute toxicity . after radiotherapy , patients were to visit the clinic every 3 months for the first 2 years and 6 months thereafter for surveillance of late complications and recurrence . adverse effects of chemotherapy and radiotherapy were assessed using the national cancer institute common terminology criteria for adverse events ver . overall survival was defined as the period from the end date of radiotherapy to the date of death . relapse - free survival was defined as the period from the end date of radiotherapy to the date of recurrence at any site or death . for a prospective and definitive analysis , the trial is designed to have 80% power in detecting a difference in the primary end point , the recurrence - free rate at 3 years , of 85% in radiotherapy alone arm versus 90% in chemoradiotherapy arm using a two - sided test at the 5% level of significance . thirteen hundred and ninety evaluable patients , with a minimum follow - up of 3 years , were required . however , it is not easy to conduct a randomized controlled trial due to the low incidence of endometrial cancer in korea . therefore , we undertook a retrospective and matching study to evaluate the role of concurrent chemoradiotherapy in endometrial cancer . propensity score matching analyses were used to compensate for the differences in baseline characteristics of age , stage , and histology . of the 48 patients who received postoperative radiotherapy ( rt ) , the propensity scores were estimated without regard to the outcome variables , with multiple logistic regression analysis . prespecified covariates were age , stage , and tumor histology were included in the non - parsimonious models for rt alone versus crt . the model was well - calibrated ( hosmer - lemeshowthe test , p=0.141 ) with reasonable discrimination ( c statistic=0.62 ) . the kaplan - meier method was used to figure out the overall survival and relapse - free survival rates . prognostic factors , such patient age , cancer antigen ( ca ) 125 , ca 19 - 9 , pt stage , histologic grade , lymphovascular invasion , and stump boost were analyzed . in a univariate analysis , the log - rank test was used to evaluate the association between survival time and prognostic factors . for the multivariate analysis , cox proportional hazards regression model was used to estimate the hazard ratio of the prognostic factors for overall survival and relapse - free survival . from march 1999 to july 2013 , a total 83 patients with stage i - ii endometrial carcinoma , who underwent curative surgery and postoperative pelvic radiotherapy with or without concurrent chemotherapy , were reviewed retrospectively . however , two patients were excluded from the study due to both patients having lung metastases during the course of radiotherapy , and one patient proved to have uterine sarcoma on examination of the permanent pathologic specimen . the patients had histologically proven adenocarcinoma arising from the uterine body , with a tumor stage of pt1n0 with one or more risk factors , such as high grade tumor , old age ( > 60 ) , lymphovascular invasion , and outer half of myometrial invasion or t2n0 . low grade represents a well or moderately differentiated histology and high grade represents poorly differentiated , papillary serous or clear - cell histology . all patients underwent open or laparoscopic hysterectomy with adnexectomy and pelvic and paraaortic lymph node dissection . external beam radiation therapy was delivered with the standard whole pelvic four - box field technique 2 to 8 weeks ( median , 3 weeks ) after surgery . two patients with postoperative pelvic abscess received a delayed radiation therapy , 6 and 8 weeks after surgery , respectively . patients were in supine position and a candle - shaped cylinder was inserted into the vagina to indicate a stump . clinical target volume included distal common and iliac lymph nodes , tumor bed , and vaginal stump . the lateral border of the anterior - posterior field was defined to be 1.5 cm from the lateral margin of the bony pelvis . in the lateral field , the anterior border was the anterior aspect of the symphysis pubis , and the posterior border was the s2/s3 interspace . the median external beam radiation therapy dose was 45 gy with a daily dose of 1.8 gy , ranging from 40 gy to 60 gy . all other patients tolerated the treatment well , and the initial planned dose was fully delivered . intracavitary brachytherapy was recommended to stage i - ii endometrial cancer patients , except to those who had toxicity grade 3 or higher during the external beam radiotherapy period . adjuvant vaginal brachytherapy was delivered with the largest fitting vaginal cylinder ( 2.5 - 4.0 cm in diameter ) . a total of 32 patients received six cycles of weekly cisplatin , in which 40 mg / m was delivered concurrently with radiotherapy . basically , radiotherapy alone after surgery was recommended to patients with stage i - ii endometrial cancer . radiotherapy with concurrent cisplatin was considered in patients with high - grade histology or lymphovascular invasion . during radiotherapy , patients were to have a weekly physical examination and complete blood count for the evaluation of acute toxicity . after radiotherapy , patients were to visit the clinic every 3 months for the first 2 years and 6 months thereafter for surveillance of late complications and recurrence . adverse effects of chemotherapy and radiotherapy were assessed using the national cancer institute common terminology criteria for adverse events ver . overall survival was defined as the period from the end date of radiotherapy to the date of death . relapse - free survival was defined as the period from the end date of radiotherapy to the date of recurrence at any site or death . for a prospective and definitive analysis , the trial is designed to have 80% power in detecting a difference in the primary end point , the recurrence - free rate at 3 years , of 85% in radiotherapy alone arm versus 90% in chemoradiotherapy arm using a two - sided test at the 5% level of significance . thirteen hundred and ninety evaluable patients , with a minimum follow - up of 3 years , were required . however , it is not easy to conduct a randomized controlled trial due to the low incidence of endometrial cancer in korea . therefore , we undertook a retrospective and matching study to evaluate the role of concurrent chemoradiotherapy in endometrial cancer . propensity score matching analyses were used to compensate for the differences in baseline characteristics of age , stage , and histology . of the 48 patients who received postoperative radiotherapy ( rt ) , the propensity scores were estimated without regard to the outcome variables , with multiple logistic regression analysis . prespecified covariates were age , stage , and tumor histology were included in the non - parsimonious models for rt alone versus crt . the model was well - calibrated ( hosmer - lemeshowthe test , p=0.141 ) with reasonable discrimination ( c statistic=0.62 ) . the kaplan - meier method was used to figure out the overall survival and relapse - free survival rates . prognostic factors , such patient age , cancer antigen ( ca ) 125 , ca 19 - 9 , pt stage , histologic grade , lymphovascular invasion , and stump boost were analyzed . in a univariate analysis , the log - rank test was used to evaluate the association between survival time and prognostic factors . for the multivariate analysis , cox proportional hazards regression model was used to estimate the hazard ratio of the prognostic factors for overall survival and relapse - free survival . the median age of the patient population in this study was 55 years and ranged from 40 to 77 years . all patients were diagnosed with stage i - ii ( pt1 - 2n0m0 ) endometrial carcinoma ( 54 with stage i and 10 with stage ii ) . the baseline characteristics of the two arms were more or less skewed in spite of propensity score matching . more patients with pt2 , lymphovascular invasion , and poorly differentiated histology have been allocated to the crt arm , rather than the rt alone arm . the median follow - up time was 42.5 months , ranging from 6 to 172 months . the 5-year overall survival rates were 90.0% for the radiotherapy arm and 91.6% for the chemoradiotherapy arm . there was no significant difference in overall survival between the two arms ( p=0.798 ) ( fig . five patients died during the follow - up period , two in the crt arm and three in the rt arm . the 5-year relapse - free survival rates were 87.2 % in the radiotherapy arm and 88.0 % in the chemoradiotherapy arm . there was no statistically significant difference in relapse - free survival between the two arms ( p=0.913 ) ( fig . three patients failed ; two failed locoregionally and one failed distantly . in the crt arm , three patients failed ; one had locoregional failure only , one had distant failure , and one had both locoregional and distant failures . age , ca 125 , pathologic t stage , histological grade , lymphovascular invasion , stump boost , and treatment modality were analyzed for relapse - free survival . table 2 shows the univariate and multivariate analyses of the factors associated with relapse - free survival . in the univariate analysis , age > 55 years ( p=0.032 ) and high - grade histology ( p=0.034 ) ( fig . as for the multivariate analysis , high - grade histology ( hazard ratio , 3.67 ; 95% of confidence interval , 2.34 to 7.65 ; p=0.045 ) was the only statistically significant prognostic factor for relapse - free survival . acute grade 3 or 4 hematologic toxicities in the chemoradiotherapy arm were significantly higher than the radiotherapy alone arm ( 12.5% vs. 37.5% , p=0.021 ) . ten patients in the chemoradiotherapy arm and 2 patients in the radiotherapy arm had acute grade 3 or higher hematologic toxicities . non - hematologic grade 3 or higher acute toxicities were as follows : four patients with diarrhea , three patients with pelvic abscess , and one patient with cystitis in the chemoradiotherapy arm ; two patients with diarrhea and one patients with pelvic abscess in the radiotherapy alone arm . one patient in the radiotherapy alone arm had small bowel obstruction 2 years after the end of radiotherapy and she received a reoperation , and three patients in the chemoradiotherapy arm had long - term grade 3 or higher toxicities of diarrhea , cystitis , and small bowel ileus . patients with grade 3 cystitis and small bowel ileus in the chemoradiotherapy arm were cured after medical and surgical treatment . however , grade 3 cystitis was observed in one patient 5 years after chemoradiotherapy . the median follow - up time was 42.5 months , ranging from 6 to 172 months . the 5-year overall survival rates were 90.0% for the radiotherapy arm and 91.6% for the chemoradiotherapy arm . there was no significant difference in overall survival between the two arms ( p=0.798 ) ( fig . five patients died during the follow - up period , two in the crt arm and three in the rt arm . the 5-year relapse - free survival rates were 87.2 % in the radiotherapy arm and 88.0 % in the chemoradiotherapy arm . there was no statistically significant difference in relapse - free survival between the two arms ( p=0.913 ) ( fig . three patients failed ; two failed locoregionally and one failed distantly . in the crt arm , three patients failed ; one had locoregional failure only , one had distant failure , and one had both locoregional and distant failures . age , ca 125 , pathologic t stage , histological grade , lymphovascular invasion , stump boost , and treatment modality were analyzed for relapse - free survival . table 2 shows the univariate and multivariate analyses of the factors associated with relapse - free survival . in the univariate analysis , age > 55 years ( p=0.032 ) and high - grade histology ( p=0.034 ) ( fig . as for the multivariate analysis , high - grade histology ( hazard ratio , 3.67 ; 95% of confidence interval , 2.34 to 7.65 ; p=0.045 ) was the only statistically significant prognostic factor for relapse - free survival . acute grade 3 or 4 hematologic toxicities in the chemoradiotherapy arm were significantly higher than the radiotherapy alone arm ( 12.5% vs. 37.5% , p=0.021 ) . ten patients in the chemoradiotherapy arm and 2 patients in the radiotherapy arm had acute grade 3 or higher hematologic toxicities . non - hematologic grade 3 or higher acute toxicities were as follows : four patients with diarrhea , three patients with pelvic abscess , and one patient with cystitis in the chemoradiotherapy arm ; two patients with diarrhea and one patients with pelvic abscess in the radiotherapy alone arm . one patient in the radiotherapy alone arm had small bowel obstruction 2 years after the end of radiotherapy and she received a reoperation , and three patients in the chemoradiotherapy arm had long - term grade 3 or higher toxicities of diarrhea , cystitis , and small bowel ileus . patients with grade 3 cystitis and small bowel ileus in the chemoradiotherapy arm were cured after medical and surgical treatment . pelvic radiotherapy still remains as an indispensible treatment option for high - risk or advanced stage endometrial carcinoma . several retrospective studies have shown significantly higher rates of locoregional recurrence if high - risk patients were not treated with radiotherapy after curative surgery . mundt et al . reported a retrospective analysis of high - risk pathologic stage i - iv endometrial carcinoma patients who were treated with chemotherapy alone . of the 43 patients , 67% relapsed , 40% had pelvic recurrence , and the 3-year pelvic relapse rate was 47% , and the pelvic cavity was the first or the only site of relapse in 31% of patients . in a study conducted by klopp et al . patients who were treated with regional radiotherapy had a significantly better 5-year relapse - free survival compared to those who received chemotherapy only ( 98% vs. 61% , p=0.001 ) . patients who received regional radiotherapy at the same time showed a better outcome in disease - specific survival ( 78% vs. 39% , p=0.01 ) and overall survival ( 73% vs. 40% , p=0.03 ) . in patients who received chemotherapy alone , therefore , it has come down to the theory that combining chemotherapy and radiotherapy might be the optimal treatment modality to reduce both locoregional and distant relapse . in our study , both the radiotherapy alone and the chemoradiotherapy arms showed excellent outcomes as adjuvant therapy in surgically treated stage i - ii endometrial carcinoma . in our retrospective analysis , overall survival and relapse - free survival of the chemoradiotherapy arm was not significantly higher than the radiotherapy alone arm . however , acute treatment hematologic toxicities were significantly higher in the chemoradiotherapy arm than in the radiotherapy alone arm . some randomized trials tested the efficacy of concurrent or sequential chemoradiotherapy in endometrial carcinoma . the first randomized clinical trial to evaluate the benefit of chemoradiotherapy was designed and tested by the gynecologic oncology group ( gog ) . however , this study was closed early due to protocol violations , small sample size , and loss of follow - up . hence , it was unable to determine whether the use of chemoradiotherapy as adjuvant therapy would have a significant effect on recurrence , progression , and survival . randomized trials comparing radiotherapy alone and chemoradiotherapy in endometrial cancer are shown in table 4 . randomized 156 patients to receive radiotherapy or sequential chemoradiotherapy . however , adjuvant chemoradiotherapy failed to show an improvement in the overall survival and recurrence rates . grade 3 or 4 bowel toxicity in the sequential chemoradiotherapy arm was higher than that of the radiotherapy alone arm ( 9.5% vs. 2.8% ) . in the nsgo 9501/eortc-55991 trial , 382 patients were randomized to receive radiotherapy with or without sequential chemotherapy . the combined treatment modality showed a 7% increase in progression free survival ( 79% vs. 72% , p=0.03 ) , but not in overall survival . currently , trials are ongoing to evaluate the role of concurrent chemoradiation or radiotherapy followed by adjuvant chemotherapy . the portec-3 trial compares pelvic radiotherapy alone with two cycles of concurrent chemotherapy during radiation , followed by four cycles of chemotherapy in stage i - ii endometrial carcinoma . this trial was designed to see if chemoradiotherapy improves the overall and relapse - free survival rates . this trial is expected to address the concerns of chemoradiotherapy and its impact over radiotherapy alone on survival . in our series , high - grade tumor histology was found to be a poor prognostic factor for relapse - free survival . prognostic factors for survival , such as tumor histology , surgical stage , depth of myometrial invasion , and presence of lymphovascular invasion in endometrial carcinoma , have been reported by other studies [ 19 - 21 ] . in the results of a retrospective study conducted by irwin et al . , high tumor grade , lower uterine segment involvement , and old age were independent poor prognostic factors for disease - free survival in a multivariate analysis . reported a retrospective analysis of 440 patients who were treated with postoperative radiotherapy . in a multivariate analysis , histologic type , myometrial invasion , and histologic grade were the prognostic factors for disease - free survival . we reported no significant impact on relapse - free survival of chemoradiotherapy over radiotherapy alone for patients with stage i - ii endometrial cancer in our study . first , our study should be understood with the consideration of inherent biases due to the nature of a retrospective study design . we evaluated just 64 cases of both chemoradiotherapy and radiotherapy alone arm . as a result there also might be a selection bias which allocated more patients with risk factors , such as pt2 , lymphovascular invasion , and poorly differentiated histology to the crt arm rather than the rt alone arm . although we executed a matching analysis to minimize selection bias of our study , we admitted that our result could be just preliminary . second , we had a shortage of patient information in our analysis and did not conduct quality of life assessments , such as sexual dysfunction and depressive disorder . therefore , a long - term follow - up of more than 5 years is indicated for the exact survival analysis in the chemoradiotherapy and radiotherapy alone arms . in our investigational matching study , postoperative adjuvant chemoradiotherapy after curative surgery did not show higher overall survival and relapse - free survival than radiotherapy alone in stage i - ii endometrial carcinoma . hence , postoperative radiotherapy alone rather than chemoradiotherapy is a standard treatment in patients with stage i - ii endometrial cancer .	purposethe purpose of this study was to compare the results of postoperative adjuvant radiotherapy ( rt ) and concurrent chemoradiotherapy ( crt ) in stage i - ii endometrial carcinoma.materials and methodswe analyzed a total of 64 patients with surgically staged i - ii endometrial carcinoma who were treated with postoperative adjuvant rt or concurrent crt between march 1999 and july 2013 . thirty - two patients who received postoperative rt alone were matched with those who received postoperative crt ( n=32 ) in accordance to age , stage , and tumor histology . overall survival and relapse - free survival , as well as toxicity of the rt and crt arms were evaluated and compared.resultsthe 5-year overall survival rate was 90.0% for the rt arm and 91.6% for the crt arm . there was no significant difference in overall survival between the two treatment arms ( p=0.798 ) . the 5-year relapse - free survival rate was 87.2% in the rt arm and 88.0% in the crt arm . again , no significant difference in relapse - free survival was seen between the two arms ( p=0.913 ) . in a multivariate analysis , tumor histology was an independent prognostic factor for relapse - free survival ( hazard ratio , 3.67 ; 95% of ci , 2.34 to 7.65 ; p=0.045 ) . acute grade 3 or 4 hematologic toxicities in the crt arm were significantly higher than in the rt alone arm ( 6.2% vs. 31.2% , p=0.010).conclusionadjuvant pelvic concurrent chemoradioherapy did not show superior results in overall survival and relapse - free survival compared to rt alone in stage i - ii endometrial carcinoma .
upper gastrointestinal ( ugi ) cancers ( gastric and esophagus cancer ) constitute a major health problem worldwide ( 1 ) . although a decreasing incidence of gastric cancer has been observed during the last decades , it remains the fourth most common cancer worldwide and the second leading cause of cancer - related death ( 2 , 3 ) . esophageal cancer is among the 10 most common malignancies worldwide , and ranks as the 6th leading cause of death from cancer ( 2 ) . it constitutes 7% of all gastrointestinal ( gi ) cancers and is one of the most lethal of all cancers ( 47 ) . ugi cancers cause 55% of all cancer - related deaths in iran , with gastric cancer being the most common and accounts for nearly 50% of all gi cancers ( 8) . a family history of cancer can increase the risk for developing cancer ( 10 ) and recognized as one of the most important risk factors in predicting personal cancer risk ( 11 ) . it is suggested that esophageal cancer was more common in both the parents and siblings of the patients with the disease ( 4 ) . ( 15 ) found that , 1 of 3 patients diagnosed with gastric cancer had a family history of stomach cancer in first- or second - degree relatives . it is also known that the risk of development of gastric carcinoma among first - degree relatives of patients with stomach cancer appears to be 2 - 3 times higher than in the general population ( 16 ) . while a few studies have been conducted on evaluation of family history of ugi cancers in iranian cancer patients , there is no data of family history of gastric and esophageal cancer in the general population . therefore , we aimed to evaluate the prevalence of positive family history of these cancers in a large population - based sample of tehran province , capital of iran . this study designed as a cross - sectional survey in general population ( 2006 - 2007 ) of tehran province ( including tehran metropolitan , and five other cities and their rural areas in tehran province ) . a total of 7,300 persons ( older than 20 years ) were sampled by random sampling on the basis of the list of postal codes ( registered in tehran central post office ) , of whom 6,700 persons agreed to participate ( response rate 92% ) . the research group were referred to each selected postal code and interviewed with all members of selected house according to questionnaire . the participants were informed that attending the interview was not compulsory and patient 's anonymity was preserved . respondents were asked if any first - degree ( father , mother , sister , brother , daughter , or son ) or second - degree ( grandparents , aunts or uncles ) relatives had gastric or esophageal cancer . the participants completed a question specifying the age of diagnosis for each relative with a history of cancer . the study samples were asked about their age and sex . the independent t - test was used to test for a difference between two independent groups on the means of a continuous variable . numeric variables are presented as mean standard deviation other parameters as frequency and percentage . we exclude 247 persons from further analyses because they reported unknown family history of cancer . totally , 6,453 respondents ( 48% male with mean ( sd ) age of 41.2 19.6 years and 52% female with mean ( sd ) age of 39.6 17.9 years , p < 0.05 ) entered to the study . the mean ( sd ) age of responders with positive fh was 37.117.1 years ; it is significantly higher than the mean age ( sd ) of those with negative fh ( 40.518.8 ) ( p < 0.05 ) . in total , 341 respondents ( 5.3% ) reported a history of ugi cancers in their relatives , 134 ( 2.1% ) in fdrs , and 207(3.2% ) in sdrs ( table 1 ) . the mean ( sd ) age at diagnosis of relatives affected with ugi cancers was 47.816.8 and 60.120 years in fdr and sdr , respectively ( p < 0.05 ) . also , 749 ( 11.6% ) people reported a fh of any cancer ( except ugi ) in fdrs , and 1091 ( 16.9% ) reported a fh of any cancer ( except ugi ) in sdrs . the mean ( sd ) age at diagnosis of relatives affected with any cancer ( except ugi ) was 57.113.1 and 55.312.3 years in fdr and sdr , respectively ( p < 0.05 ) . prevalence of first and second degree relative family history of cancer according to sex of responders the prevalence of first and second degree relative fh of ugi cancers vs. all cancer ( except ugi cancers ) according to sex of responders is shown in table 1 . in our sample , female with fh of ugi cancers , 56 ( 1.7% ) reported having a fdr history and 164(4.9% ) reported having only a sdr history . regarding to male responders , 78(2.5% ) reported having a fdr and 99 ( 3.2% ) having a sdr affected with ugi cancers . observed difference between male and female reporting fdr with ugi cancers was significant ( p < 0.05 ) . table 2 shows the distribution of fh in fdrs and sdrs according to age of affected relatives ( < 50 , > 50 and both age groups ) . of those responders with positive fh , 27 ( 20.1% ) and 13 ( 6.3% ) have at least one affected person diagnosed at age < 50 years in fdrs or sdrs , respectively . distribution of responders with first or second degree relatives according to diagnostic age of affected person ( < 50 , > 50 and both ages < 50 and > 50 years ) family history assessment is gaining importance as a research tool , a clinical marker of increased risk for disease , and a potential tool for initiating aggressive cancer screening and prevention ( 10 , 17 ) . population - based data on the prevalence of having fh of ugi cancers are still scant . this study is the first manuscript on the prevalence of positive fh of cancer among general population of iran . in this study we estimated the prevalence of having fh of ugi cancers in iranian general population . overall , approximately 5.3% of respondents reported at least one first or second relative with ugi cancers . the mean age of responders with positive fh was significantly higher than the mean age of those with negative fh . data on the prevalence of a positive fh of healthy subjects ( not affected with cancer ) may be derived from control groups in case - control studies . the reported fh for gastric or esophagus cancer in the control groups varied in these studies from 4.9 to 12.3 % ( 12 , 13 , 1822 ) . information on family history was self reported , and it is possible that some of subjects may tend to recall a family history of gastric or esophageal cancers more accurately than others . a review of studies evaluating the accuracy and completeness of reporting family history in relatives of cancer patients and controls found satisfactory results for family history in fdrs of breast , colon and prostate cancer , and less so for endometrial and ovarian cancer ( 23 ) . a study found that respondents those aged greater than 75 years were significantly more likely to give a false - negative report of their cancer history than those aged 4564 years ( 24 ) . another study found the overall sensitivity and specificity of self - reported breast cancer to be quite high ( 25 ) . females who were older , less educated , or of nonwhite race / ethnicity had the lowest sensitivities ( 24 , 2628 ) . goldberg ( 29 ) showed that a number of participants may have felt that cancers diagnosed years and decades ago were unimportant and not worth reporting , since they had survived and moved on . alternatively , the observed association between time since diagnosis and self - reported cancer history may reflect a period effect with respect to patient - physician communication and disclosure of cancer diagnosis ; evidence suggests that , in many instances , cancer diagnoses were not communicated clearly , if at all , in the past , but over time , patient - physician communication has improved ( 30 ) . however , most validations have not examined underreporting , and reported confirmation rates from previous studies . found that positive predictive values tended to be better in studies concerning fdrs compared to second - degree relatives ( 23 ) . first , our study is that we did not verify independently the accuracy of the self - reports of a family history . another limitation is that population - based and cross - sectional data gathering has own problems . it is also reported that prevalence of fh depends on of family size , and unfortunately we did n't have any information of size of family for participants . because of randomization in sample selection , our study population is representative of total population of iran . these samples were drown - up from urban and rural areas of tehran province , capital of iran . most of the previous researches conducted on evaluation of the prevalence of a fh among fdr . in this study , we also considered the prevalence of a fh of cancer in sdr . in this research work participants were asked to complete a question on age at diagnosis of cancer in their relatives . this may be help us to estimate the at risk groups for cancer development in total population and encourage them to participate in early diagnosis and screening program for cancers . in conclusion , our findings showed that the reported prevalence of fh of ugi cancers was relatively low and varied by specific respondent characteristics such as age and sex . however , the estimates of prevalence presented here are likely to be conservative compared with actual prevalence because of self - reported data gathering . since suggested that those with fh in their fdrs at young age and those with two or more affected relatives are at high risk to develop cancer ( 10 , 30 ) , family history taking in clinical settings is useful for evaluation of the risk for healthy individuals .	aimthe present study aimed to evaluate the prevalence of positive family history of these cancers in a large population - based sample of tehran province , capital of iran.backgroundupper gastrointestinal ( ugi ) cancers ( gastric and esophagus cancer ) constitute a major health problem worldwide . a family history of cancer can increase the risk for developing cancer and recognized as one of the most important risk factors in predicting personal cancer risk.patients and methodsthis study designed as a cross - sectional survey in general population ( 2006 - 2007 ) of tehran province . totally 7,300 persons ( age > = 20 years ) sampled by random sampling on the basis of the list of postal , of whom 6,700 persons agreed to participate ( response rate 92% ) . respondents were asked if any first - degree ( fdr ) or second - degree ( sdr ) relatives had gastric or esophageal cancer.resultstotally , 6,453 respondents ( 48% male ) entered to the study . the mean age of responders with positive fh was significantly higher than those with negative fh ( p < 0.05 ) . in total , 341 respondents ( 5.3% ) reporting a history of ugi cancers in their relatives , 134(2.1% ) in fdrs , and 207(3.2% ) in sdrs.conclusionour findings showed that the reported prevalence of fh of ugi cancers was relatively low and varied by specific respondent characteristics such as age and sex . however , the estimates of prevalence presented here are likely to be conservative compared with actual prevalence because of self - reported data gathering .
cystitis glandularis ( cg ) is defined as glandular metaplasia of bladder mucosal and submucosal urothelium . small changes of cg are commonly found in specimens from the bladder wall taken from cancer and other diseases or at autopsy . in most cases , these changes are microscopic and clinically insignificant . however in some patients it may lead to a significant proliferation of epithelial cells with the formation of macroscopically visible cysts of different size ( cystitis cystica ) or changes similar to bladder cancer ( cystitis glandularis ) . it is thought that chronic irritation of the bladder mucosa ( infection , bladder stones , indwelling catheterization , bladder exstrophy ) is a major risk factor for cg . patients with advanced cg usually complain of hematuria , lower urinary tract symptoms ( luts ) , or symptoms associated with the obstruction of the upper urinary tract . cystoscopy allows giving the initial diagnosis of cg , but the final diagnosis requires pathological examination . treatment of focal lesions generally causes no major problems and usually involves removal by transurethral resection . in the case of extended lesions with recurrent hematuria and urinary tract obstruction the only solution may be to perform a cystectomy with orthotopic neobladder . in this paper we present a case of 45-year - old man with the extensive form of cg causing complete urinary retention . a 45-year - old male came to the regional urological clinic with a diagnosis of recurrent cystitis . pollakiuria , urgency ( every 20 minutes ) , nocturia ( every hour ) , feeling of incomplete emptying of the bladder , and a significant decreasing in urine flow . previously , the patient received a 7-day course of oral antibiotic therapy ( ciprofloxacin 2 x 500 mg ) with no improvement . ultrasonography showed extensive irregular thickening of the bladder walls , up to 20 mm in greatest dimension , with the presence of two cystic lesions ( 32 mm and 21 mm ) with anechoic content and a thin hyperechogenic wall ( fig . urography showed a normal course of both ureters with moderate dilation in the distal part ( fig . 2 ) . during the cystoscopy two cystic changes were found in the neck of the bladder on the anterosuperior wall . the first with a diameter of about 30 mm and the second with a diameter of about 15 mm . 3 ) . mucosa throughout the bladder was significantly swollen , hemorrhagic , and particularly irregular within the trigone of the bladder ( fig . 4 ) . during cystoscopy , biopsy of the bladder mucosa was performed . during waiting for pathological results , transurethral resection of all exophytic cystic lesions with biopsy of the bladder mucosa was performed . pathological examination of both specimens ( cyst and bladder mucosa ) showed cystitis cystica glandularis ( fig . two weeks after surgery the patient noticed significant improvement in urinary flow without feeling of incomplete emptying of the bladder . maximum urinary flow in uroflowmetry performed 6-weeks after surgery was 34.8 ml / s ( vv = 142 ml ) and residual urine volume was about 15 ml . bladder ultrasound showed persistent thickening of the walls up to 15 mm , without cystic changes . a. the lesion is well circumscribed in the edematous and inflamed vesical mucosa and contains some cystically dilated structures . b. larger magnification shows columnar lining cells devoid of mucus and goblet cells . possible risk factors are : chronic irritation of the bladder mucosa due to infection or mechanical irritation ( bladder stones , indwelling catheters , bladder exstrophy ) . it is thought that hormonal disturbances , the response to toxic substances in the urine , and deficiency of vitamins may also play a role in the development of cg . it is probably caused by lymphostasis in the pelvis and the increased concentration of proteins in the intercellular fluid of the bladder mucosa . in some patients , resulting from actions of the above - mentioned factors , von brunn 's nests appear in mucosal and submucosal layers of the bladder . they consist of foci of invaginated hyperproliferative urothelial cells , which further undergo cystic or glandular transformation . typical cg is characterized by the presence of structures covered by cubic or columnar epithelium and unchanged urothelial cells . a general characteristic for the both types is the ability to over - produce mucins , which create the mucous - filled structures . in typical cg , muc1 is commonly present in the unaltered urothelium and plays an important role in the interaction of cells . on the other hand , in intestinal cg muc5ac and muc2 muc5ac is one of the major mucin secretors of gastric mucosa , and muc2 is present in the small and large intestine . a few case reports regarding neoplastic transformation of cg into adenocarcinoma or urothelial carcinoma were published . it seems that the intestinal cg has a higher risk of malignant transformation . on the other hand , foci of cg quite often coexist with bladder cancer ( urothelial carcinoma , adenocarcinoma , squamous cell carcinoma ) and are diagnosed in tumor biopsy specimens . in most cases however , some patients complain of hematuria and luts of varying degrees like pollakiuria , urgency , and dysuria . in case of a trigonal location of the lesions , obstructive symptoms may appear . the extensive form of cg can also cause compression of the intramural ureteral segments with dilation of the upper urinary tract and development of ureterohydronephrosis and chronic renal failure . such cases often present symptoms associated with pyelonephritis resulting from a primary or secondary infection of the urinary system . most of the cg lesions , both asymptomatic and symptomatic , are detected during urinary tract ultrasound . however , due to lack of characteristic features of cg ( exophytic papillary lesions , irregular thickening of the bladder wall ) , the ultrasound only allows recommendation for further evaluation . the additional examinations include urography , which allows assessing the degree of obstruction of the upper urinary tract and secretory function of the kidneys , and ct and mri , which allow assessing wall thickening of the bladder and exclude the presence of pelvic lipomatosis . treatment is based on the elimination of possible etiological factors that cause chronic irritation of the mucosa of the bladder : long - term antibiotic therapy for urinary tract infections , treatment of bladder calculi , and intermittent catheterization in patients with neurogenic bladder . cases of endovesicular administration of hydrocortisone were described and , although it caused a reduction in symptoms , changes in the bladder persisted . the long - term oral indomethacin therapy also results in only partial improvement . in the presence of obvious exophytic lesions the method of choice is to perform transurethral resection ( tur ) of the lesions [ 6 , 8 ] . this results in a complete disappearance or significant relief of symptoms and patients usually do not require additional treatment . in patients with extensive form of cg ablation of the entire bladder mucosa using a neodymium : yag laser ( nd : yag ) was performed . however , treatment is partially effective and may be complicated by cirrhosis of the bladder . in the case of recurrent cg , which leads to obstruction of the upper urinary tract and the only one solution may be to perform cystectomy with orthotopic neobladder . there are no recommendations regarding follow - up of patients with cg in the literature . due to the low risk of malignant transformation this paper presents a case describing treatment of a patient with symptomatic cg . due to the location of the exophytic lesions ( neck of the bladder ) , the patient had significant luts , which gradually increased , and were the cause of acute urinary retention . immediately after surgery the patient noticed significant improvement in urine flow , and uroflowmetry performed 2-weeks after surgery confirmed the normal flow of urine and lack of residual volume . during the 2-month follow - up there was no relapse in the bladder neck , although irregular thickening of the wall still remained . luts , in the majority of young patients , are caused by urinary tract infection and disappear after antibiotic treatment . however cystoscopy with simultaneous biopsy helped to determine the nature of cystic lesions and plan further treatment .	cystitis glandularis ( cg ) is defined as glandular metaplasia of bladder urothelium . in most cases the course of cg is asymptomatic . however , some patients complain of hematuria and lower urinary tract symptoms ( luts ) of varying degrees . we present a case of 45-year - old man with an extensive cg causing acute urinary retention . although it was initially treated as an infection , prompt ultrasound and cystoscopy helped to establish the diagnosis . transurethral resection of the cyst with biopsy of the bladder mucosa was then performed . immediately after surgery the patient noticed significant improvement in urine passing . during the 2-month follow - up there was no relapse .
the problem of being overweight and obesity is one of the important predictors of cardiovascular diseases . the world health organization predicts that the number of overweight people will be around 2.5 billion in 2015 . nowadays , the global pandemy of obesity is also a major problem in developing countries such as iran . around 34% of iranian women thomson et al . , ( 1999 ) , presented 16 different terms for this word , including : weight dissatisfaction , body image , body image satisfaction , body satisfaction , appearance evaluation , appearance knowledge , fear of physical condition and so on . body image is the picture of our own body in our mind . actually , the way in which the body appears to ourselves is our body image . body dissatisfaction is a negative distortion of one 's body which is especially mentioned by women.[58 ] many studies have shown links between body image and health . weight is a major component which influences body image . more than 80% of women aged between 40 - 60 years are on a diet to lose or to maintain their weight . in a study in the united states 38.3% of normal - weight women thought they were overweight . most of the women are on unhealthy regimens in order to achieve an ideal weight and they feel overweight even with a normal figure . this problem can become very important when we observe that the trend towards a western lifestyle is becoming widespread in developing countries due to bombardment of media 's propaganda , satellite advertisements , movies , the internet and other events , following the phenomenon of globalization . while the issue of dieting and body satisfaction has been deeply inspected in the western world , there are only few studies in this regard in developing countries such as iran . so , this study intended to determine the relationship between perceived body satisfaction and weight control behaviors . subject : this cross - sectional study was done on 408 female employees at isfahan university and isfahan university of medical sciences during 2012 . after obtaining the numbers of women who worked in different departments , appropriate percentage of female employees were selected in a way that the sample included employees from all the various educational and administrative sections in the faculties and departments of the two universities . after the approval of the proposal by the ethics committee and coordinating with the related units , the goals of research were explained to the female personnel , satisfactions forms were given to them , questionnaires were distributed , and then the next day , the questionnaires were collected . inclusion criteria were : ( 1 ) willing to participate in the study and ( 2 ) lack of serious physical defect . this included age , marital status , education , occupation , job location , number of children , history of plastic surgery , and self - evaluation of income . body satisfaction tool was a self - administered questionnaire designed for self - assessment of women 's satisfaction with different aspect and parts of the body including weight , chest , abdominal , waist and hip circumference , height , and overall body appearance . the overall scores varied between 0 - 28 . acquiring a higher total score indicated a higher level of body satisfaction . dieting behaviors was assessed using the standard questionnaire named weight control behavior scale ( wcbs ; french , perry , leon , and fulkerson , 1995 ) . the wcbs is a measure of various weight loss behaviors ; it contains a healthy dieting behavior ( 10 items ) and an unhealthy dieting behavior ( 8 items ) subscale . considering cultural adaptation , some questions were deleted from this questionnaire . for each weight loss behavior , participants were asked to indicate how often they had used each strategy in the past six months to try to lose weight . each dieting strategy was rated on a likert scale that ranges from 0 to 3 ; 0 indicated they had never used that strategy , 1 indicated that they sometimes used the strategy , and 2 showed that they the internal correlation for healthy behavior ( low consumption of high - fat , high - calorie food , reducing snacks and exercise . ) was 0.93 and for unhealthy behavior ( use of various medications mitigating appetite , eliminate a main food ) was 0.81 . reliability of the tool : to determine the reliability of the tool , the questionnaire was completed by 30 women employees of the two universities who were later excluded from the study . the obtained internal correlation for the scales varied from 0.70 to 0.84 which was acceptable . completing the questionnaire took about 15 min , while receiving the questionnaires , their height and weight were measured . their height was obtained by taking the average of two readings of height in meters using a portable stadiometer , and their weight was recorded in the scale of kilogram and measured using a weight capacity pre - calibrated digital scale . body mass index ( bmi ) was determined by dividing weight ( kg ) by height square ( m ) . bmi category served as a classification of weight status and the following categorical values was used ( centers for disease control and prevention , 2008 ) : ( 1 ) those with bmi less than 18.5 kg / m were considered as underweight , ( 2 ) normal weight status was considered as having a bmi from 18.5 kg / m to 24.9 kg / m , ( 3 ) overweight status was defined as having a bmi from 25.0 kg / m-29.9kg / m , ( 4 ) obesity was described as having a bmi at or greater than 30.0 there was a question about the numbers of regimens during the last six months , and their willingness to do something about their weight was measured in another question . finally , the collected data was analyzed by spss version 18 using the independent t - test , one - way anova , pearson correlation coefficient , and spearman correlation analysis to determine the relationship of body satisfaction scores with dietary intake and weight control behavior in women . to determine the differences in body image score between women employees of the two universities we used t - test . one - way anova was used to distinguish body satisfaction differences between women employees with different marital status . pearson correlation coefficient was used to clarify the relationship of body satisfaction score with age and parity . spearman correlation was used to clarify the relationship of body satisfaction with educational status , occupational status and self - evaluation of income . this included age , marital status , education , occupation , job location , number of children , history of plastic surgery , and self - evaluation of income . body satisfaction tool was a self - administered questionnaire designed for self - assessment of women 's satisfaction with different aspect and parts of the body including weight , chest , abdominal , waist and hip circumference , height , and overall body appearance . the overall scores varied between 0 - 28 . acquiring a higher total score indicated a higher level of body satisfaction . dieting behaviors was assessed using the standard questionnaire named weight control behavior scale ( wcbs ; french , perry , leon , and fulkerson , 1995 ) . the wcbs is a measure of various weight loss behaviors ; it contains a healthy dieting behavior ( 10 items ) and an unhealthy dieting behavior ( 8 items ) subscale . considering cultural adaptation , some questions were deleted from this questionnaire . for each weight loss behavior , participants were asked to indicate how often they had used each strategy in the past six months to try to lose weight . each dieting strategy was rated on a likert scale that ranges from 0 to 3 ; 0 indicated they had never used that strategy , 1 indicated that they often used the strategy and 3 showed that they always did the tasks . the internal correlation for healthy behavior ( low consumption of high - fat , high - calorie food , reducing snacks and exercise . ) was 0.93 and for unhealthy behavior ( use of various medications mitigating appetite , eliminate a main food ) was 0.81 . reliability of the tool : to determine the reliability of the tool , the questionnaire was completed by 30 women employees of the two universities who were later excluded from the study . the obtained internal correlation for the scales varied from 0.70 to 0.84 which was acceptable . completing the questionnaire took about 15 min , while receiving the questionnaires , their height and weight were measured . their height was obtained by taking the average of two readings of height in meters using a portable stadiometer , and their weight was recorded in the scale of kilogram and measured using a weight capacity pre - calibrated digital scale . body mass index ( bmi ) was determined by dividing weight ( kg ) by height square ( m ) . bmi category served as a classification of weight status and the following categorical values was used ( centers for disease control and prevention , 2008 ) : ( 1 ) those with bmi less than 18.5 kg / m were considered as underweight , ( 2 ) normal weight status was considered as having a bmi from 18.5 kg / m to 24.9 kg / m , ( 3 ) overweight status was defined as having a bmi from 25.0 kg / m-29.9kg / m , ( 4 ) obesity was described as having a bmi at or greater than 30.0 there was a question about the numbers of regimens during the last six months , and their willingness to do something about their weight was measured in another question . finally , the collected data was analyzed by spss version 18 using the independent t - test , one - way anova , pearson correlation coefficient , and spearman correlation analysis to determine the relationship of body satisfaction scores with dietary intake and weight control behavior in women . to determine the differences in body image score between women employees of the two universities we used t - test . one - way anova was used to distinguish body satisfaction differences between women employees with different marital status . pearson correlation coefficient was used to clarify the relationship of body satisfaction score with age and parity . spearman correlation was used to clarify the relationship of body satisfaction with educational status , occupational status and self - evaluation of income . women had a mean age of 36.12 7.67 years and mean height of 159.67 5.68 cm . the mean bmi was 25.41 4.21 kg / m , which falls into the overweight range . 14.1% of participants were obese , 35.3% overweight , 47.6% normal weight , 3.0% underweight . in other words , around 50% of the women were obese or overweight . socio - demographic characteristics of the study population mean body satisfaction score was 54.07 20.33 . prevalence of dissatisfaction regarding different areas of the body there was an inverse correlation between body satisfaction and bmi ( r = 0.64 , p = 0.001 ) . in addition , weight satisfaction was low , 75% of the women were dissatisfied with their weight and nearly more than half of the respondents ( 60.5% ) reported a desire to lose weight . less than one tenth of the women ( 7.4% ) reported that they did not desire special attempts to lose weight ; 25.5% of the participants desired to maintain their weight and only 6.6% wanted to gain weight ; 15.2% of women had been on a diet at least for one time , 7.1% had been on a diet for two times and 6.1% had been on a diet for three times or more during the past two years ; 71.6% did not use any special diet during the past two years . pearson correlation test showed a significant inverse relationship between body satisfaction and healthy and unhealthy dieting behaviors ( r = 0.19 , p = 0.001 ) . bmi and body satisfaction were associated with their participation in healthy and unhealthy dieting behaviors . spearman correlation test showed a weakly significant relationship between body satisfaction and attempt to lose weight during the past two years ( r = 0.22 , p = 0.001 ) and a significant relationship between body satisfaction and the desire to lose weight ( r = 0.45 , p = 0.001 ) and appearance evaluation ( r = 0.57 , p = 0.001 ) . also , spearman correlation test showed significant correlation between evaluation of bodies and dieting behaviors ( r = 0 . 31 , p = 0.001 ) [ tables 3 and 4 ] . correlations among bmi , body satisfaction , healthy and unhealthy dieting behaviors correlations among body satisfaction , appearance evaluation and number of attempts to lose weight during the past 2 years the most practiced healthy dieting behavior was increased consumption of fruit and vegetables as 47.4% ( n = 194 ) reported that they had practiced this behavior either often or always . the second healthy dieting behavior was the increasing exercise 26.1% ( n = 107 ) . the prevalence of other practices were : reducing high - fat meat ( 24.4% , n = 100 ) ; decreasing sweets and junk food ( 23.2% , n = 95 ) ; eliminating snacks ( 21.5% , n = 88 ) ; decreasing the amount of food in each meal ( 18.7% , n = 77 ) consuming low - carbohydrate food ( 17.1 , n = 77 ) ; and consuming low - fat food ( 14.7 , n = 60 ) . the most practiced unhealthy behavior was skipping dinner to lose weight ; almost 8.8% , n = 36 ) reported practicing this behavior either often or always . the second most practiced unhealthy behavior was skipping breakfast ; 8.1% reported this behavior ( n = 33 ) . of all the participants , 2.7% ( n = 11 ) reported that they used medications either often or always and 1.2% ( n = 11 ) fasted for many hours to lose weight . increasing smoking to lose weight chi square test showed a significant correlation between bmi catagory with desire to lose weight and self evaluation weight ( p = 0.001 ) . more than half the women ( 51.7% ) overestimated their weight and 5.7 thought that they were obese ; 26.6% of normal weight thought they were overweight ; 41.7% of thin women thought they were about the right weight ; 67.2% obese women thought they were only a little overweight [ table 5 ] . many studies have found a similar pattern , in such a way that the presence of a significant negative association between body mass index ( bmi ) , and body dissatisfaction could be seen . also , a positive association between body dissatisfaction and behaviors such as dieting to control weight was proved.[4141822 ] in our study those with a high body weight , and low body satisfaction followed their diet more . these results were on the same lines as those of the bayyari and markey studies . our result indicated that 36.3% of normal - weight women thought they were overweight , . thin or obese individuals mostly misunderstood their weight and 67.2% of obese women imagined they were just overweight . it was revealed that even educated women misunderstood obesity or being overweight , so it 's necessary to focus on this problem by increasing health literacy . in the present study , 41.7% of thin or normal women desired to lose weight . it seems , like the western culture , the value of thinness is increasing in our society and obesity is considered as an anti - norm . our study showed that the women who evaluated their bodies as obese or overweight were more likely to engage in both healthy and unhealthy dieting behaviors than those who were relatively satisfied with their bodies . in fact , in our study , what causes women focus on their food was their imagination about their not their objective bmi group . the current study women who were more dissatisfied with their bodies , participated more in healthy or unhealthy dieting behaviors ( although in our study this correlation was weak ) . that means when these women did not like the size of the different parts of the body , they attempted to exterminate this defect by resorting to healthy and unhealthy dieting behaviors . this result was stronger in other studies such as the bayyari , markey , suka , and neumark - sztainer studies , so participants who were heavy or assumed themselves overweight or obese were less satisfied with their bodies and had a greater desire to diet and were engaged in dieting more frequently than those who were relatively thin.[14162628 ] the mean age of the women studied was younger than our study and this difference in results could be related to age difference . in the current research the women studied were middle - aged and according to the said studies , adolescents focus on their bodies more than others . also the most strategy of weight control was increasing the consumption of fruit and vegetables and the unhealthiest behavior was skipping dinner to lose weight . this result was inconsonant with anton 's study , therefore the women was more dissatisfied with their bodies used less fruit and vegetables . other studies in our society have shown women consumed less than five or more servings of fruits and vegetables . also , in jackson 's , hofmann 's study the most healthy behavior for weight control was exercising , and taking diet pills or fasting was the most unhealthy behavior . this result could be related to differences of age , group of studied people , differences of culture or the place of study . also , like other studies in our country , the prevalence of physical activity in our study was low too . on the other hand , with a higher bmi , we observed omission of a number of meals to lose weight . it can be stated that practicing unhealthy dieting behaviors was not common among this population . so the prevalence of smoking to lose weight was very low , while king , croghan and cavallo pointed to this strategy as one of the weight control behaviors among women.[3436 ] perhaps this variation can be attributed to cultural differences . it is fortunate that our society 's women did not believe in smoking as a dieting strategy . despite what there was in our study , greater prevalence of this practice was observed by tami 's study . this result may be related to differences in age and educational level in different studies . the prevalence of being overweight or obese ( bmi > 25 ) in the current study was similar to the kelishadi et al . the prevalence of obesity in adults was reported to be 20 - 25% in some countries such as the united states and it is now increasing in developing countries such as iran . therefore , the principles of a healthy diet such as consuming a diverse diet full of fruits , vegetables , functional foods and moderate amount of whole grains and low - fat dairy should be considered in any weight reduction program . the present study has been based upon self - report measures of eating patterns ; there was no independent support for the self - report data . secondly , generalization of the findings in this study may be limited to female employees , with high educational level . it is recommended that this study is done in other age group . to the best of our knowledge this is the first study to explore the relationship between body dissatisfaction and levels of dieting behavior in women employees in our society . the present study has been based upon self - report measures of eating patterns ; there was no independent support for the self - report data . secondly , generalization of the findings in this study may be limited to female employees , with high educational level . to the best of our knowledge this is the first study to explore the relationship between body dissatisfaction and levels of dieting behavior in women employees in our society . due to the importance of the prevention of obesity and the necessity of having a normal bmi in order to prevent subsequent complications , precise self - evaluation of the body is very important . so we should train our society and focus on designing and conducting public health programs , especially for women since lack of proper knowledge of normal weight will lead to unhealthy lifestyles .	background : considering the importance and prevalence of obesity and the desire to lose weight , especially among women , this study intended to investigate the relationship between body mass index , body satisfaction , and weight control behavior among women employees in isfahan.methods:an analytical cross - sectional study was conducted among 408 women who were selected by stratified random sampling method among employees at isfahan university and isfahan university of medical sciences during 2012 . the data collection tool was a multidimensional questionnaire which comprised two sections : demographic tool and body satisfaction tool ( 7 items ) , weight control behavior scale ( 18 items).results : age , marital status , educational level , and multiparity were significantly correlated with body size satisfaction . seventy - five participants were dissatisfied with their weight and 60.5% reported a desire to lose weight ; 92.15% of women studied had participated in a healthy dieting behavior and 10.8% of them had participated in an unhealthy one during the past six months . there was an inverse correlation between body satisfaction and body mass index ( bmi ) ( r = 0.64 , p = 0.001 ) . ninety percent of participants had at least one of the dieting behaviors . there was an inverse significant relationship between body satisfaction and dieting behaviors ( r = 0.19 , p = 0.001).conclusions : due to the importance of the prevention of obesity and the necessity of having a normal bmi in order to prevent subsequent complications , precise self - evaluation of body size can be used to focus on designing and conducting public health programs , especially for women .
alpha-1-antitrypsin deficiency ( aatd ) is an autosomal recessive disorder caused by several mutations in the alpha-1-antitrypsin ( aat ) gene ( serpina1 ) . the most important clinical manifestations of aatd include copd in adults and liver disorders in children as well as in adults.1 liver disease is mediated by aggregation of misfolded aat molecules to polymers that accumulate within the hepatocytes.2 this results in a reduction in the circulating levels of aat , an excess of neutrophil elastase ( the main substrate for aat ) , and therefore , pulmonary tissue destruction . the serpina1 gene is located on the long arm of chromosome 14 and is organized in three nonencoding ( ia , ib , and ic ) exons and four ( ii , iii , iv , and v ) encoding exons.3 at least 120 variants of the serpina1 gene have been identified , and these variants are codominantly inherited and closely associated with specific serum aat levels . the most common normal aat allele is the m variant , and the most frequent deficient variants in caucasian populations are z ( g / a , glu342lys ) and s ( a / t , glu264val).4,5 nevertheless , several aat alleles , other than the z and s variants , are associated with significantly reduced or absent serum aat levels . these deficient variants are called rare , with little being known about their epidemiology and even less about the associated clinical phenotypes.68 the mmalton variant is a rare deficiency variant that differs from the normal allele by deletion of the entire codon ( ttc ) for the residue phe at position 51/52 ( exon ii ) . it is associated with severe aatd and hepatic inclusions due to the formation of polymers9,10 and is characterized by a normal isoelectrophoretic pattern,11 which may , at first , be confounded with an m protein . in a retrospective review of aatd studies performed in our laboratory , a remarkably high frequency of the deficient i and mmalton alleles was observed , which together account for 54% of all the rare aat variants in spain ( 34% for i and 20% for mmalton).7 while the i variant causes moderate aatd ( 60%70% of normal level ) , the mmalton causes a deficiency similar to the z allele and is also considered the most common cause of severe aatd in sardinia ( italy)8 and tunisia,12,13 where it accounts for 60% and 35% of the rare variants , respectively . mmalton represents 8% of the rare variants in switzerland.7 the current laboratory testing for aatd involves the determination of a combination of serum aat levels , aat phenotyping by isoelectrofocusing ( ief ) , and an allele - specific genotyping assay for detecting the most prevalent deficiency alleles ( z and s).14 nevertheless , most rare variants can only be detected by molecular biology techniques , such as genome sequencing , which is not available in all routine laboratories.15 moreover , since mmalton and other rare variants are not easily recognizable , this may contribute to misclassification of many of these cases , with the subsequent underestimation of their true frequency.16 for this reason , in our laboratory , we have recently developed a rapid real - time polymerase chain reaction ( pcr ) and melting curves assay designed for the detection of the mmalton allele.17 this method detects the mmalton mutation by analysis of the melting point of one of the two adjacent fluorescently labeled probes . this last probe hybridizes over the mutation position and matches perfectly with the mutated sequence ( deletion of an entire ttc codon ) . this technology is optimal for working with small amounts of dna , such as dried blood spot ( dbs ) samples and even with residual dna present in serum samples . this technology has been included in the diagnostic algorithm of aatd used in our laboratory , as follows : aat levels are determined , and in cases with concentrations < 1.16 g / l,18 the phenotype is determined by ief . a specific range of aat levels was established for each of the major phenotypes;19 therefore , if aat values are in accordance with the phenotype observed , the laboratory results are considered definitive . if aat levels are not within the range established for their phenotype and the phenotype contains one or two m alleles , the mmalton genotype is determined by the allele - specific genotyping assay . in addition , s and z alleles are determined by the allele - specific genotyping method in order to test possible s and z variants not correctly characterized by phenotyping . if these variants are not detected , exonic sequencing of the serpina1 gene is performed.17 this method consists of amplification of the four encoding exons of the serpina1 gene by pcr followed by sanger automated sequencing . serum samples are needed for the determination of aat levels and ief , while dna extracted from whole blood samples is necessary for sequencing . currently , dna is usually obtained from dbs samples14,20 or even from serum samples ( residual dna ) . nevertheless , when there is discordance between aat levels and the ief phenotype pattern , an additional whole blood or dbs sample is usually required for exonic sequencing of the serpina1 gene . this sample requirement represents an additional venopuncture or finger puncture resulting in a significant delay in aatd diagnosis . besides , this additional sample can sometimes not be obtained for different reasons : the patient does not return to the doctor s office for the results , delay between results and new blood extraction , or disabled individuals . therefore , we have proposed a fast allele - specific genotyping assay for the detection of mmalton , the most prevalent deficient rare variant in our population . for this purpose , the same serum sample used for the determination of aat levels and phenotyping was processed . this methodology is applicable to all deficient alleles and can be adapted to other populations and their more prevalent rare variants.21 the aim of this study was to test the usefulness of this new algorithm for aatd mmalton detection used in our laboratory . this is a retrospective review of all aatd determinations performed at the clinical laboratory of hospital vall dhebron ( barcelona ) over the 2 years after this mmalton allele - specific methodology became available . we performed a retrospective revision of the results obtained in all the aatd studies carried out in our laboratory over a 2-year period . all samples were processed according to the following algorithm : aat serum levels were determined and samples with aat levels below the established cutoff ( 1.16 g / l ) were characterized by phenotyping . in case of discrepancy , the mmalton allele - specific genotyping assay was performed . all discordant aatd cases were analyzed by sequencing of the entire encoding region of the serpina1 gene . serum samples were used to complete the diagnostic algorithm except in the case of serpina1 sequencing in which whole blood samples were required . the study was approved by the ethics committee of the vall dhebron hospital ( barcelona , spain ) ; as this was a retrospective study , and all samples were anonymized , the committee did not require informed consent . the study was conducted in accordance with the principles of the declaration of helsinki , guidelines for good clinical practice , and in full conformity with relevant national regulations . aat levels were determined in serum samples by nephelometry ( siemens , bnii instrument , munich , germany ) . in order to facilitate the interpretation of the results , each laboratory has reference values for aat concentrations in serum samples obtained from a healthy population.22 phenotyping was performed using the semiautomatic hydrasys system and the hydragel 18 a1at isofocusing kit ( sebia , evry , france),23 designed for the qualitative detection and characterization of the different aat phenotypes . genotyping was performed when there was a discrepancy between aat levels and the phenotype observed . in this study , we focused on the search for mmalton variants . therefore , the candidate samples to be analyzed by the mmalton allele - specific genotyping assay were patients with a phenotype showing one or two m alleles , deficient aat serum levels , and without other deficiency alleles detectable by phenotyping . the protocol for mmalton allele - specific genotyping has been described in detail elsewhere.17 in cases in which the mmalton allele was not found , direct sequencing was performed after pcr amplification of all the encoding exons ( ii v ) using a whole blood sample . we performed a retrospective revision of the results obtained in all the aatd studies carried out in our laboratory over a 2-year period . all samples were processed according to the following algorithm : aat serum levels were determined and samples with aat levels below the established cutoff ( 1.16 g / l ) were characterized by phenotyping . in case of discrepancy , the mmalton allele - specific genotyping assay was performed . all discordant aatd cases were analyzed by sequencing of the entire encoding region of the serpina1 gene . serum samples were used to complete the diagnostic algorithm except in the case of serpina1 sequencing in which whole blood samples were required . the study was approved by the ethics committee of the vall dhebron hospital ( barcelona , spain ) ; as this was a retrospective study , and all samples were anonymized , the committee did not require informed consent . the study was conducted in accordance with the principles of the declaration of helsinki , guidelines for good clinical practice , and in full conformity with relevant national regulations . aat levels were determined in serum samples by nephelometry ( siemens , bnii instrument , munich , germany ) . in order to facilitate the interpretation of the results , each laboratory has reference values for aat concentrations in serum samples obtained from a healthy population.22 phenotyping was performed using the semiautomatic hydrasys system and the hydragel 18 a1at isofocusing kit ( sebia , evry , france),23 designed for the qualitative detection and characterization of the different aat phenotypes . genotyping was performed when there was a discrepancy between aat levels and the phenotype observed . in this study , we focused on the search for mmalton variants . therefore , the candidate samples to be analyzed by the mmalton allele - specific genotyping assay were patients with a phenotype showing one or two m alleles , deficient aat serum levels , and without other deficiency alleles detectable by phenotyping . the protocol for mmalton allele - specific genotyping has been described in detail elsewhere.17 in cases in which the mmalton allele was not found , direct sequencing was performed after pcr amplification of all the encoding exons ( ii v ) using a whole blood sample . during the 24 months from january 2014 to december 2015 , 654 subjects were screened . a total of 273 aat deficiency studies were performed in our clinical laboratory in samples with serum aat levels < 1.16 g / l ( range : 0.181.15 g / l ) following the algorithm described earlier . samples with one or two m alleles and aat levels discordant with that phenotype were analyzed using the mmalton allele - specific genotyping assay . we detected 49 samples with these characteristics ; 7.5% of the total samples and 18% of those with low aat concentrations . the rare mmalton variant was detected in a heterozygous fashion ( aat levels ranging from 0.46 eight also carried a normal m variant ( 0.60.9 g / l ) and one sample carried an s allele ( 0.46 , family screenings of previously detected m / mmalton and s / mmalton individuals were performed . in these studies , two relatives of each patient this allowed the identification of four additional mmalton variants in heterozygosity : three m / mmalton and one s / mmalton ( value range : 0.560.86 g / l ) . of the 40 remaining deficient samples with the mm and ms phenotypes and without mmalton alleles , we analyzed 24 by serpina1 exonic sequencing . the results showed 22 cases of mm without exonic mutations , with a range of serum concentrations of 0.641.02 g / l . nineteen of these 22 samples had aat serum levels close to the limit established ( range of values : 0.961.02 g / l ) . we have introduced a new algorithm of aatd diagnosis including the mmalton allele - specific genotyping assay . we determined the serum aat concentrations and phenotypes in all the samples attended in the hospital with a request for aatd diagnosis over a period of 2 years . the discrepancy between the ief phenotype and aat serum levels led to suspicion of the presence of rare alleles in several cases.24,25 the mmalton allele - specific genotyping assay was applied in samples that showed one or two m alleles and discordant aat serum levels , and we detected this rare variant in 18% of the cases . using this method , we avoided the sequencing technique , which is quite labor intensive and time consuming . little is known about the epidemiology of the non - s- and non - z - deficient aat variants , due to their low prevalence . a retrospective study of samples obtained in barcelona from 1998 to 2010 showed that among these variants , mmalton represented 20% of cases.7 it must be kept in mind that the mmalton variant is difficult to interpret without molecular studies.26 for this reason , it is important to complement the diagnostic algorithm of aatd with simple molecular biology methods such as real - time pcr melting curves . this technique is considered one of the fastest and most sensitive and reproducible methods.20,27 indeed , the extremely high sensitivity of this technology allows the analysis to be performed with the very low amount of dna recovered from dbs samples or with the residual dna that remains in standard clinical serum samples . the incorporation of the allele - specific genotyping assay for the mmalton variant in the diagnostic algorithm of aatd results in a faster and cheaper approach to diagnose this rare allele . in addition , the use of the same serum samples available for the determination of aat levels and ief avoids a new appointment by the patient and an additional venopuncture . this practice allows a faster diagnosis and avoids the significant delay in aatd diagnosis when the serpina1 gene sequencing method is needed . population studies indicate that aatd is an underdiagnosed condition , and prolonged delays in diagnosis are common.28 recommendations of the world health organization and the european respiratory society advocate screening programs for the detection of patients with aatd.29 in the spanish population , there is a high frequency of i and rare mmalton aat variants,7 mmalton being the more deficient than the i variant . in this case , the combination of this technique with the habitually used allele - specific genotyping assay for detecting the s and z variants20 in large - scale screening programs may increase the number of individuals with aatd who are actually diagnosed with this disease in spain . genotyping using real - time pcr has the advantage that a definitive diagnosis is given , and it can be adapted for automation . moreover , routine genotyping procedures may be cheaper than other methods for large screening programs . for this reason , it would be of interest to implement a simple allele - specific genotyping assay to study the most prevalent rare variants in each region . this could be done with the i variant in ireland , in which this allele accounts for 90% of the total of rare variants,30 or in finland , where the mmalton variant has not been found , but the f allele is the most prevalent rare variant in the finnish population.31,32 other cases are certain regions of italy , where mmalton and mprocida are more prevalent than z.8 bornhorst et al33 reported the m / i phenotype to be associated with aat levels ranging from 0.80 however , the f variant presents aat levels similar to the m variant but has reduced functional ability to inhibit neutrophil elastase compared to the normal allele.34 in our case , we found the mmalton variant in combination with the m allele with aat levels ranging from 0.6 most of the m / mmalton individuals described have normal pulmonary functions , while z / mmalton patients often have a high risk of developing emphysema , especially smokers.35,36 mprocida is another rare deficiency variant with ief mobility similar to the m allele , which has been reported to produce 3%4% of the normal amount of aat.37,38 in this study , we have described a new diagnostic algorithm that has been used for 24 months with the samples received for aat study . this new algorithm was computerized and provided definitive results even using serum samples . in this sense , it should be kept in mind that serum samples are used for routine determination of aat levels and phenotyping . therefore , the use of these samples for specific genotyping avoids the significant delay in diagnosis when a new whole blood sample is required for further molecular characterizations . a new appointment with the patient requires weeks or even months , while the use of the same serum sample significantly accelerates the routine diagnostic process . in fact , a significant number of patients did not provide a second sample for analysis in our study . the detection of mmalton variants is relevant because patients with homozygote mmalton or z / mmalton genotypes with evidence of emphysema are candidates to receive augmentation therapy . even some cases of s / mmalton or in combination with other deficient alleles that show serum aat levels < 11 m would fulfill the internationally accepted criteria for initiation of this therapy.29 the incorporation of the mmalton allele - specific genotyping assay in the diagnostic algorithm of aatd could allow the clinical characterization of mmalton individuals who , to date , are not well characterized . our results show the strong need to develop standardized algorithms to obtain conclusive data of the real incidence of rare aat variants in each country or region .	background and objectivesalpha-1-antitrypsin deficiency ( aatd ) is associated with a high risk for the development of early - onset emphysema and liver disease . a large majority of subjects with severe aatd carry the zz genotype , which can be easily detected . another rare pathologic variant , the mmalton allele , causes a deficiency similar to that of the z variant , but it is not easily recognizable and its detection seems to be underestimated . therefore , we have included a rapid allele - specific genotyping assay for the detection of the mmalton variant in the diagnostic algorithm of aatd used in our laboratory . the objective of this study was to test the usefulness of this new algorithm for mmalton detection.materials and methodswe performed a retrospective revision of all aatd determinations carried out in our laboratory over 2 years using the new diagnostic algorithm . samples with a phenotype showing one or two m alleles and aat levels discordant with that phenotype were analyzed using the mmalton allele - specific genotyping assay.resultswe detected 49 samples with discordant aat levels ; 44 had the mm and five the ms phenotype . in nine of these samples , a single rare mmalton variant was detected . during the study period , two family screenings were performed and four additional mmalton variants were identified.conclusionthe incorporation of the mmalton allele - specific genotyping assay in the diagnostic algorithm of aatd resulted in a faster and cheaper method to detect this allele and avoided a significant delay in diagnosis when a sequencing assay was required . this methodology can be adapted to other rare variants . standardized algorithms are required to obtain conclusive data of the real incidence of rare aat alleles in each region .
cutaneous metastasis of lung tumours are rare , occurring in 0 - 4% of cases ( 1 ) . they indicate a very poor prognosis ; mean survival has been reported as 5 - 6 months ( 2 ) . metastases to the digits may initially arise in bone , before enlarging locally to invade skin , or arise in skin first ( 3 ) . we describe the case of a non - small cell lung cancer presenting as metastasis in the great toe , demonstrating how advanced the disease may be at the time of presentation and how metastases may be mis - diagnosed . a 62-year - old male was admitted for investigations following a 6-week history of increasing shortness of breath , weight loss , and swellings of the right great toe and right elbow . his toe had felt hot for 6 weeks , and in the latter 2 weeks had discharged pus , become itchy and developed a burning sensation before becoming swollen . he had developed a productive cough , a reduced exercise tolerance and had recently given up smoking . the patient s medical history included chronic obstructive pulmonary disease , idiopathic pulmonary fibrosis and cerebrovascular disease . he was on appropriate medication including aspirin , his alcohol intake was minimal and he was a retired plasterer . examination showed a swollen , foul smelling raw area at the distal phalanx of right great toe with overlying slough . there was erythema and pain initially thought to be consistent with infection from an ingrowing toenail ( fig . a firm , 3 cm wide , rubber - like swelling was noted at the lateral aspect of the right elbow that was not tethered to skin , but was fixed to underlying structures . fine crackles were heard at the right lung base and there was an enlarged right supraclavicular lymph node and associated digital clubbing . 2 ) showed destruction of the distal phalanx with adjacent soft tissue swelling , and of the right elbow showed soft tissue swelling over the lateral aspect with possible cortical destruction of the lateral epicondyle ( fig . 3 ) . radiograph of right great toe demonstrating osteolysis of distal phalanx with adjacent soft tissue swelling but no periosteal reaction . a ct scan showed an 8.8 cm soft tissue mass occluding the right lower lobe bronchus ( fig . 4 ) , bilateral nodules suspicious of pulmonary metastases , and lymphadenopathy in the right hilar , paratracheal and anterior mediastinal regions . in addition there were foci of reduced attenuation in the liver and thoracic skeletal metastasis with imminent cord compression at t4 . biopsy of this provided a histological diagnosis of squamous cell carcinoma . punch biopsies of the ulcerated great toe swelling also showed squamous cell carcinoma . the presumed metastasis at the right elbow was not confirmed histologically as it was asymptomatic and the focus of treatment was elsewhere . a staging of t4n3m1b ( stage iv ) non - small cell lung cancer was made following multidisciplinary discussion . the patient was referred to plastic surgeons for palliative amputation of the toe , which was undertaken 4 weeks following histological diagnosis . the patient reported an improvement in pain at the amputation site at first follow - up 1 week post - operatively . due to a performance status of who grade 3 the patient was not a candidate for palliative chemotherapy , but five fractions of palliative radiotherapy were administered urgently to the t4 spinal metastasis , which allowed the patient to remain ambulant until his death 2 months following histological diagnosis . this survival time was shorter than the previously reported 5 - 6 months from detection of cutaneous metastases ; some patients in the quoted studies had received chemotherapy(2 ) . lung cancer is known to metastasize to all organ systems of the body ( 4 ) , though skin is only the 12 most common site in non - small cell tumours ( 1 ) . a review of 579 cases of lung cancer elicited cutaneous metastases in 16 ( 2.8% ) of which all had accompanying metastases in other organs ( 5 ) . in the latter study , a median survival of 4 months was observed following development of cutaneous metastases . as demonstrated well by this case , 20 - 60% of cutaneous metastases present before or at the same time as the primary lung tumour ( 2 ) . metastasis to the digits is thought to mainly arise in the bone first , before enlarging locally to invade the skin but it can occur in skin first ( 3 ) . the presentation is variable , but swelling , erythema , pain and fluctuance may be considered typical . hence cutaneous metastases from many primary tumours are often confused with infection , inflammation , cysts , osteomyelitis and gout among other conditions ( 6 ) , thereby delaying diagnosis . it is not clear whether the metastasis in this patient s toe arose in the skin , bone , or other tissue . the histology report from the amputation stated that the tumour invades bone , implying that it s tissue of origin was outside the bone . a series of 133 digital metastases from various types of primary tumour showed only 8% occurred as solitary metastases in the great toe , compared to 86% in the fingers ( 3 ) . the rarity of digital metastasis is thought to be due to the relative absence of red marrow in the digital bones . the mechanism by which lung cancer metastasises to distal peripheries is thought to be via extension through the valves of the vertebral venous system , to lumbar spinal and iliofemoral veins in which incompetent valves allow distal passage of emboli , aided by gravity ( 3 ) . interestingly , the tumour in this case seemed to almost exclusively involve the right side of the patient s body , which given the above proposed mechanism , may not be entirely due to chance . it has also been suggested that chemotactic factors released following trauma may contribute to migration of tumour emboli and adherence to injured tissue ( 7 ) , and the great toe is a frequently injured site . this case highlights how advanced lung cancer may be at the time of presentation , with multiple widespread distant metastases presenting in a narrow time period . cutaneous metastases and those in the digits indicate a very poor prognosis , with palliative treatment frequently being the only option . a diagnosis of metastatic disease should be considered in patients with risk factors for , or known cancer , particularly when microbiological anaylsis and antibiotic treatment for presumed infection has failed to improve symptoms . prompt diagnosis and management of these metastases can dramatically improve a patient s quality of life and should be strived for .	a 62-year - old man presented with a 6-week history of shortness of breath , weight loss and painful swelling of the great toe . examination elicited an ulcerated swelling of the right great toe , thought to be an ingrowing toenail . chest crackles and an enlarged supraclavicular lymph node were detected . biopsies of the toe swelling and bronchi both showed squamous cell carcinoma , confirmed radiologically as stage iv lung cancer . the patient was referred for palliative amputation of the toe . cancer metastases to the digits may arise in bone , before enlarging to invade skin , or arise in skin first . 20 - 60% of cutaneous metastases present before or at the same time as the primary lung tumour . cutaneous metastases and those in the digits indicate very poor prognosis . their prompt diagnosis and management can dramatically improve a patient s quality of life and should be strived for .
the crystal structures of the sh2 and sh3 domains of cd2 were obtained from the pdb database ( i d : 1hnf ) , and the transmembrane helical part of cd2 was built using the vmd package . hence , we simulated residues 25235 of cd2 . for numbering of the residues , we used the uniprot scheme . however , because all n - linked glycans share the common pentasaccharide core ( glcnac2man3 ; see figure s1 ) , this primary sugar core is here attached to the n - terminus of residues 89 , 141 , and 150 ( figure s9 ) using the protocol described elsewhere . the present study shows that even this primary sugar core that is an appropriate choice for simulation purposes to demonstrate the effects of glycosylation has a major influence on cd2 orientation and presentation . the effects of glycosylation can be expected to strengthen with longer and more specific glycans . the multicomponent lo membrane used in our simulations was comprised of 35.3 mol % sm ( sm d16:1/16:0 ) , 43.0 mol % dopc , and 28.7 mol % chol . as a control system in the ld phase , we used a single - component dopc bilayer . the number of lipids ranged between 508 and 982 , and the number of water molecules was between 85 000 and 220 000 . the refined opls all - atom force field was used for sm and dopc , and the standard opls all - atom force field was employed for the protein and the sugars together with the opls - compatible tip3p water model . rahman barostat were used to maintain the temperature and pressure at 310 k and 1 atm . a semi - isotropic pressure coupling with a compressibility of 4.5 10 bar was used in the npt ensemble . long - range electrostatic interactions were incorporated through the particle mesh ewald ( pme ) method with a cutoff of 1 nm ( between real and reciprocal space descriptions ) , and the same cutoff was also used for lennard - jones interactions . for each of the systems , we carried out three independent 500 ns simulations . the first 300 ns of the 500 ns trajectories was considered as an equilibration period based on convergence of the ecd orientation distributions ( see the si ) , and the last 200 ns was used for analysis unless mentioned otherwise . the results of the three independent simulations were used as the basis for error analysis . the results of the three simulations , for each of the systems considered , were found to be consistent with each other .	proteins embedded in the plasma membrane mediate interactions with the cell environment and play decisive roles in many signaling events . for cell cell recognition molecules , it is highly likely that their structures and behavior have been optimized in ways that overcome the limitations of membrane tethering . in particular , the ligand binding regions of these proteins likely need to be maximally exposed . here we show by means of atomistic simulations of membrane - bound cd2 , a small cell adhesion receptor expressed by human t - cells and natural killer cells , that the presentation of its ectodomain is highly dependent on membrane lipids and receptor glycosylation acting in apparent unison . detailed analysis shows that the underlying mechanism is based on electrostatic interactions complemented by steric interactions between glycans in the protein and the membrane surface . the findings are significant for understanding the factors that render membrane receptors accessible for binding and signaling .
chronic granulomatous disease is a hereditarily determined illness , characterized by inability of the body 's phagocytic cells to destroy certain microorganisms . as a consequence , patients with cgd have an increased susceptibility to infections caused by certain bacteria and fungi ( frequently staphylococcus aureus , and aspergillus species , respectively ) . this insufficiency in microbial killing is usually caused by a defect in the nicotinamide adenine dinucleotide phosphate ( nadph ) oxidase enzyme complex . nadph is the enzyme that generates the microbicidal respiratory burst [ 2 , 3 ] . the deficiencies of oxidative metabolism are usually detected with either the nitroblue tetrazolium ( nbt ) dye test or the dihydrorhodamine ( dhr ) oxidation test . these tests are based on the ability of products of oxidative metabolism to alter the oxidation states of reporter molecules so that they can be detected microscopically ( in the case of nbt ) or by flow cytometry ( in the case of dhr ) . however , nbt test , although more conventionally used , is sometimes not sufficiently sensitive . therefore , many investigators have preferred the use of the dhr test . in cgd , phagocytes manage to ingest bacteria normally ; however they can not destroy them . patients usually have an elevated incidence of mucosal inflammatory disorders such as colitis , enteritis , and gastric outlet obstruction . cgd is an extremely rare disease that occurs in about 1 in every quarter of a million live births . a libyan boy first presented to the pediatrics department of tripoli medical centre ( libya ) when he was 3 months old suffering from loose stool with streaks of blood . he also had frequent episodes of fever at first presentation to the hospital ; later , he developed persistent cervical lymphadenitis and failed to gain weight . the boy is the 3rd child ( of 4 children ) from related libyan parents . he was born in the 35th week of gestation with a birth weight of 2400 grams . the first child of the family ( a girl ) had a similar medical history , with persisting pneumonia , suspected tuberculosis , and anemia . however , his younger sister was found to be a carrier of the disease ; but otherwise healthy and has no significant susceptibility to infections up to the present date . the patient presented to the university children 's hospital of bonn ( germany ) at the age of 10 months ; and because of a positive tuberculin skin test and previous history of bcg vaccination , an infection with bcg was suspected . the patient was later transferred to charite children 's hospital of berlin to continue his treatment , and further investigations were carried out . a lymph node of the left axilla was surgically removed , and a culture from the biopsy grew bacteria . these findings have lead to a suspicion of a primary immune deficiency disorder . at the age of 12 months , he again showed recurrent episodes of pneumonia , diarrhea and enlargement of lymph nodes . a few months later , following an immunological profile for his case , a negative nbt and dhr assay was detected . the final diagnosis of chronic granulomatous disease was confirmed by molecular analysis , which revealed a defect in the p22-phox component of the cyba gene . the results of his investigations were as follows : stool analysis revealed the presence of clostridium difficile ; however , no parasites were found . the excised cervical lymph nodes ( ln ) yielded the growth of enterococcus faecalis and klebsiella pneumoniae on culture . there was no growth of mycobacteria on culture of fluids from the bronchial lavage performed . gastroduodenoscopy and coloscopy showed redness and swelling of the mucous membrane of the stomach and the colon , respectively . serum antibody levels were as follows : igg 1697 mg / dl , iga 161 mg / dl , igm 100 mg / dl . indicating that humoral immunity of the patient was functioning normally . normal values were detected for sodium , potassium , calcium , chloride , creatinine , transaminases , ldh and alkaline phosphatase . ultrasound imaging ( usi ) showed enlarged cervical ln ( maximum diameter 22 mm ) , and slightly enlarged mesenteric ln ( 11 mm ) . furthermore , the liver was moderately enlarged ( figure 1 ) . ultrasound image showing a mild hepatomegaly . x - rays of the chest showed that the left diaphragm was lower than the right , and the left lung was smaller than the right , with central infiltrates on both sides . a high - resolution computed tomography ( ct ) scan of the chest confirmed the x - ray readings and showed hypoplastic left lung with diffuse dystelectasis ; however , no other specific infiltrates were found ( figure 2 ) . high - resolution ct - scan showing a left hypoplastic lung . magnetic resonance imaging ( mri ) of the chest confirmed the usi findings and revealed multiple enlarged cervical ln , and one large mediastinal ln on the right side in addition to a nonspecific mass situated on the right paravertebral area at the level of chest vertebral body number 11 and 12 . positron emission tomography ( pet ) showed focally intense signals of the upper lobe of the right lung ( paravertebrally ) and in the glandula submandibularis ( cervical ln ) , and a less intense signal in the inferior lobe of the left lung ( figure 3 ) . pet image showing intense signals on the upper lobe of the right lung ( yellow arrow ) . pet - ct fusion technique which allows accurate correlation of great anatomic details ( from the ct scan ) and informative metabolic information ( from the pet scan ) was used , and it confirmed the ct scan findings ( figure 4 ) . genetic analysis of the patient 's nadph oxidase revealed a deletion of 7 base pairs ( bp ) in exon 5 on both alleles of the p22-phox gene . the bp deleted were from 323 to 329 ( the investigation was done in dresden , germany ) . pet - ct fusion scan showing the location of the focally intense signals from the upper lobe of the right lung paravertebrally , and the less intense signals from the left lung , indicating a hypoplastic left lung . , it is estimated that cgd affects approximately one infant per quarter of a million live births [ 6 , 8 ] . worldwide , however , the incidence of the disease varies among the populations studied , with variations from 1 case per 160 thousands individuals to 1 case per 1 million people . surprisingly , for libya , and after a thorough literature search , we confirm that this is the first reported case of cgd . four genes have been connected to cgddthe cybb gene , encoding the gp91-phox subunit ; the cyba gene , encoding for p22-phox ; the ncf1 gene , encoding p47-phox ; and the ncf2 gene which encodes for p67-phox . cgd is frequently inherited ( 60% of cases ) in an x - linked recessive manner where most patients are boys , who have hemizygous mutations on the x - linked gene coding for gp91-phox . however , among the other subtypes of cgd , the autosomal recessive forms ( 40% of cases ) may be associated with milder disease . the extent to which environmental factors and secondary genetic defects may influence the course of the disease a wide variety of molecular defects have been described in the genes for the gp91-phox component , the p22-phox component , and the p67-phox component . these defects include frame shifts ; deletions ; and nonsense , missense , spliceregion , and regulatory region mutations . the protien , p40-phox , has been involved in the regulation of the nadph oxidase , but no individual with a mutation in this protein has been found so far . a new variant of cgd has been described ; this form is caused by an inhibitory mutation in rac2 , which regulates activity of the neutrophil respiratory burst and actin assembly . mutations in the genes for p67-phox ( ncf2 ) and p22-phox ( cyba ) are usually the rarest , accounting for fewer than 10% of cases of cgd . morbidity due to infections remains significant , particularly in those with the x - linked type . currently , the yearly mortality rate is a little more than 1.5% per year for persons with autosomal recessive cgd and over 5% for those with x - linked cgd the long - term survival rates of patients who present with symptoms after the end of their first year of age is significantly better than that of patients whose illness starts in infancy . fortunately , since the introduction of prophylactic antibacterials and anti - fungals the prognosis for patients with cgd has greatly improved , with patients frequently living to see their 30th and 40th birthdays [ 12 , 13 ] . in our case and because of the detection of clostridium difficile in stool and enterococcus faecalis and klebsiella pneumoniae in the excised cervical lymph node the patient 's condition has improved greatly and the degree of inflammation ( as monitored by the level of crp and leukocyte count ) has decreased . although generally , the patient became better , nevertheless , fresh blood spots were still noticeable in stools . because nonspecific colitis was assumed to be associated with his condition , he was treated with oral prednisone at an initial dose of 1 mg / kg body weight ( bw ) however , the two anal fissures which were found on physical examination , might explain the fresh blood streaks of the stool in some of the instances . while in germany , the patient was vaccinated with pneumococcus vaccine and with the measles , mumps and rubella ( mmr ) a few months later . therefore , with the previous vaccines he took ( in libya ) his vaccination status is now considered complete . in view of the fact that patients with cgd usually have recurrent bactarial and fungal infections , which may lead to abscess formation in the skin , lymph nodes and viscera , our patient was put on a lifelong antibiotic ( cotrimoxazole , 5 mg tmp / kg bw ) and antimycotic ( itraconazole 10mg / kg bw ) prophylaxis plan . the last respiratory system examination revealed a clear chest with normally breathing lungs devoid of infections . his parents are pleased with the outcome of the treatment so far , and are constantly monitoring his condition with regular follow - up visits to the hospital . alternative methods of treating cgd include interferon - gamma injections , white blood cells ( wbc ) infusions , and bone marrow transfusion ( bmt ) . gammainterferon , although widely used in the usa , in europe it is usually preserved for the difficult and persistent infections . the route of administration ( an injection given twice weekly ) is also a drawback . transfusion of wbc from healthy donors is sometimes used but not preferred for its obvious side effects in the form of transfusion reactions that can occur especially after repeated transfusions . gene therapy is also an option for cgd cases , where patients own stem cells are removed and the defective genes replaced with normal genes before reinjecting them back to the patient . by presenting this typical case of cgd , the authors advise that primary immune deficiency diseases , particularly cgd , should be considered in cases of chronic unresponsive infections , especially in children . after confirming the diagnosis , all cases of cgd should be put on a life - long aggressive antibiotic and antifungal prophylaxis treatment ; and for infections , special antibiotic and antifungal therapy regimes should be started for periods up to 3 times longer than that for patients with other similar conditions . early detection and treatment of such congenital disorders will certainly decrease the many complications that may arise in the course of the disease . furthermore , cgd patients should have frequent and regular consultations with their health care providers , and have access to cgd resources for information in the form of booklets and websites dedicated to the disease .	chronic granulomatous disease ( cgd ) is a primary immune deficiency disorder of the phagocytes . in this disorder , phagocytic cells ( polymorphonuclear leukocytes and monocytes ) can not produce active oxygen metabolites and , therefore , can not destroy the ingested intracellular bacteria . clinically , patients with cgd usually have recurrent bacterial and fungal infections causing abscess and granuloma formation in the skin , lymph nodes and visceral organs.in this report , we present a boy from libya with a rare autosomal recessive trait of cgd ( defect of p22-phox ) who has chronic lung disease following multiple severe pneumonia attacks . the case we present suffered from bloody diarrhea since the third month of his life . he also had recurrent episodes of fever , and later , developed persistent cervical lymphadenitis and failure to gain weight . cgd is a very rare condition worldwide . it is also not recognized here in libya , and usually not in the list of differential diagnosis for chronic pulmonary infections . we advise that pediatricians and general practitioners who treat chronic cases of lung diseases ( with or without chronic diarrhea ) should consider primary immunodeficiency disorders in the hope that early diagnosis and treatment may prevent chronic complications especially of the respiratory tract . furthermore , we state that , to the best of our knowledge , this is the first documented case of cgd from libya .
gestational diabetes mellitus ( gdm ) , defined as glucose intolerance with onset during pregnancy , is a common disease affecting approximately 6% to 7% of pregnant women . pregnancies complicated by gdm have maternal and fetal implications , including increased preeclampsia , rate of operative delivery , and subsequent diabetes mellitus as maternal complications . fetal risks include macrosomia , shoulder dystocia , other birth traumas , neonatal hypoglycemia , and long - term sequelae such as obesity and impaired intellectual achievement . to prevent these adverse outcomes , optimal screening and diagnostic criteria must be adequate , timely , and efficient . most clinicians have used the 2-step method that was introduced in 1964 by o'sullivan and mahan for gdm screening and diagnosis . the 2-step method remains the recommendation of the american congress of obstetricians and gynecologists ( acog ) . a 1-step method , introduced by the hyperglycemia and adverse pregnancy outcomes study in 2008 , however , has become an alternative option for gdm screening and diagnosis . the 1-step method allows earlier gdm diagnosis and treatment , thereby decreasing the risks associated with the disease . despite an association with adverse outcomes , optimal screening and diagnostic criteria for gdm , including both the 1- and 2-step methods , remain controversial . furthermore , both approaches , although validated by extensive research and endorsed by experts , are extremely demanding . the oral glucose tolerance test ( gtt ) , whether it is 75 or 100 g , is a costly and cumbersome test , both for patients and clinicians . we identified several risk factors associated with adverse outcomes , and using those risk factors , we developed a screening tool for predicting adverse outcomes of gdm . because the gdm diagnostic tests are imperfect , our model suggests an alternative approach , which is more practical , efficient , and patient- and clinician - friendly . this is a retrospective cohort study conducted via medical record review of patients admitted between march 2001 and april 2013 at the severance hospital , seoul , south korea . the institutional review board of the yonsei university health system approved the protocol of this study ( project no : 420141031 ) . exclusion criteria were preexisting type 1 or type 2 diabetes and diagnosis with gdm at < 24 weeks gestation . multiple gestations , anomalous fetuses , and patients with chronic hypertension were also excluded . in our institution , we have used the 2-step method following the acog recommendation . gestational diabetes mellitus was defined as 2 or more of the 4 values ( fasting , 1 hour , 2 hours , or 3 hours ) being abnormal on the 100-g , 3-hour gtt in a patient with an abnormal 50-g , 1-hour glucose challenge test ( gct ) , for which fasting is not required . abnormal values for the 100-g , 3-hour gtt were defined as fasting blood glucose ( fbg ) 95 , 1-hour blood glucose 180 , 2-hour blood glucose 155 , and 3-hour blood glucose 140 mg / dl . an abnormal value for the 50-g , 1-hour gct was blood glucose 140 mg / dl . adverse outcomes included neonatal hypoglycemia , hyperbilirubinemia , and hyperinsulinemia ; admission to the neonatal intensive care unit ; large for gestational age , gestational insulin therapy ; and preeclampsia or gestational hypertension . hypoglycemia was defined as blood glucose < 40 mg / dl by using the heel stick within 2 hours of birth and before the first nonbreast - feeding , and hyperbilirubinemia was defined as a bilirubin level > 5 mg / dl . hyperinsulinemia was defined as an insulin level > 10.7 u / ml . large for gestational age was defined as birth weight above the 90th percentile compared with gestational age based on sex - specific and race - specific norms . preeclampsia was diagnosed according to the criteria of the acog practice bulletin : new onset of blood pressure 140/90 mm hg on 2 separate readings taken 6 hours apart after 20 gestational weeks and proteinuria 300 mg/24 hours . data are reported as the mean ( sd ) for continuous variables . for univariate analysis , the t test was used to compare continuous variables . for multivariate analysis , we used multivariate models of logistic regression that included all risk factors that were significantly associated in the univariate analysis . we developed a model using multiple logistic regression including body mass index ( bmi ) , calculated as weight ( kg)/height squared ( m ) , and fbg . we evaluated diagnostic abilities including not only accuracy , sensitivity , and specificity but also area under the curve ( auc ) . to compare the performance of our model with the 2-step method , we selected as a cutoff value , the point on the receiver operating characteristic curve closest to the upper left corner . , chicago , il ) was used for statistical analyses , all reported p values were 2-tailed , and a p value < 0.05 was considered statistically significant . between march 2001 and april 2013 , among 3434 women who came to the hospital , 802 women had an abnormal 50-g , 1-hour gct , of whom 306 were diagnosed with gdm and 496 were found to not have gdm ( the false - positive group ; fig . in contrast , 240 women ( 48.4% ) in the false - positive group had an adverse outcome ( table 1 ) . in the false - positive group , women with adverse outcomes had significantly higher bmi at entry and fbg in both univariate and multivariate analyses ( table 2 ) . in the gdm group , women with adverse outcomes had significantly higher glucose levels after gct and gtt only in the univariate analysis ( table 3 ) . gct = glucose challenge test , gdm = gestational diabetes , gtt = glucose tolerance test . number of patients with or without adverse outcomes false - positive group with or without adverse outcomes gestational diabetes mellitus group with or without adverse outcomes table 4 describes our logistic regression model derived from 2 variables , bmi at entry and fbg , which were significantly different in women in the false - positive group with adverse outcomes . our model was well calibrated ( hosmer lemeshow goodness - of - fit test , p = 0.27 ) . the conventional 2-step method predicted gdm adverse outcomes with an auc of 0.610 , accuracy of 59.1% , sensitivity of 47.6% , and specificity of 74.4% . in contrast , our model predicted gdm adverse outcomes with an auc of 0.642 , accuracy of 61.3% , sensitivity of 57.2% , and specificity of 66.9% ( table 5 ) . figure 2 shows the receiver operating characteristic curves of the conventional 2-step method and our logistic regression model for predicting gdm adverse outcomes . odds ratios for predicting gestational diabetes mellitus adverse outcomes using the multiple logistic regression comparison of the performance of the conventional 2-step method and logistic regression in predicting gestational diabetes mellitus adverse outcomes receiver operating characteristic curves of logistic regression and conventional 2-step method for predicting gestational diabetes mellitus adverse outcomes . gdm = gestational diabetes mellitus , lr = logistic regression , roc = receiver operating characteristics . our logistic regression model performed better in predicting gdm adverse outcomes than the conventional 2-step method using only bmi at entry and fbg . our model is cost - effective , patient - friendly , and convenient because these 2 variables are simple to measure and require only 1 venipuncture . the gtt in the conventional 2-step method requires 4 venipunctures and a wait period of 3 hours . with our model moreover , our model compensates for the drawbacks of the 2-step method and corresponds to the strengths of the 1-step method . the reasoning behind the shift from the 2 step to the 1 step is the ability of the 1-step method to allow earlier gdm diagnosis and treatment , which results in decreased risk associated with the disease , and its ease of administration , convenience for patients and clinicians , and its diagnostic accuracy . use of the 1-step method began after release of the hyperglycemia and adverse pregnancy outcomes study findings in 2008 , and is recommended in the 2010 recommendations of the international association of diabetes and pregnancy study group and by the american diabetes association in 2011 . our model is more convenient than the 1-step method because it avoids the 1- and 2-hour postload measures and there is no need to wait at the hospital . moreover , because there is no gct step , gdm diagnosis and treatment can be as fast as the 1-step method . higher bmi at entry and fbg were selected as important risk factors for adverse outcomes . torloni et al performed a systematic review of the literature and a meta - analysis and found out that the maternal prepregnancy bmi is directly associated with the risk of developing gdm . insulin resistance seems to play a central role among changes in maternal metabolism caused by obesity . there is a report that pregnancy further exacerbated defects of insulin receptors and postreceptors associated with obesity . in addition to insulin resistance , inflammation also might be related to the mechanism of obesity in relation to gdm . although the pathogenesis of gdm is not clearly understood , several studies found higher levels of serum c - reactive protein , interleukin-6 , and ferritin in gdm , suggesting that gdm is associated with systemic inflammation . obesity is usually accompanied by inflammation because of the secretion of proinflammatory cytokines by adipocytes . thus , women with higher bmi have abundant adipocytes that produce excessive proinflammatory cytokines and might lead to the development of gdm . owing to the probable pathogenesis of obesity in association with gdm as mentioned earlier , bmi at entry was selected as a significant factor for predicting gdm adverse outcomes . another significant factor for predicting gdm adverse outcomes was elevated fbg , which indicates insulin resistance and impaired insulin secretion . an elevated fbg suggests an underlying pathology of gestational diabetes and does not change throughout gestation , which offers another advantage in its use . during the years , fbg has been widely used as a screening test for gdm , because of its advantages of being less expensive , reproducible , and universally easily administered . most studies have focused on investigating the optimal factors and its values in predicting gdm . the main strength of our study is its focus on investigating performance for predicting gdm adverse outcomes and suggesting a more efficient and effective prediction model . first , because our institution does not perform the 1-step method , we could not compare the performance of that method with our model . second , our model might have been fit to the study population ; therefore , future studies in other populations are needed to verify this model . our logistic regression model performed better than the conventional 2-step method in predicting gdm adverse outcomes . furthermore , using only bmi at entry and fbg in our model , we attained a practical , inexpensive , efficient , more reproducible , easier , and patient- and clinician - friendly approach . further studies should be targeted to evaluating our model in other very high - risk populations , including overweight , obese , african american , and hispanic women .	abstractgestational diabetes mellitus ( gdm ) is a common disease in pregnancy causing maternal and fetal complications . to prevent these adverse outcomes , optimal screening and diagnostic criteria must be adequate , timely , and efficient . this study suggests a novel approach that is practical , efficient , and patient- and clinician - friendly in predicting adverse outcomes of gdm . the authors conducted a retrospective cohort study via medical record review of patients admitted between march 2001 and april 2013 at the severance hospital , seoul , south korea . patients diagnosed by a conventional 2-step method were evaluated according to the presence of adverse outcomes ( neonatal hypoglycemia , hyperbilirubinemia , and hyperinsulinemia ; admission to the neonatal intensive care unit ; large for gestational age ; gestational insulin therapy ; and gestational hypertension ) . of 802 women who had an abnormal 50-g , 1-hour glucose challenge test , 306 were diagnosed with gdm and 496 did not have gdm ( false - positive group ) . in the gdm group , 218 women ( 71.2% ) had adverse outcomes . in contrast , 240 women ( 48.4% ) in the false - positive group had adverse outcomes . women with adverse outcomes had a significantly higher body mass index ( bmi ) at entry ( p = 0.03 ) and fasting blood glucose ( fbg ) ( p = 0.03 ) . our logistic regression model derived from 2 variables , bmi at entry and fbg , predicted gdm adverse outcome with an area under the curve of 0.642 , accuracy of 61.3% , sensitivity of 57.2% , and specificity of 66.9% compared with the conventional 2-step method with an area under the curve of 0.610 , accuracy of 59.1% , sensitivity of 47.6% , and specificity of 74.4% . our model performed better in predicting gdm adverse outcomes than the conventional 2-step method using only bmi at entry and fbg . moreover , our model represents a practical , inexpensive , efficient , reproducible , easy , and patient- and clinician - friendly approach .
several reports have been published on late - acquired incomplete stent apposition ( isa ) after drug - eluting stent ( des ) implantation , particularly sirolimus - eluting stent ( ses).1 ) most incidences of late - acquired isa induced by positive vascular remodeling were of the focal type and occurred in a single vessel . however , multivessel isa with extensive forms have not been reported as yet . in the present case study , extensive late - acquired isa occurred throughout two vessels , namely the left anterior descending artery ( lad ) and the right coronary artery ( rca ) . a 45-year - old male patient was admitted with effortinduced chest pain for a month . the electrocardiogram was sinus bradycardia without any ischemic changes such as changes in st - t . however there was significant st segment depression at bruce protocol stage 1 during the treadmill test . a multidetector computed tomogram revealed total occlusion with positive vascular remodeling at proximal lad and middle rca . the cag revealed a total occlusion in distal rca and significant stenosis in mid rca and proximal to mid lad ( fig . the patient underwent percutaneous coronary intervention ( pci ) at the mid to distal rca with a 3.0 by 33 mm cypher ( cordis , miami lakes , fl , usa ) with 20 atmospheres { minimal lumen diameter ( mld ) 2.72 mm}. to revascularize the lad lesion , dess were successfully implanted with a 3.5 by 23 mm cypher and 3.0 by 23 mm cypher with 20 atmospheres covering proximal to mid lad with overlapping technique ( mld 2.93 mm ) . intravascular ultrasound ( ivus , atlantis plus 40 mhz ; boston scientific , natick , ma , usa ) was then performed with a pullback speed of 0.5 mm / sec from distal to middle rca and from middle to proximal lad . nine months later , a scheduled follow - up cag was performed ( figs . 2 and 3 ) . a subsequent ivus showed that there was remarkable separation between the stent struts and the vessel wall indicating the extensive development of the lateacquired isa in the stented segments of both lad and rca . the maximum and mean depth of isa in lad was 0.89 mm and 0.5 mm respectively , while that of isa in rca was 1.09 mm and 0.63 mm respectively . the length of isa in lad and rca was 34.2 mm and 26.2 mm respectively . the patient received 100 mg of aspirin as well as 75 mg of clopidogrel as part of the antiplatelet therapy . there was no evidence of any major adverse cardiac events during a 25-month followup period , post detection of late - acquired isa . isa is the ivus finding of lack of contact between stent struts , not overlying a side branch , and the underlying arterial wall . late isa may be due to either persistence of acute isa occurring at the time of stent implantation or late - acquired isa only being detected during a follow - up analysis.2 ) many cases have previously reported the incidence of late - acquired isa after des implantation.2 - 4 ) such phenomena were more frequently observed after ses implantation.5 ) the present case study was also about the stent fracture and late - acquired isa after ses implantation . however , this was a rare occurrence of extensive late - acquired isa in multiple vessels with the evidence of stent fracture . several mechanisms of late - acquired isa have been postulated , including : 1 ) positive vascular remodeling with or without an increase in plaque area , 2 ) plaque or thrombus dissolution primarily in patients presenting acute coronary syndromes , 3 ) chronic stent recoil , 4 ) decrease in plaque , and 5 ) isa not being recognized at implantation and only being detected during followup analyses.2)3)6 ) hong et al.7 ) reported that the incidence of late - acquired isa after des implantation during a 6-month follow - up analysis was 12.1% and the average length of isa was 3.91.4 mm . in their study , the majority of late - acquired isa cases were focally distributed . unlike the isa pattern frequently observed , our case showed late - acquired isa occurring throughout all treated segments , which conformed to the so - called " extensive isa " pattern in both lad and rca . in the ivus analysis of lad , the mean external elastic membrane ( eem ) volume was 13.9 mm / mm at post stenting and 16.6 mm / mm at follow - up . in rca , the mean eem volume was 12.9 mm / mm at post stenting and 16.0 mm / mm at follow - up . there was a significant interval change in the mean eem volume but not in the mean stent volume in lad ( 7.5 mm / mm at post stenting vs. 8.0 mm / mm at follow - up ) and rca ( 7.0 mm / mm at post stenting vs. 6.8 mm / mm at follow - up ) . as compared to the poststenting , the mean eem volume at follow - up was increased in both lad and rca ( 19% and 24% , respectively ) . this suggested that positive vessel remodeling along with all treated segments was the main mechanism of our late - acquired isa case . the main cause of our extensive isa remains unclear , but we postulate that hypersensitivity against the drug or polymer may contribute to this phenomenon . additional ivus finding of the present case was that stent fracture occurred in mid to distal rca segment , which had high angulations.8 - 11 ) we were unable to convincingly determine whether the stent fracture was associated with isa or not . however , there was no difference in the lumen area between segments of isa and stent fracture . we can assume that fracture occurred after the whole drug had been released since there was no in - stent restenosis found in fractured segments , and the lumen area in fractured segments was almost same with the overall lumen area . however , according to recent findings , this therapy may exert potential benefits if it is continued for longer than one year in patients with des.12 ) thus , we assume that dual antiplatelet therapy over a one - year duration should be considered in the case of isa . the present case study calls for more attention to late - acquired isa after des implantation . as des is frequently used for coronary heart disease , more careful long - term follow - up studies are required to assess the significance of late - acquired isa and to establish reasonable therapeutic guidelines that may include antiplatelet therapy .	late - acquired incomplete stent apposition ( isa ) is frequently observed after drug - eluting stent ( des ) implantation . most incidences of late - acquired isa induced by positive vascular remodeling were of the focal type and occurred in a single vessel . we present an unusual case of a 45-year - old male subject diagnosed with late - acquired isa that occurred in multiple vessels .
dacryoadenitis is a rare disorder that is caused by an obstruction occurring anywhere between the conjunctiva and the lacrimal ductules . if improperly treated , dacryoadenitis can result in orbital abscess or cellulitis . as a result , patients should be monitored very closely and iv antibiotics should be administered if worsening is noted . we present a case report on an extremely rare condition , bilateral dacryoadenitis , which required treatment with iv antibiotics . the diagnostic and therapeutic approaches that should be considered in cases of dacryoadenitis are discussed in this case report . a 24-year - old male presented to the emergency department ( ed ) with complaints of bilateral eye pain , redness , and photosensitivity for two days . the patient had been evaluated at another hospital the previous day where he was treated with ciprofloxacin eye drops and neomycin / polymyxinb / hydrocortisone ophthalmic suspension . his orbital examination revealed chemosis , injected sclera , edema of the upper and lower lids , and yellow discharge bilaterally [ figures 1 and 2 ] . erythema and edema are greatest over the lateral one - third of the upper eyelids ( photo was taken after the application of flouroscein and with the patient 's written permission ) chemosis and injection of the sclera are present ( photo was taken after the application of flouroscein and with the patient 's written permission ) laboratory analysis revealed an elevated white blood cell count of 13,800 k / ul . a computed tomography ( ct ) scan of the orbits was performed which showed bilateral enlargement and inflammation of the lacrimal glands without evidence of orbital cellulitis [ figures 3 and 4 ] . after evaluation by an ophthalmology physician the patient was discharged home on ofloxacin eye drops and oral amoxicillin / clavulanic acid . the patient was instructed to discontinue his previous antibiotics and to follow up for reassessment . coronal ct image showing bilateral lacrimal gland inflammation ( arrows ) axial ct image showing bilateral lacrimal gland inflammation ( arrows ) the patient was reevaluated 4 days later and his symptoms had worsened . it was recommended that the patient be admitted for intravenous antibiotics as well as further diagnostic assessment to better determine the etiology for his symptoms . blood cultures drawn from the patient 's ed visit 4 days prior had no growth . rapid plasma reagin , cytomegalovirus immunoglobulin m antibody , measles antibody , and blood cultures were all negative . the patient 's cytomegalovirus immunoglobulin g antibody and mumps immunoglobulin g antibody tests were positive suggesting previous but not active infection . the patient 's monospot test was negative ; however his epstein barr virus viral capsid antigen immunoglobulin g was positive in a 1:640 ratio suggesting previous exposure but no acute infection . thyroid - stimulating hormone , thyroid stimulating immunoglobulin , neutrophil cytoplasmic antibody , rheumatoid factor , antinuclear antibody , angiotensin- converting enzyme levels , and a chest radiograph were also performed to determine if the patient 's inflammation was secondary to an autoimmune process . the patient 's symptoms were felt to be secondary to a bacterial infection so he was treated with intravenous vancomycin and piperacillin / tazobactam . he was successfully discharged home on hospital day 3 with oral trimethoprim / sulfamethoxazole and ofloxacin ophthalmic drops . dacryoadenitis occurs in only one in 10,000 ophthalmic cases and may be acute , subacute , or chronic . it is most common in children and young adults but may been seen in all age groups . patients generally present with redness , tenderness , warmth , and swelling of the lateral third of the upper eyelid . a mucopurulent discharge , localized conjunctival injection , and chemosis may also be present . impaired vision , limited ocular movement , and pain with movement suggest an orbital cellulitis . dacryoadenitis may be caused by an obstruction occurring anywhere between the conjunctiva and the lacrimal ductules . when it occurs acutely , it is usually due to infection , with viral etiologies such as mumps , measles , influenza , mononucleosis , herpes , and cytomegalovirus occurring most commonly . infections may also be caused by streptococcus pyogenes , haemophilus influenza , neisseria gonorrhea , chlamydia trachomatis , and treponema pallidum . infection may spread to the lacrimal gland hematogenously , transneuronally , from the conjunctiva or via traumatic injury . other causes of dacryoadenitis include neoplasms such as lymphoma and lacrimal gland tumors . in rare cases , these patients should be evaluated for an infectious process but also screened carefully for systemic diseases and autoimmune disorders such as sarcoidosis , wegener 's granulomatosis , grave 's disease , and sjogren 's syndrome . severe cases of dacryoadenitis should initially be treated with a broad spectrum intravenous antibiotic such as cefazolin or ticarcillin / clavulanate which covers gram positive and gram negative organisms . antibiotic selection can later be tailored to culture results . in areas in which methicillin - resistant s aureus ( mrsa ) is present , vancomycin is recommended . patients with suspected mrsa should receive oral treatment with sulfamethoxazole - trimethoprim , clindamycin , or linezolid . viral infections can be managed symptomatically with warm compresses and oral non - steroidal anti - inflammatory medications . if symptoms continue for more than 2 weeks , a lacrimal gland biopsy should be considered . if improperly treated , dacryoadenitis can result in lacrimal gland or orbital abscess , orbital cellulitis , lid ptosis , and adhesions . patients should be followed up very closely and iv antibiotics should be administered if worsening is noted . continued diagnostic testing is mandatory in patients who remain symptomatic .	acute dacryoadenitis is an uncommon condition that involves inflammation of the lacrimal gland . in rare instances , dacryoadenitis may be bilateral . a delay in proper treatment of an otherwise simple case of dacryoadenitis may lead to significant soft tissue morbidity such as cellulitis , lacrimal gland abscess , or orbital abscess . we report the case of a 24-year - old male who presented to the emergency department with acute bilateral dacryoadenitis . the patient 's symptoms did not respond to oral antibiotics and he subsequently required admission for intravenous antibiotics . during his hospitalization the patient had diagnostic testing to try to determine the etiology for his symptoms . the unique aspects of managing a case of bilateral dacryoadenitis as well as treatment recommendations are discussed in this case report .
warfarin has been the mainstay of oral anticoagulation therapy for the prevention of thromboembolic stroke in patients with atrial fibrillation ( af ) . its major drawbacks have been the need for frequent monitoring and numerous interactions with other drugs and food . the food & drug administration ( fda ) in the usa approved dabigatran etexilate ( de ) in 2010 for stroke prophylaxis in patients with non - valvular af . it does not need any monitoring or dietary restrictions and has far fewer drug interactions . dabigatran significantly lowers the risk of thromboembolic stroke and the risk of spontaneous intracranial hemorrhage is less than one - third the rate with warfarin . but the testing for its anticoagulant effect is not routinely available and not unlike some other anticoagulants , dabigatran does not have a specific antidote either . both these factors limit our ability to effectively manage patients on dabigatran when they present with a life - threatening bleed . as we discuss here , dialysis may have an important role in clearance of this drug from the systemic circulation . a 64-year - old electrician presented with headache and mild confusion following a fall from his work truck . his medical history was significant for non - valvular af , hypertension , immune thrombocytopenic purpura and diabetes mellitus . he had taken pradaxa that morning . at presentation , he was mildly confused but otherwise neurologically and hemodynamically intact . imaging revealed an occipital skull fracture , right subdural hematoma with 7 mm shift and bilateral subarachnoid blood . the basic metabolic panel and complete blood count were normal except for a platelet count of 49 10 per liter ( normal range 140440 10 per liter ) . the patient 's platelet count 2weeks prior to this admission was 36 10per liter . the prothrombin time ( pt ) was 14.9 s ( normal range 9.612.4 s ) , the international normalized ratio ( inr ) was 1.4 s ( normal < 1.2 ) and the activated plasma thromboplastin time ( aptt ) was 44.3 s ( normal range 22.334 s ) . reversal of dabigatran 's anticoagulant effect was imperative . previous review of the literature confirmed that there was no proven antidote to dabigatran and that dialysis was probably the only reliable means of drug removal from the systemic circulation . a temporary dialysis catheter was placed and the patient commenced heparin - free hemodialysis within 90min of arrival . we used an optiflux 200 dialyzer for 3 h with a blood flow of 300350 ml / min , dialysate flow of 500 ml / min and a 141 mmol / l sodium , 3 mmol / l potassium , 35 mmol / l bicarbonate and 2.5 mmol / l calcium bath . the patient tolerated the procedure well though he was intubated halfway through dialysis due to the declining neurological status . post - dialysis , repeat head computerized tomography showed worsening subdural hematoma with a 15 mm midline shift . he was then urgently taken to the operating theater for craniectomy and evacuation of hematoma . subsequent imaging showed extensive right intracerebral bleeding , bilateral hemispheric infarction and signs of herniation . table 1.pre- and post - dialysis clotting profile ( all values are in s)pre - dialysispost - dialysispt ( range 9.612.4)14.915.7aptt ( range 22.334)44.349.3ect ( range 22.629)125.583.1 pre- and post - dialysis clotting profile ( all values are in s ) for over 50 years , warfarin has been the mainstay of oral anticoagulation especially in the chronic management of thromboembolism . but difficulty in achieving the optimal anticoagulant effect , narrow therapeutic window necessitating frequent monitoring of the inr , significant interindividual variability in pharmacological response and numerous drug and food interactions are major limitations to its use . parenteral factor xa inhibitors such as fondaparinux are not practical in chronic management of thromboembolism . direct thrombin inhibitors such as argatroban and lepirudin suffer from the same handicap and are restricted to treatment of heparin - induced thrombocytopenia . de is an oral prodrug that is rapidly absorbed from the gastrointestinal tract and converted by serum esterase to its active form dabigatran . the latter is a synthetic , non - peptide , direct , competitive inhibitor of thrombin . it inhibits both clot - bound and circulating thrombin and decreases thrombin - stimulated platelet aggregation . peak serum concentrations are reached an hour after oral administration when fasting and 3h after a fatty meal . it has a half - life of 1217 h and does not undergo hepatic elimination . twice daily dosing provides a predictable and reliable anticoagulant effect that is not impacted by food or drugs and does not need frequent monitoring . dabigatran at a dose of 150 mg twice daily has been associated with lower rates of stroke , systemic embolism and hemorrhagic stroke than warfarin . the re - ly study reported a higher risk of major bleeding with warfarin when compared with dabigatran 110 mg and no difference in the dabigatran 150 mg group . moreover , in a subsequent independent analysis , the case fatality rate per major bleed was numerically lower in those treated with dabigatran than warfarin ( although the number of deaths was too few to perform meaningful statistical analyses ) . this observation may be due to less severe bleeding on dabigatran or because , despite effective reversal of warfarin , correction of coagulation parameters may not have a significant impact on patient survival . as would be expected , bleeding risk has been shown to be specifically higher in the elderly and those with low body weight [ 9 , 10 ] . the dose should be reduced by 50% for those with a glomerular filtration rate ( gfr ) between 15 and 30 ml / min/1.73 m and the drug should be avoided in those with a lower gfr . fda guidelines recommend the assessment of renal function prior to starting dabigatran and annually in those > 75 years and also those with a gfr of < 50 ml / min/1.73 m . aptt , thrombin time ( tt ) , activated clotting time ( act ) and ecarin clotting time ( ect ) have been advocated for monitoring dabigatran 's anticoagulant effect . aptt and tt are both sensitive to the anticoagulant activity of direct thrombin inhibitors , but may underestimate their true anticoagulant activity . in our patient others have shown that this parameter was in the normal range in acutely bleeding patients on dabigatran . tt is of limited value in an overdose as coagulometers are unable to measure it when dabigatran concentration exceeds 600 ng / ml ( steady - state concentration is much lower at 200 ng / ml after multiple doses of the drug ) . ecarin is a metalloprotease isolated from the venom of the saw - scaled viper echis carinatus . ect has a linear relationship to dabigatran 's serum concentration and is hence suitable for monitoring its anticoagulant effect . but this test is not widely available and the result takes several days to be reported [ 14 , 15 ] . rapid thromboelastography ( rteg ) is a point - of - care test that has been used to assess the anticoagulant activity of dabigatran , but has not been validated for this purpose . another challenge in managing patients on dabigatran who are bleeding is the lack of a specific antidote . while vitamin k , fresh frozen plasma , factor viia and prothrombin complex concentrate can all be used to reverse the effect of warfarin , none of these have been proven to reverse anticoagulation due to dabigatran . one study did recommend the use of recombinant activated factor viia for patient bleeding on dabigatran , but a subsequent systematic review demonstrated that this off - label use of the product does not lower mortality and indeed increases the risk of thromboembolism . this review does not indicate if some or all of the included patients were on anticoagulants . however , in a critically ill patient such as ours , it may be prudent to use recombinant activated factor viia or prothrombin complex concentrates in tandem with dialysis . the only strategy for hastening the removal of dabigatran may be dialysis [ 20 , 21 ] . it is a small molecule with a molecular weight of only 628 g / mol . but , the volume of distribution of dabigatran is 5070 l , suggesting that some of it is distributed into the tissues and may not be immediately accessible at dialysis necessitating longer and repeated treatments . the few case reports where dialysis was instituted show mixed results from this intervention [ 21 , 22 ] . the use of dialysis is also limited by the lack of universal availability . in patients with ich three hours of hemodialysis seems to have improved the ect in our patient and possibly reduced the risk of ongoing bleeding . that this patient had a preserved gfr and a decline in the ect may be a reflection of normal renal excretion of dabigatran , but it is possible that the aptt that should have improved in tandem with ect went up slightly due to administration of fluids and also because patients with major intracranial bleeding may sometimes develop disseminated intravascular coagulation . thus , the role of dialysis in dabigatran - associated life - threatening bleeding needs further evaluation . it is possible that longer and repeat dialysis sessions may be more effective , but this remains to be proven . continuous renal replacement therapies ( crrt ) may also be more useful in this setting . this is because of their ability to provide sustained clearance and to minimize intra - dialytic hypotension a major pitfall of intermittent hemodialysis . thus , even though there is not enough evidence to support its use , dialysis should be considered in patients with life - threatening bleeding on dabigatran and those with dabigatran overdose especially if they have reduced renal function . we conclude that dabigatran is a potent oral direct thrombin inhibitor that , despite lack of testing for monitoring its anticoagulant effect and a specific antidote , is a real , and potentially superior , alternative to warfarin . given the current state of the evidence on treating patients with dabigatran - related bleeding , further work is needed to assess the role of currently available therapeutic modalities including dialysis and for the development of potential new treatments .	dabigatran , marketed as pradaxa ( boehringer ingelheim ) in the usa , is a direct thrombin inhibitor that holds great promise . it has been shown to reduce the risk of stroke and venous thromboembolism with similar if not greater efficacy than warfarin and with far fewer side effects . however , like other anticoagulants , it can cause severe bleeding complications and lacks a specific antidote with proven efficacy . the patient presented here was on dabigatran and sustained a traumatic intracranial hemorrhage ( ich ) . the ich continued to progress despite prompt initiation of 3h of hemodialysis in an effort to remove the offending drug from the circulation . through this case report , we highlight the challenges of anticoagulation with dabigatran including the lack of routine testing for monitoring its effect and of a specific antidote . we also discuss the potential role of dialysis in treating patients with life - threatening bleeding on dabigatran .
it is now relatively safe for an attempt at vaginal birth after cesarean section ( vbac ) . vbac is thus being recommended as a relatively safe way of decreasing the ever rising rate of cesarean delivery globally.1,2 vaginal delivery is associated with fewer risks , requires less anesthesia , poses a lower potential for postpartum morbidity , involves a shorter hospital stay , is more affordable , and encourages earlier and better bonding between mother and infant.3 these advantages are significant , especially in our resource poor setting where sociocultural aversion to cesarean delivery is common.4 successful vaginal birth has been reported in 60%80% of cases reported in published studies of women attempting vaginal birth after a previous cesarean section.59 the recommended criteria1012 for selecting candidates suitable for vbac are potentially applicable in tertiary centers in developing countries , but there are many inadequacies in meeting them in our environment.13 such inadequacies may include the unavailability of anesthesiologists and neonatologists on demand during labor / delivery , lack of adequate blood transfusion services , and at times a lack of other basic facilities and the personnel needed to carry out a timely cesarean section . perhaps because of this , anecdotal evidence shows that the attitude of many obstetricians towards practice of vbac appears guarded . meanwhile , a cesarean section rate of over 25% has been reported in our study center.14,15 there is a growing concern over the rising rate of cesarean section in developing countries ; cesarean section rates have been significantly linked to the practice of vbac.13,15 in nigeria many women are poor and may not easily afford cesarean delivery . however , previous studies show that the problem associated with attempting vbac in nigeria includes a lack of facilities for continuous fetal monitoring and inadequate manpower and facilities for emergency cesarean section , which result in long decision operation intervals.9,1317,19 the aim of this study was to describe the maternal and perinatal outcomes of delivery in women with one prior cesarean section seen at a tertiary hospital in enugu , south east nigeria . the study was carried out in the obstetric unit of the university of nigeria teaching hospital ( unth ) , ituku - ozalla , enugu , southeast nigeria , a referral center for high - risk obstetric cases in the south eastern part of the country and beyond . the hospital provides both general and specialist services to the people of enugu state and its environs . it conducts about 1,500 deliveries annually , with a reported cesarean section rate of 25% . this was a prospective study involving consecutive eligible women with one previous cesarean section at the university of nigeria teaching hospital , ituku ozalla , enugu , southeast nigeria . there was individual counseling of each woman recruited for the study , after which her written consent was obtained . women who had one previous cesarean section and were in the second or third trimester of pregnancy were recruited in the study . each study participant was given a number ( or tag ) to enable specific follow - up . women who had two or more cesarean sections or a uterine scar ( myomectomy or uterine rupture ) were excluded . women who had cesarean sections for a reason that would typically lead to another cesarean delivery in subsequent pregnancies ( recurrent indications ) were also excluded . such recurrent indications included women with radiological evidence of pelvic contraction , previous classical cesarean section , and previous repair of vaginal fistula . selection of candidates for vbac by the obstetrician was based on the 2004 american college of obstetricians and gynecologists ( acog ) recommendation10 which specified that such a candidate should have no more than one prior lower segment cesarean delivery , clinically adequate pelvis , no other uterine scars or previous rupture , and the availability of obstetricians and anesthetists and other requisite personnel to monitor the active labor and carry out a timely cesarean delivery . data obtained included bio data , sociodemographic characteristics , details of the previous cesarean section , mode of delivery , outcome , and possible complications of each delivery . all women included in the study were followed through delivery and for at least 48 hours after delivery . each labor was monitored closely using a partogram and regular auscultation of the fetal heart at least once every 30 minutes . for the single case of induction of labor , cervical ripening was done by intra cervical catheter and the entire process of induction was physically monitored by a senior obstetrician . syntocinon was used for the labor induction and also for the few patients who had augmentation . all data collected were keyed into statistical package for social science ( ibm corporation , armonk , ny , usa ) computer software version 17.0 for analysis . maternal and perinatal outcomes were compared between women who had vbac , those who had elective repeat cesarean delivery , those who had successful vbac , and those who had failed vbac . categorical variables were compared using the chi - square test ( or fisher s exact test where appropriate ) . the study was carried out in the obstetric unit of the university of nigeria teaching hospital ( unth ) , ituku - ozalla , enugu , southeast nigeria , a referral center for high - risk obstetric cases in the south eastern part of the country and beyond . the hospital provides both general and specialist services to the people of enugu state and its environs . it conducts about 1,500 deliveries annually , with a reported cesarean section rate of 25% . this was a prospective study involving consecutive eligible women with one previous cesarean section at the university of nigeria teaching hospital , ituku ozalla , enugu , southeast nigeria . there was individual counseling of each woman recruited for the study , after which her written consent was obtained . women who had one previous cesarean section and were in the second or third trimester of pregnancy were recruited in the study . each study participant was given a number ( or tag ) to enable specific follow - up . women who had two or more cesarean sections or a uterine scar ( myomectomy or uterine rupture ) were excluded . women who had cesarean sections for a reason that would typically lead to another cesarean delivery in subsequent pregnancies ( recurrent indications ) were also excluded . such recurrent indications included women with radiological evidence of pelvic contraction , previous classical cesarean section , and previous repair of vaginal fistula . selection of candidates for vbac by the obstetrician was based on the 2004 american college of obstetricians and gynecologists ( acog ) recommendation10 which specified that such a candidate should have no more than one prior lower segment cesarean delivery , clinically adequate pelvis , no other uterine scars or previous rupture , and the availability of obstetricians and anesthetists and other requisite personnel to monitor the active labor and carry out a timely cesarean delivery . data obtained included bio data , sociodemographic characteristics , details of the previous cesarean section , mode of delivery , outcome , and possible complications of each delivery . all women included in the study were followed through delivery and for at least 48 hours after delivery . each labor was monitored closely using a partogram and regular auscultation of the fetal heart at least once every 30 minutes . for the single case of induction of labor , cervical ripening was done by intra cervical catheter and the entire process of induction was physically monitored by a senior obstetrician . syntocinon was used for the labor induction and also for the few patients who had augmentation . all data collected were keyed into statistical package for social science ( ibm corporation , armonk , ny , usa ) computer software version 17.0 for analysis . maternal and perinatal outcomes were compared between women who had vbac , those who had elective repeat cesarean delivery , those who had successful vbac , and those who had failed vbac . categorical variables were compared using the chi - square test ( or fisher s exact test where appropriate ) . two thousand six hundred and ten women delivered in the center during the study period , out of which 395 had one previous cesarean section . the prevalence of women with one previous cesarean section in this study was therefore 15.1% . of 370 women who consented to participate in the study , only 355 women presented for delivery in the study center and their data were used for analysis . the sociodemographic characteristics of the study population indicate that the average age of the women was 32.14.7 years ( range : 2344 years ) . most of the patients were married ( 98.6% ) , christian ( 95.8% ) , and of igbo ethnicity ( 98.6% ) . the leading indications for the one previous cesarean were prolonged labor in 90 women ( 25.4% ) , pre - eclampsia in 55 ( 15.5% ) , and 35 each of failed induction and abnormal lie ( 9.9% ) . table 2 summarizes the comparison of maternal and fetal outcomes between women who had vbac and those who had elective repeat cesarean section . the majority of the women ( 320 , 90.1% ) preferred to have vaginal delivery despite the one previous cesarean section . as regards the planned mode of delivery determined by the obstetrician , 190 ( 53.5% ) of the women were assessed as suitable to allow an attempt at vaginal birth ; of these 190 women 95 ( 50.0% ) eventually had successful vaginal birth , while the remaining 95 ( 50.0% ) failed to deliver successfully via vbac and delivered by emergency cesarean section . of the remaining 165 ( 46.5% ) women , 130 ( 35.7% ) had elective repeat cesarean section while thirty - five ( 9.8% ) women had emergency cesarean section for other obstetric indications ( apart from failed vbac ) . there was no case of uterine rupture , neonatal , or maternal deaths recorded in any group . table 3 summarizes the comparison of maternal and perinatal outcomes between women who had successful trial of vbac and those who had failed attempt at vbac . apgar scores of less than seven in the first minute were significantly more frequent amongst those with vaginal delivery when compared to those with elective repeat cesarean section ( p=0.00 ) . apgar scores less than seven in the first minute were more frequent in those with failed vbac than vaginal delivery ( successful vbac ) ; however , the difference was not statistically significant ( p=0.082 ) ( table 3 ) . among those allowed trial of labor , 30 ( 31.6% ) failed vbac had birth weight 4.0 kg or greater compared to none ( 0% ) of those with successful vbac ( p=0.03 ) . the prevalence of one previous cesarean section of 15.1% in this study is higher but still comparable to the 11.9% documented in a private hospital in lagos , nigeria.7 this marginally higher prevalence may be due to the fact that high risk cases including previous cesarean section are referred to and managed in our teaching hospital . the clear preference for vaginal delivery ( 90.1% ) among the women was expected due to strong cultural aversion for cesarean delivery , which even formal education does not seem to alter.4 only about half ( 53.3% ) of the women with one previous cesarean section were allowed to attempt vbac despite a high cesarean section rate in the hospital and overwhelming preference for vaginal delivery by the women . this percentage is high compared to the less than 10% recorded in enugu in 1989.13 our results are similar to the reports from benin , nigeria17 but much lower than the 70.7% in a recent similar study in lagos.7 the lower proportion of women allowed to attempt a vbac in this study may be due to a combination of reasons . the medical , legal , and ethical concerns related to the responsibility of monitoring a high risk labor after a previous cesarean section without the appropriate tools and facilities may be a factor . necessary conditions including personnel especially anesthetists and neonatologists and the facilities needed for emergency cesarean section ( eg , blood for transfusion ) are often not available on demand . the absence of adequate facilities for electronic fetal monitoring in the study center makes fetal monitoring in labor less intensive . another factor may be the relocation of the hospital to its permanent site about 21 kilometers from the city where senior obstetricians are resided . this may have lowered the threshold for elective cesarean section as a precautionary measure . out of those allowed to attempt vbac , this finding is higher than the 34% success rate recorded in pakistan18 and benin , nigeria,17 but much lower than the more recent studies from ibadan and lagos that had successful vaginal deliveries in the range of 60%80%.7,19 these higher success rates are similar to a previous report from enugu.9 it must be noted that these local studies with higher success rates had increased fetal and maternal complications compared to our finding . the lower success rate of vbac in this work may be due to early and abrupt recourse to emergency cesarean section occasioned by medical or legal concerns and conceivable complications in the face of suboptimal facilities for intrapartum care . only one woman in our series had induction of labor and three had cautious augmentation of labor , all with good outcome . it may be that more liberal application of induction and augmentation of labor when indicated may have increased the rate of successful vbac in our hospital . a study to determine the safety and impact of liberal use of induction and augmentation of labor in women with one previous cesarean in this area good fetal and maternal outcomes of labor were recorded among women who had trial of vbac in this study with no case of uterine rupture or perinatal and maternal deaths . previous retrospective reviews from nigeria had recorded one to five cases of uterine rupture and one to three cases of neonatal death with no maternal death but with higher rates of successful vbac.7,9 in those reviews , a greater proportion of women with one previous cesarean were allowed trial of vbac than in this study , and this may explain the inability of this study to detect these complications . the strengths of this work include its prospective design which enabled verification of data directly from the women . the main weakness was that the study involved only one center ( due to logistic reasons ) ; it may have been better to have more participating institutions to get a wider picture in the zone and country . in conclusion , it remains a challenge to strike a balance between concern for safety and the need to decrease cesarean section rates . due to limited facilities for fetal monitoring , most obstetricians tended to have a low threshold for elective repeat cesarean section ; hence , only about half of women with a previous cesarean were allowed attempt at vbac . this is a sharp contrast to the less restrictive guidelines by both the royal college of obstetricians and gynaecologists and the acog.20,21 in the recently revised document acog recommended that trial of labor after previous cesarean delivery ( tolac ) is safe and appropriate for most women with previous cesarean delivery , including those with two previous lower segment transverse incisions , twin pregnancy , and those with unknown type of scar . together with a recent meta - analysis22 that reported over 70% successful vaginal births after two cesareans , all these recommendations may not influence practice in our environment in the near future unless there is improvement in our intrapartum care in terms of personnel , facilities , and our capacity to carry out a timely cesarean section .	backgroundobstetricians in developing countries appear generally reluctant to conduct vaginal delivery in women with a previous cesarean because of lack of adequate facilities for optimal fetomaternal monitoring.objectiveto describe delivery outcomes among women with one previous cesarean section at a tertiary hospital in southeast nigeria.methodsthis was a prospective observational study to determine maternal and perinatal outcomes of attempted vaginal birth after cesarean sections ( vbac ) following one previous cesarean section . analysis was done with spss statistical software version 17.0 for windows using descriptive and inferential statistics at 95% level of confidence.resultstwo thousand six hundred and ten women delivered in the center during the study period , of whom 395 had one previous cesarean section . a total of 370 women with one previous cesarean section had nonrecurrent indications , of whom 355 consenting pregnant women with one previous cesarean section were studied . a majority of the women ( 320/355 , 90.1% ) preferred to have vaginal delivery despite the one previous cesarean section . however , only approximately 54% ( 190/355 ) were found suitable for trial of vbac , out of whom 50% ( 95/190 had successful vbac . ninety - five women ( 50.0% ) had failed attempt at vbac and were delivered by emergency cesarean section while 35 women ( 9.8% ) had emergency cesarean section for other obstetric indications ( apart from failed vbac ) . there was no case of uterine rupture or neonatal and maternal deaths recorded in any group . apgar scores of less than 7 in the first minute were significantly more frequent amongst women who had vaginal delivery when compared to those who had elective repeat cesarean section ( p=0.03).conclusionmost women who had one previous cesarean delivery chose to undergo trial of vbac , although only about half were considered suitable for vbac . the maternal and fetal outcomes of trial of vbac in selected women with one previous cesarean delivery for non - recurrent indications were good . obstetricians in this area should do more to allow vbac in women with one previous cesarean section for nonrecurrent indications .
all surviving members of the 2012 outbreak in jordan , their exposed contacts , and their household members who were identified serologically as either mers - cov positive or indeterminate were asked to consent to further participation . participants were asked to provide a follow - up serologic specimen so we could compare 34-month results with 13-month results . specimens were prepared by the jordan central public health laboratory ( amman , jordan ) and tested at the us centers for disease control and prevention ( atlanta , ga , usa ) . antibody titers in serum samples were determined by an anti mers - cov nucleocapsid indirect elisa and by mers - cov ( hu / jordan - n3/2012 strain ) indirect ifa ( 1 ) . the presence of neutralizing antibody titers was determined by microneutralization with live mers - cov ( hu / jordan - n3/2012 strain ) in a biosafety level 3 laboratory as described previously ( 1 ) . neutralization titers were defined as the reciprocal of the highest serum dilution completely protecting the vero cell monolayer from cytopathic effect in at least 1 of 3 parallel wells . of the 15 surviving persons with > 1 positive serologic test result , 13 ( 87% ) consented to follow - up testing . all 7 ( 100% ) surviving persons with > 2 positive serologic test results 13 months after the mers - cov outbreak also consented . each of these 7 persons was considered to be a probable mers - cov case - patient according to world health organization criteria ; each had had a symptomatic acute respiratory infection during the outbreak period and documented , unprotected exposure to > 1 person with a case confirmed by reverse transcription pcr . for the 7 probable case - patients , elisa titers at 34 months ranged from < 400 to 1,600 , representing reduced antibody titers compared with the 13-month estimates ( 4006,400 ) for all but 1 person ( table ) . a nurse who worked in an intensive care unit ( participant 06 ) and cared for confirmed case - patients during the outbreak was the only participant for whom elisa indicated a consistent titer of 1,600 at both times . * ifa , immunofluorescence assay ; ind , indeterminate ; mn , microneutralization assay ; na , not applicable ; nd , not documented ; neg , negative ; pos , positive . symptomatic but refused hospitalization . classified overall as being serologically negative at 34 mo . of these 7 participants , 6 ( 86% ) had neutralizing antibody titers ranging from 20 to 80 at the 34-month follow - up evaluation , and only 2 ( 29% ) had any decrease in neutralizing antibody titers over time . of the 7 participants for whom ifa results were positive at 13 months , 4 ( 57% ) had positive results at 34 months . neutralizing antibody titers against middle east respiratory syndrome coronavirus ( hu / jordan - n3/2012 strain ) among 7 surviving case - patients at 13 and 34 months after the 2012 outbreak in jordan . for the 8 surviving participants whose serologic results at 13 months were indeterminate , 6 ( 75% ) consented to further testing . of these , 3 ( 50% ) reported having had no respiratory symptoms , 1 reported having had mild respiratory symptoms , and 2 had been hospitalized with respiratory infections ; all 6 had had documented , unprotected exposure to > 1 case - patient . each of these 6 persons had negative serologic test results at 34 months and continue to be considered negative overall . antibodies against mers - cov , including neutralizing antibodies , persisted in 6 ( 86% ) of 7 persons 34 months after the 2012 mers - cov outbreak in jordan . the observed persistence of these antibodies contributes to the understanding of individual immune responses to mers - cov infection , of population - based immunity in regions where mers - cov outbreaks have occurred , and to efforts for developing effective vaccines and therapeutics to counter mers - cov infections . notwithstanding improvements in public health awareness and infection control practices in affected countries on the arabian peninsula and in the middle east , emergence of the virus ( e.g. , its introduction to south korea and the resultant epidemic of 2015 ) ongoing . mers - cov continues to pose grave risks to international healthcare and socioeconomic systems ( 6 ) . it has been hypothesized that mild or asymptomatic mers - cov infections are potentially associated with lower levels of mers - cov neutralizing antibodies over time ( 7 ) . all 7 case - patients reported here had respiratory symptoms , were relatively young , and had few underlying medical conditions ( table ) . any association between our mers - cov antibody results and clinical severity is therefore difficult to assess . nonetheless , of the 5 persons for whom chest radiographs showed substantial changes within 3 days of symptom onset , each remained positive by microneutralization ( > 20 ) 34 months after the outbreak . although some similarities in the short - term development of antibodies against mers - cov and sars - cov ( e.g. , seroconversion 23 weeks after illness onset ) have been observed ( 8,9 ) , longer term serum antibody kinetics of these infections have not yet been compared . after sars - cov infection , robust igg titers were observed through the second year but declined substantially during the third year after infection ( 10 ) . our finding of generally reduced but persistent mers - cov antibody responses even at 34 months suggests the potential for longer lasting antibody - mediated protective immunity against reinfection . however , whether such long - lasting antibodies can prevent reinfection or affect clinical outcome has yet to be examined . diverse individual antibody test results allude to a potential role of genetic factors in explaining observed differences in immunologic responses to mers - cov exposure and infection . the times at which mers - cov antibodies were measured in our study were chosen because of logistics and field practicalities . although limited outbreaks of mers - cov have occurred in jordan since 2012 , contact tracing efforts by investigators in jordan lead us to believe that these persons were not subsequently exposed . the observed elisa titers and neutralizing antibody titers support this supposition ; otherwise , we would expect increases resulting from a booster effect after secondary exposure and infection . to further assess the duration and resiliency of mers - cov antibodies in human populations , continued follow - up serologic evaluations of these persons	to determine how long antibodies against middle east respiratory syndrome coronavirus persist , we measured long - term antibody responses among persons serologically positive or indeterminate after a 2012 outbreak in jordan . antibodies , including neutralizing antibodies , were detectable in 6 ( 86% ) of 7 persons for at least 34 months after the outbreak .
cosmetic eye - whitening procedures for the treatment of chronic conjunctival hyperemia have recently become a topic of interest due to accumulating evidence of severe postoperative complications associated with their practice.1,2 a large proportion of patients have undergone the so - called regional conjunctivectomy procedure in south korea , along with many patients in the us who have undergone a similar procedure known as i - brite ( boxer wachler vision institute , beverly hills , ca , usa ) . each respective procedure entails resection involving significant amounts of bulbar conjunctiva with or without resection of tenon s capsule components within the nasal and temporal regions of the palpebral fissure , with concomitant intraoperative and postoperative mitomycin c ( mmc ) administration topically , subconjunctivally , or both.1 there are a multitude of worrisome postoperative complications that have been reported in association with these procedures , including , but not limited to : scleral thinning with or without calcified plaques , diplopia induced by fibrovascular growth , necrotizing scleritis , and elevated intraocular pressure ( iop).1,2 herein , we report a case of bilateral scleral thinning with associated calcified plaque formation in a patient who had undergone a cosmetic eye - whitening procedure 4 years prior to presentation . a chart review of a single patient who received a cosmetic eye - whitening procedure at an outside institution was performed . medical records detailing preoperative , intraoperative , and postoperative findings were obtained from outside providers . information including , but not limited to , preoperative signs and symptoms , intraoperative procedure , patient demographics , visual acuity , medication regimen , complications , and postoperative complication related therapy were obtained through the record review . a thorough examination was performed , including quantification of best corrected and uncorrected visual acuities , measurement of iop , and slit - lamp and dilated fundus examinations , and slit - lamp photography . a literature review was performed through pubmed from 1980 through 2014 using the search terms ocular , conjunctivectomy , regional conjunctivectomy , i - brite , eye - whitening , bevacizumab , along with associated cross - referencing from relevant articles . as a result of various inter - investigational discrepancies associated with reported complications , aggregation of patients ( rather than total eye quantity ) was used for quantitative analysis . a 33-year - old male with a previous history of chronic conjunctival hyperemia and past surgical history of right - sided congenital ptosis repair at age 5 years and bilateral cosmetic eye - whitening procedure was referred to our institution for evaluation and management of bilateral scleral thinning . during the patient s previously performed cosmetic procedure , subsequent to conjunctival excision , unilateral cauterization of the left eye was required to achieve hemostasis . intraoperative 0.02% mmc was also administered topically to the nasal conjunctivae of each eye for a duration of 1 minute . three weeks prior to presentation at our institution , the patient was treated with topical moxifloxacin for an episode of conjunctivitis within the left eye . during a follow - up visit , the patient developed anterior uveitis for which topical steroids were prescribed ; despite this treatment , the inflammation did not resolve . at this point , the patient was then referred to our quaternary care facilities for further evaluation . on initial presentation , the patient did not have any associated pain or other ocular symptoms , and demonstrated uncorrected visual acuity of 20/30 in the right eye and 20/70 in the left eye ; tonometric evaluation was not performed . slit - lamp examination revealed bilateral nasal scleral thinning and corresponding regional scleral avascularity , which was more prominent in the left eye . associated overlying calcific plaque formation and prominent accompanying epithelial irregularities visualized with fluorescein staining were also present ( figures 1 and 2 ) . bilateral pronounced enlargement of nasal and temporal limbal vessels was observed , along with unilateral accumulation of pigment on the anterior lenticular capsule and detached synechiae within the left eye . despite this finding although there was high suspicion that these findings were attributable to the patient s previously performed eye - whitening procedure , a full immunologic work - up was initiated ( antinuclear antibodies ( ana ) , perinuclear anti - neutrophil cytoplasmic antibodies ( p - anca ) , cytoplasmic anti - neutrophil cytoplasmic antibodies ( c - anca ) , angiotensin - converting enzyme ( ace ) , rheumatoid factor ( rf ) ) to exclude any potential immunologic etiologies . the patient was prescribed 100 mg doxycycline orally and 1,000 mg ascorbic acid orally to be taken twice per day , and 3.5 g erythromycin ointment to apply nightly , along with aggressive administration of lubricating ointment , preservative - free artificial tears , and autologous serum tears . the left - sided calcific plaques were removed at the slit lamp and transported to ocular pathology for further analysis ( figure 3 ) . histopathologic analysis along with hematoxylin and eosin ( h&e ) as well as alizarin red stains confirmed the presence of calcium amongst regular scleral collagen lamellae . considerable subjective relief of ocular irritation occurred after plaque removal , as a result of conjunctival re - epithelialization . serum tears were continued , and all epithelial defects resolved with continual aggressive utilization of preservative - free artificial tears and ointment over a 6-week period . during the patient s last visit , the bilateral scleral thinning remained unchanged . this patient will continue to be followed closely for the foreseeable future and may eventually require amniotic membrane transplantation and/or tectonic graft placement in the event of regression . methodology associated with each respective cosmetic eye - whitening procedure ( regional conjunctivectomy and i - brite ) involves dissection and excision of the greater portion of conjunctiva / tenon s capsule segments along with associated vasculature within the palpebral fissure . varying combinations of 5-fluorouracil ( specific to i - brite ) , bevacizumab , and 0.02% mmc either topically or subconjunctivally are administered intraoperatively with the stated objective of preventing excessive scar formation and recurrent vascular neogenesis . postoperative administration of 0.02% mmc is then prescribed to be used four times daily for a period of 27 days contingent on preexisting thickness of the resected conjunctival tissue.1 bevacizumab is used routinely during i - brite , and had been recently implemented during performance of regional conjunctivectomy , with 350 out of 1,713 patients having received the drug in a recent study.2 cosmetic ocular - whitening procedures have been associated with complication rates as high as 82.9%.2 postoperative complications demonstrate a high degree of variability with respect to subtype , severity , latency interval , and management technique ( table 1 ) . collective incidence of complications previously reported in the literature include : scleral thinning ( 1.8% ) , calcific plaque formation ( 2.9% ) , fibrovascular proliferation ( 13% ) , diplopia ( 1.2% ) , elevation of iop ( 4.2% ) , and recurrence of conjunctival hyperemia ( 2.1% ) ( table 2 ) . elevation of iop has been attributed to prolonged use of postoperative topical corticosteroids , specifically fluorometholone acetate.1 other reported complications include lymphangiectasis ( 0.1% ) , extraocular muscle fiber exposure ( 0.04% ) , dry eye ( 0.6% ) , chronic conjunctival epithelial defects ( 1% ) , decreased vision ( 1.8% ) , granuloma formation , and corneal ulceration.13 in 2011 , the korean ministry of health and welfare issued an order for discontinuation of the regional conjunctivectomy procedure,2 and the american society of cataract and refractive surgery issued a clinical alert in march 2014 recommending use of alternative procedures for treatment of conjunctival hyperemia.3 to date , reported cases of necrotizing scleritis and other complications resulting from ocular - whitening procedures have taken place shortly following the procedure , up to an observed period of nearly 4 years.18 within larger case series reports , average onset of diplopia and granuloma following initial cosmetic surgical intervention have been observed to occur with relative rapidity ; ensuing after 1.6 and 2 months , respectively.1,4 kwon et al documented a case series of patients with scleral thinning and calcification , some of whom showed similarities to the patient currently being discussed , that were first treated for complications anywhere from 1236 months after surgery.7 however , the date of their initial presentation was less well - described . all generally associated postoperative complications have been reported to occur predominantly within the first 412 months following surgery.1,2 interestingly , our patient presented 4 years after his performed procedure , denoting to our knowledge , one of the most protracted documented latency intervals associated with this procedure . in addition to creating a predisposition for the development of necrotizing scleritis / infectious scleritis,9,10 mmc has been known to cause scleral ulcerations / calcifications , corneal edema , limbal stem cell deficiency , and iritis , with a highly variable postoperative onset of symptoms , ranging from days to years.1122 mmc is an alkylating agent that covalently crosslinks dna , and leads to the inhibition of dna , rna , and protein synthesis.23 it follows that regions of the ocular surface that have been exposed to mmc will therefore experience protracted wound healing along with impaired revascularization , particularly with intraoperative addition of bevacizumab ( a recombinant humanized monoclonal antibody prohibiting interaction of vascular endothelial growth factor - a with its associated receptors ) . areas of avascularity in regions exposed to intraoperative and postoperative mmc during pterygium excision have been observed to persist for longer than 7 years post - operatively.24 in addition , a decreased density of goblet cells within affected conjunctival regions has also been documented.21 by virtue of the normal physiologic relative avascularity of the sclera , it is solely dependent on the adjacent blood supply provided by the choroidal and episcleral vessels.25 disruption of this crucial vascular network can result in necrosis of scleral tissue accompanied by a prospective possibility for several catastrophic consequences . development of ischemic sclerae as a postoperative complication of cosmetic eye - whitening procedures is thought to constitute the underlying etiology for the resultant ocular morbidities ( conjunctival epithelial defects , scleral thinning / plaque formation , fibrovascular adhesions ) that have been documented in postoperative patients.4 indeed , regions of avascularity typify associated pathological alterations that were observed within our patient , as well as other previously recognized cases of scleromalacia.5,6 intraoperative cauterization with resultant injury to episcleral vessels will undoubtedly result in a certain degree of vascular compromise within the immediate subjected area . it has been logically inferred that performance of intraoperative cauterization will likely result in increased probability of vascular impotence , resulting in scleral ischemia.1,6 notably , our patient received intraoperative unilateral cauterization within the left eye , which correlates with the increased severity of scleral thinning observed . on account of the collective antiproliferative augmentative effects resulting from combined use , use of intraoperative 5-fluorouracil ( an antimetabolite that disrupts function of rna and dna ) , bevacizumab , and mmc should be avoided , especially after instances of required intraoperative cauterization . avoidance of postoperative mmc is also recommended due to resultant prolonged wound healing associated with its use . after definitive exclusion of autoimmune and infectious etiologies , an appropriate therapeutic approach can be instituted . therapeutic options for noninfectious , nonimmunologic causes of necrotizing scleritis vary depending upon severity and progression . conservative treatment may be instituted at the clinician s discretion for a discrete period of time to monitor for signs of regression or progression . this may aid in significant improvement of symptoms , sparing the patient from undergoing unnecessary surgical intervention . application of ethylenediaminetetraacetic acid ( edta ) may also be an option for treatment of calcific plaques . implementation of autologous serum tears may also be considered , especially for cases demonstrating recalcitrance of conjunctival epithelial defect resolution . in more severe cases , additional treatment options available for utilization include placement of amniotic membrane grafts , autologous conjunctival flaps , or simultaneous placement of both.7,8,2629 other forms of tissue media available for scleral surface repair include sclera , cornea , pericardium , fascia lata , dermis , and cartilage.2937 after initiation of treatment , close patient follow - up should be continued for a prolonged period of time to monitor for progression or recurrence of symptoms . this specific population of postoperative patients remains at significant risk for potentially catastrophic postoperative complications several years following initial surgical intervention . it is of particular importance that a thorough surgical history is obtained from every patient in order to anticipate and properly manage any of several ensuing sequelae that may later occur . cosmetic ocular whitening procedures such as regional conjunc - tivectomy and i - brite have exhibited exceedingly high rates of complications . varying combinations of topical or subconjuncti - val mmc , 5-fluorouracil , and/or bevacizumab can cause regional avascularity , which may lead to scleral thinning and calcific plaque deposition , in addition to several other concerning ocular findings . despite this , more work needs to be done in order to determine which combinations of the abovementioned medications achieve an acceptable safety profile . physicians are encouraged to follow these patients closely , particularly during the first few postoperative months , to monitor for abnormalities that may develop . patient correspondence is imperative , as complications may occur 4 or more years after surgery .	purposeto report a case of delayed - onset bilateral scleral thinning and calcium deposition following a cosmetic ocular - whitening procedure ( i - brite).methodsa 33-year - old male patient with a history of right - sided ptosis repair and left - sided anterior uveitis had previously undergone bilateral i - brite treatment for chronic conjunctival hyperemia . four years after the procedure , the patient was referred to our institution with bilateral scleral thinning and overlying calcific depositions . a literature review was performed through pubmed from 1980 through 2014 using the search terms cosmetic , ocular , conjunctivectomy , regional conjunctivectomy , i - brite , eye - whitening , scleritis , necrotizing scleritis , anterior uveitis , mitomycin c , 5-fluorouracil , and bevacizumab , along with associated cross - referencing from relevant articles.resultsexamination of the patient revealed bilateral necrotizing scleritis within the nasal region of both eyes . calcified plaques were also present within the areas of scleromalacia , along with epithelial defects demonstrated with fluorescein staining . although evidence of previous intraocular inflammation was apparent within the left eye , there were no active signs of inflammation evident within either eye on initial presentation . complication rates reported in the literature include : scleral thinning ( 1.8% ) , calcific plaque formation ( 2.9% ) , fibrovascular proliferation ( 13% ) , diplopia ( 1.2% ) , elevation of intraocular pressure ( 4.2% ) , and recurrence of conjunctival hyperemia ( 2.1%).conclusioncosmetic ocular whitening procedures have an attendant high complication rate , and have been associated with several adverse postoperative complications , which have in turn generated several reservations regarding the veritable benefit of the procedure . many postsurgical complications may demonstrate delayed apparition , varying from several months to several years after primary surgical intervention as in the case reported here .
every clinician has experienced the failure of a restoration , be it loosening of a crown , loss of an anterior class v restoration , or leakage of a composite restoration . the procedure is much the same for any such failure , namely , removal of residual adhesive or luting agent and recementation of the restoration . the clinical notes will describe the problem as , commonly , adhesive or cohesive failure based on a simple classification system such as that in figure 1 . adhesion and cohesion are terms that are often confused although these subjects are discussed in many standard texts in dental biomaterials science [ 13 ] . there are also many excellent texts and monographs on adhesion , cohesion , and interfacial reactions [ 46 ] together with a comprehensive treatment in the on - line encyclopedia , wikipedia . since adhesion and cohesion play a very important role in the use of luting agents , an in - depth discussion is appropriate in view of the communications presented in this issue . adhesion but the most apposite here is the molecular attraction exerted between the surfaces of bodies in contact . the most pertinent here is the molecular attraction by which the particles of a body are united throughout the mass . in other words , adhesion is any attraction process between dissimilar molecular species , which have been brought into direct contact such that the adhesive clings or binds to the applied surface or substrate . the postsurgical complication of adhesions , involving soft tissues , will not be discussed here . in contrast , cohesion is an attraction process that occurs between similar molecules , primarily as the result of chemical bonds that have formed between the individual components of the adhesive or luting agent . thus , cohesion may be defined as the internal strength of an adhesive due to various interactions within that adhesive that binds the mass together , whereas adhesion is the bonding of one material to another , namely , an adhesive to a substrate , due to a number of different possible interactions at the adhesive - substrate surface interface . when a restoration is cemented or bonded to a tooth , adhesive forces bind the luting agent to the restoration on one side and to the tooth on the other side with cohesive forces operating within the luting agent itself , figure 3 . the characteristics of chewing and bubble gums clearly indicate the difference between cohesion and adhesion . gum holds together during mastication because of good cohesion and , in the case of bubble gum , enables the gum to be blown into a bubble . these materials , however , exhibit poor adhesion in that they do not readily stick to the teeth , oral tissues , or other surfaces , unless mechanical effects intervene . if , for example , gum while being chewed can lodge into undercuts or between teeth , it can get locked in and may be torn away from the bulk of the gum , that is , mechanical interlocking of the gum within the interproximal area is greater than the cohesive strength of the gum . likewise , chewed gum does not stick well to smooth surfaces such as glass or polished metal because of its poor adhesion . however , if the masticated and softened gum is pressed onto a rough surface , the gum will distort and flow into gaps , rugosity and voids in the surface such that it sticks , often very tightly , to that surface , as most of us know when we try to scrape discarded gum off the soles of our shoes . likewise , zinc phosphate cement has good cohesive strength but exhibits poor adhesion to smooth surfaces . in particular , it does not bond , chemically to surfaces and its bonding or adhesion , that is , its application as a luting agent , is possible only through mechanical interlocking at the interface with the restoration and that with the tooth . zinc phosphate cement , however , does possess good cohesive strength , even in thin films , so that when used as a luting agent for restorations subject to high masticatory stresses , it can support elastic deformation . in every situation involving an adhesive and a substrate , the combination of adhesion and cohesion determines the overall bonding effectiveness . the adhesive bond will fail if the adhesive separates from the substrate or there is internal breakdown of the adhesive ( i.e. , cohesive failure ) , figure 1 . the cohesive strength of a luting agent or adhesive , regardless of its chemical composition , is determined by a number of molecular forces : the chemical bonds within the adhesive material , chemical bonds due to crosslinking of the polymer(s ) within a resin - based material , intermolecular interactions between the adhesive molecules , and mechanical bonds and interactions between the molecules in the adhesive . these molecular interactions , really intermolecular forces , affect the properties of the uncured ( unset ) adhesive , typically the consistency , flow properties , and viscosity of the adhesive . when the adhesive sets or cures to a solid mass , solidification occurs through bonds formed between the molecules in the adhesive , through formation of new bonds and by strengthening of existing bonds . this overall process typically consists of crosslinking of short chain molecules to form longer chains and/or formation of 3-dimensional networks of molecular chains . the latter is the common mechanism involved in the setting of zinc oxide - based dental cements . it follows from this that the cohesive strength of an adhesive is significantly affected by the curing conditions and , when curing / setting occurs under suboptimal conditions , the adhesive will lack cohesive strength . suboptimal conditions during the setting or solidification process is a common concern in restorative dentistry and all luting agents , regardless of composition and characteristics , must be protected against the effects of oral fluids prior to and during the curing process to avoid detrimental effects on the setting reactions . ingress of saliva and oral fluids into the adhesive during the setting process will adversely affect the curing reactions of both inorganic and organic adhesive materials , commonly reducing strength , bonding efficiency and the degree of cure . thus , fluid ingress will not only imperil the integrity and efficacy of the adhesive - substrate interactions at both the tooth and restoration interfaces but also decrease the cohesive strength of the adhesive . the latter effect is important because the maximum load a bond can withstand in clinical practice as well as in laboratory strength tests may be dictated primarily by the cohesive strength of the adhesive , that is , under loading , the bond fractures due to cohesive failure of the adhesive rather than failure of the adhesive - substrate bond . in other words , the cohesive strength of the adhesive , and not the adhesion between adhesive and the substrate , may be the limiting factor in bond strength tests and in clinical practice . adhesion is the propensity of dissimilar particles and/or surfaces to adhere or bond to one another and can be divided into three basic types , table 1 . the intermolecular forces produce specific adhesion although this can really be divided into three different types , namely , chemical adhesion , dispersive adhesion , and diffusive adhesion , to which are added mechanical effects in effective adhesion . however , a distinction must be made between weak intermolecular interactions and strong chemical bonds . although chemical bonds can form in a few substrate / adhesive combinations , for example , epoxy resin and aluminum , they are generally uncommon in dentistry except for those that occur between carboxylate - based luting agents and the calcium within dental hard tissues . when there are chemical bonds within the adhesive joints , they can account for up to 50% of all interactions although the long - term stability of these bonds is usually dependent on their resistance to moisture . in addition to the intermolecular and chemical adhesion forces , micromechanical adhesion also can be involved in the overall adhesion phenomenon . in such cases , the adhesive can effectively cling to a roughened substrate surface and increase overall adhesion , for example , chewing gum attached to the soles of our shoes . the strength of the adhesion between two materials depends on the interactions between the two materials , and the surface area over which the two materials are in contact . as a result , materials that wet against each other tend to have a larger contact area than those that do not , however , wetting depends on the relative surface energies of the adhesive and substrate materials . low surface energy materials such as poly(tetrafluoroethylene ) or ptfe and silicone materials do not wet and are resistant to adhesive bonding without special surface preparation , hence the use of these polymers to manufacture nonstick cookware and other nonstick surfaces . wetting is the ability of a liquid to form an interface with a solid surface and the degree of wetting is evaluated as the contact angle formed between the liquid and the solid substrate surface . this is determined by both the surface tension of the liquid and the nature and condition of the substrate surface . the smaller the contact angle and the lower the surface tension of the liquid , the greater the degree of wetting , that is , the droplet of liquid will spread across the substrate surface provided the latter is clean and uncontaminated , as shown in figure 4 . a clean surface allows good wetting , that is , the contact angle is close to 0 , figure 4(a ) . there will be a greater contact angle ( is greater than 0 but less than 90 , i.e. , 0 < < 90 ) with a slightly contaminated surface , figure 4(b ) , and the contact angle between the liquid and a contaminated surface or one with low surface energy will exceed 90 , figure 4(c ) . the latter condition is sometimes referred to as dewetting and the liquid will form droplets on the substrate surface . the contact angle is a function of both dispersive adhesion ( the interaction between the molecules in the adhesive and those of the solid , as discussed later ) and the cohesion within the liquid adhesive . if there is strong adhesion to the substrate surface and weak cohesion within the liquid , there is a high degree of wetting , often termed lyophilic conditions . conversely , a combination of weak adhesion and strong cohesion , referred to as lyophobic conditions , results in high contact angles and poor wetting of the substrate surface , that is , droplets form on the surface rather than a film of fluid . a small contact angle indicates more adhesion is present because there is a large contact area between the adhesive and the substrate , resulting in a greater overall substrate surface energy and a high interactive force between the liquid and the substrate . when the surface is wetted , the contact angle is less than 90 ( < 90 ) , the substrate has high surface energy and the adhesion forces between substrate and liquid are greater than the cohesive forces within the adhesive ( i.e. , the surface tension of the liquid , ) and the liquid can spread over the substrate surface . if the surface has low energy ( or is contaminated ) , > 90 and cohesion within the adhesive can exceed the adhesion between liquid and substrate such that there is poor wetting or dewetting , with the liquid forming droplets on the surface . surface scientists express things in a different way and refer to interfacial tension using the terms liquid - air interfacial tension , la ( i.e. , the liquid 's surface tension ) , solid - liquid interfacial tension , sl ( i.e. , the surface tension between the solid and the liquid , which approximates to the surface adhesion between liquid and solid ) and the solid - air interfacial tension , sa ( i.e. , the surface tension between the solid and air , which approximates to the surface energy of the solid ) , figure 5 . surface tension is commonly expressed as dyne / cm , although it should really be given in the recommended si units of n / m or j / m . figure 6 indicates the relative surfaces tensions of some common liquids . for a contact angle of , these entities are related by young 's equation , ( 1)lacos=sasl . if there is complete wetting of the substrate surface , that is , when = 0 and cos = 1 , young 's equation indicates that la = sa sl or la sa . in other words , if the surface tension of the adhesive ( la ) is less than the surface energy of the substrate surface ( sa ) , the adhesive will spread over the substrate . for maximum adhesion , the adhesive must completely cover or spread over the substrate , that is , effectively wet it . the contact angle between the adhesive and the substrate is , therefore , a good indicator of adhesive behavior . the value of sa when cos = 1 is the critical surface energy ( cse ) and equals the value of sl when the liquid just spreads over the surface . the critical surface tension of several materials is shown in figure 7 . the very large difference in cse between say glass and ptfe and polyethylene indicates the difficulty of bonding to the two resins . wetting of the surface occurs when the adhesive surface tension ( sl ) is less than the critical surface energy . this is often expressed as the adhesion quotient which requires the substrate surface energy ( sa ) to exceed the surface tension of the adhesive liquid ( sl ) by 10 dyne / cm . if the reverse is true , that is , ( sl sa ) , surface wetting is poor , adhesion is reduced and the adhesive tends to pull away from the surface during the curing process . the take home message here is that the adhesive liquid must wet the substrate surface and such factors as surface contamination , surface conditioning , presence of moisture , and the adhesive used all affect the adhesion between substrate and adhesive . a small contact angle indicates more adhesion is present because there is an interactive force between the liquid and solid phases . if the adhesive and substrate can form a compound at their interface or union , the ionic or covalent bonds that are formed result in a strong bond between the two materials . a weaker bond is formed when there is hydrogen bonding , that is , a hydrogen atom in one molecule is attracted to an electron - donor atom such as nitrogen or oxygen in another molecule . thus , when the surface atoms of an adhesive and substrate form ionic , covalent , or hydrogen bonds , chemical adhesion occurs . however , it can be seen that whereas the strengths of these chemical bonds can be high , figure 8 , their lengths are short and therefore for bonding to occur , surfaces with the potential for chemical bonding must be brought very close together and remain in this proximity for the bond to be stable . although the average lengths of hydrogen bonds are comparable to those of covalent and ionic bonds , they are an order of magnitude weaker . in the case of dental cements , zinc polycarboxylates provide some chemical bonding between the carboxylate molecule of the cement and hydroxyapatite mineral in the tooth , whereas bonding with zinc phosphate cements is wholly mechanical in nature . in dispersive adhesion or physisorption , the surfaces of two materials are held together by van der waals forces . the latter are the attractive forces between two molecules , each of which has a region of small positive and negative charge such that the molecules are polar with respect to the average charge density of the molecule ; it should be noted that there may be multiple poles ( regions of greater positive or negative charge ) with larger and/or more complex molecules . if these positive and negative poles are an inherent property of a molecule , they are known as keesom forces , whereas polarity , that is , a transient effect due to random electron motion within the molecules that cause a temporary concentration of electrons in one region are known as london forces . london dispersion forces , which result from statistical quantum mechanics , are particularly useful in adhesion because they arise without the need for either the adhesive or the substrate surface to have any permanent polarity . although van der waals bond lengths are longer than those of other molecular forces , see figure 8 , they are still short in absolute terms so that these forces only act over very small distances . about 99% of the work required to break van der waals bonds is performed once the joined surfaces are separated by more than a nanometer and , as a result , the effectiveness of adhesion due to chemical or dispersive bonding is limited . once a crack is initiated , it propagates easily along the interface because of the brittle nature of the interfacial bonds and , consequently , greater contact surface areas often provide little difference in the measured adhesion . some materials may merge or intermingle at the bonding interface by diffusion , typically when the molecules of both materials are mobile and/or soluble in each other , which typically is the case with polymer chains where one end of a molecule can diffuse into the other material . this form of interaction , known as interdigitation , occurs when a resilient denture liner is processed onto an acrylic resin denture base , or when a fractured denture is repaired with acrylic resin . in such cases , bonding arises from the mutual solubility and interactions between methyl methacrylate ( monomer ) in the repair ( or liner ) material and the surface of the poly(methyl methacrylate ) or acrylic base with diffusive adhesion ( bonding ) resulting from sections of polymer chains from the applied material interdigitating with the substrate surface . however , the mobility of the polymers strongly influences their ability to interdigitate to achieve diffusive bonding . cross - linked polymers are less capable of diffusion and interdigitation because of their restricted mobility whereas non - cross - linked polymers have greater mobility and interdigitate more readily . these differences account for the fact that it is easier to bond a resilient liner to a recently processed acrylic base , or even during processing of the denture base , because the acrylic resin has a greater surface reactivity , that is , there is greater mobility of its surface polymer chains , than when attempting to reline a denture base diffusive adhesion is also the mechanism involved in sintering as , for example , when metal or ceramic powders are compressed and heated so that atoms diffuse from one particle to the next to produce a solid mass . diffusive bonding occurs when atoms from one surface penetrate into an adjacent surface while still being bound to their surface of origin . this is the mechanism involved in the fusing of porcelain to metal in the fabrication of a pfm crown . since diffusive adhesion requires interaction of atomic species between two surfaces , the greater the time that the two surfaces can interact , the more diffusion occurs and , accordingly , the stronger the adhesion is between the two surfaces . when uncured , adhesives are fluid and they can flow over the substrate , filling the voids , rugosity , and pores of the surface and attach or this is often referred to as micromechanical adhesion and is shown schematically in figure 9 . micromechanical adhesion is the primary mechanism for luting of restorations to teeth with dental cements and probably also contributes significantly to bonding achieved with resin - based adhesives as , for example , in fissure sealants and direct bonding of restorative resins . the effectiveness of micromechanical adhesion is determined in large part by the wetting of the substrate by the luting agent in that poor wetting of the substrate by the luting agent will inhibit good apposition of cement and substrate . further , the luting agent must be able to flow into the surface voids , and so forth , and for this process to occur , the adhesive must have a low viscosity . water , for example , has a viscosity of 1 centipoise ( cp ) and that of alcohol is 1.2 cp . many other fluids , however , have much higher viscosities , for example , 9.22 cp for eugenol ( oil of cloves ) , 1490 cp for glycerin and ~10cp for honey , and the very large difference in the viscosities of honey and water explains why water flows far more readily than honey . it should be noted that the si units for viscosity are pa s ( pascal seconds ) and are equivalent in magnitude to often quoted cp values . inevitably , micromechanical adhesion of a luting agent to a surface is not simply a matter of wetting ( i.e. , contact angles ) and the rheological or flow properties of the adhesive . other factors also enter into micromechanical adhesion , notably the electrostatic forces ( both attractive and repulsive ) that may be operating between the adhesive and the micro - topography of the substrate as well as a property of the applied fluid known as thixotropy . a thixotropic fluid is one that under the action of mechanical forces such as stirring , vibration , and even kneading will temporarily transform to a state that has a lower viscosity and which exhibits better flow than the fluid in its static state . thixotropic behavior is an important characteristic for endodontic ( root canal ) sealants which are required to flow into a root canal , often under vibration . further , thixotropy is often incorporated into industrial and domestic paints by additives such as silicic acid and is probably present in various dental adhesive and cement formulations . thixotropy , when present in an adhesive , provides certain advantages to the overall adhesion system . in particular , when a thixotropic adhesive is applied to a substrate surface , it will remain in place , even on vertical surfaces . further , because adhesive flow is determined in part by the mechanical forces imposed on the adhesive , there can be greater control of the adhesive film thickness combined with improved flow into the microtopography of the substrate surface . it follows from the above that the adhesive bonded to a substrate often has a modified molecular structure at the bonding interface . this interfacial region is known as the adhesion zone ( figure 9 ) and is characterized by the changes in the adhesive ( and sometimes in the substrate ) that may arise from the bonding interactions . the transition zone , the region between the bonding interface and the bulk of the adhesive , is the area over which the chemical , mechanical , and optical properties of the adhesive differ from those of the bulk adhesive . it varies in thickness , from a few nanometers up to a few millimeters , with the thickness depending on the nature of the substrate surface , the chemical composition , and physical characteristics of the adhesive being applied and the curing conditions . where there are thick transition zones and/or narrow adhesion zones , the behavior of the entire bonding interface may be dependent on the properties of the transition zone because the properties , notably strength , of the adhesive may be impaired because of inadequate cohesion within the adhesive . it is considerations such as these that determine , at least in part , the selection of the optimum luting agent for the various combinations of luting agents and restorations that were discussed by pameijer in his review of luting agents . adhesive dentistry , whether it is the cementation ( or luting ) of a restoration to a prepared tooth or restoration with a composite resin , involves the application and curing of an adhesive at the interface between tooth tissue and the restorative material . consequently , all of the aspects of adhesion and cohesion discussed above are involved in this process . restoration with a composite material has three principal steps . the first is the creation of microporosity in enamel or dentin by acid etching either through application of an etchant or by the in situ action of an etchant / primer / adhesive . the second step is the application of a primer / adhesive which wets and penetrates the created microstructure although because the surface energies of etched enamel and etched dentin differ , different primers are required for the two substrates . finally , a resin is applied to the primed surface so that when polymerized in situ , it micromechanically ( i.e. , there is mechanical adhesion ) interlocks with the substrate microporosity together with a degree of chemical bonding , with some materials exhibiting better chemical adhesion than others . bonding to dentin presents greater problems than to enamel because it has a high organic content , a non - uniform composition and it is permeated by tubules . further , after mechanical treatment , a 315 m thick , featureless , and poorly adherent smear layer of organic debris will form . while this smear layer can provide pulpal protection by reducing dentin permeability bonding to dentin involves three stages , namely , conditioning , priming , and bonding , although some commercial bonding systems combine two or more stages into a single step . the conditioning stage involves modifying or removing the smear layer by acidic conditioners , the precise approach being determined by the bonding system used . priming is the key step in dentin bonding because it promotes interactions between hydrophobic restorative resins and hydrophilic dentin . primers ( dentin bonding agents ) are bifunctional molecules , one end being a methacrylate group that bonds to resin and the other a reactive group that reacts with dentin . thus , primers are coupling agents , that is , they are bifunctional molecules that primarily bond to calcium but may also interact with collagen . the bonding ( adhesive ) agent is a fluid resin that flows over and wets the primed surface to form an effective bond when cured in situ . it should be noted that many manufacturers combine many of the conditioning , priming and bonding steps in their systems . if the primer and conditioner are combined as with self - etching primers , the smear layer is incorporated within the primer that directly contacts the dentin and constitutes the adhesive zone . an advantage with self - etching primers is that the dentin is maintained in a moist condition throughout the bonding procedure although enamel etching with such systems is less effective than with phosphoric acid treatment . alternatively , the primer and adhesive may be combined so that the applied material will infiltrate the collagenous network created by conditioning to form a hybrid ( resin - infiltrated reinforced ) layer . although high bond strengths ( 20 mpa ) to dentin may be achieved , bond failures commonly involve cohesive fracture of the dentin such that these systems are not infallible . they tend to be technique and material sensitive and may require successive treatments for optimal bonding . further , regardless of high bond strengths , which suggest good adaptation to the dentin , good bonding and the absence of leakage are not synonymous and no system provides consistent leak - free restorations . it follows from the above discussion that the performance of an adhesive in the luting of a restoration to a tooth will be dictated by a multiplicity of factors . ideally , laboratory bond strength test values and the resistance of luted restorations to clinical loads will be maximized when the propagating crack that causes bond failure has to travel through the adhesion zone rather than the bulk adhesive . in other words , optimal retention is achieved when adhesion rather than the cohesive strength of the adhesive determines the overall strength of the bond . nevertheless , the mechanical properties of the luting agent often can have a marked impact on the resistance of the luted restoration to applied forces when the thickness of the cement film is markedly greater than the width of the adhesion zone , as noted by in vivo determinations of cement film thicknesses beneath restorations .	the phenomena of adhesion and cohesion are reviewed and discussed with particular reference to dentistry . this review considers the forces involved in cohesion and adhesion together with the mechanisms of adhesion and the underlying molecular processes involved in bonding of dissimilar materials . the forces involved in surface tension , surface wetting , chemical adhesion , dispersive adhesion , diffusive adhesion , and mechanical adhesion are reviewed in detail and examples relevant to adhesive dentistry and bonding are given . substrate surface chemistry and its influence on adhesion , together with the properties of adhesive materials , are evaluated . the underlying mechanisms involved in adhesion failure are covered . the relevance of the adhesion zone and its importance with regard to adhesive dentistry and bonding to enamel and dentin is discussed .
multiple sclerosis ( ms ) is known as an immune - mediated multifocal demyelinating disorder , which consists of components of neuroinflammation and neurodegeneration.1 it mostly occurs between the ages of 18 and 45 and has a substantial economic and social burden.1,2 the most common symptoms include spasticity , fatigue , balance problems , weakness , sensory disturbance , neurogenic bladder and bowel , cognition impairment , depression , pain , sexual disorder , emotional lability , blurred vision , and speech difficulties , in the order of their frequency of occurrence.1,2 compared with the 60%80% prevalence rate of spasticity due to the involvement of the pyramidal system , the involuntary movement disorders due to the lesion of the extrapyramidal system , such as myoclonus , spasmodic torticollis , paroxysmal dystonia , chorea , and ballism are very rare in patients with ms.2 in this case report , we describe a woman with relapsing - remitting ms , complicated with disabling paroxysmal dystonia , which responded very well to the 4-day administration of acetazolamide . this 40-year - old woman had unremarkable past medical , family , and movement disorder history . in july 2005 , at age 34 , the patient initially presented with blurred vision , slurred speech , dysphagia , gradual onset of left hemiparesis , and ataxia . surveys of infectious diseaes were all negative , including rapid plasma regain , venereal disease research laboratory , human immunodeficiency virus , and cultures . the visual and somatosensory evoked potential indicated a right optic nerve lesion and right thoracolumbar myelopathy , respectively . magnetic resonance imaging revealed multiple lesions at the cerebellum , brainstem , corpus callosum , as well as at the cervical and thoracic spinal cord . the above findings in this patient met the criteria for the diagnosis of multiple sclerosis . , she suffered from left optic neuritis , weakness in four extremities with spasticity , left paresthesia , neuropathic pain over her chest and back , and impaired proprioception . after pulse steroid therapy , she was transferred to an acute rehabilitation ward for further training . seven days after the onset of this ms relapse , involuntary movement developed in her left hand , which consisted of nonrhythmic posture of alternative flexion and extension of the fingers , abduction and adduction of the thumb , opponens of the thumb and little finger , flexion , and extension of the wrist . the involuntary posture was usually aggravated by voluntary motion , attenuated at rest , and might subside temporarily while she slept . it did not involve the proximal upper limbs , lower limbs , trunk , face , and neck . there were no other neurological deficits , such as conscious disturbance , aura , and fatigue , indicating seizure disorders . the paresis , spasticity , and neuropathic pain improved after the treatments with steroid , interferon beta-1b , baclofen , and imipramine ; however , the paroxysmal dystonia persisted despite the above treatments and occupational therapy . , she accepted the prescription of oral acetazolamide ( 250 mg , three times a day ) 17 days after the onset of dystonia . the patient felt progressive reduction in the amplitude of the disabling dystonic movement at first , then in the frequency of symptoms . , she could continue the rehabilitative programs , and her hand functions were resumed to premorbid status . there was no recurrence of dystonia in the following 17 months of follow - up . paroxysmal dystonia is characterized by intermittent onset of sustained muscle contractions causing patterned twisting and repetitive movements or abnormal postures that might last seconds to hours.1,3 the idiopathic paroxysmal dystonia can be subdivided into kinesigenic , nonkinesigenic , hypnogenic , and exercise - induced forms.3 there were also some acquired paroxysmal dystonia , induced by the lesions involving the basal ganglion , extrapyramidal tract system , or thalamus.3,4 a widely accepted mechanism of dystonia is the increased motor cortical excitability by altered thalamic signaling.5 paroxysmal dystonia is rare in patients with ms . the pathogenesis of paroxysmal dystonia in patients with ms remains unknown.4 several mechanisms have been proposed , including axonal irritability secondary to the release of inflammatory mediators,4 a transversely spreading ephaptic activation of axons within the partially demyelinated lesion at any level in the motor fiber tracts , which was supported by a transcranial magnetic stimulation study.5 beyond the typical localizations , there were some sporadic case reports disclosing the demyelinated plaques in the cervical spinal cord , midbrain , medulla , internal capsule , cerebral peduncle , and even some cases without anatomic evidence in patients with ms.1,2,4,5 some investigators explained that the nature of lacking anatomic evidence for ms may be caused by secondary immune components , such as antibasal ganglia antibodies.6 the reported features of paroxysmal dystonia in ms were frequent attacks ( several times daily ) , short duration ( seconds to minutes mostly ) , and stereotyped posture of unilateral dystonia of the arm , leg , and sometimes the face and neck , which could not be attributed to an epileptic origin.2,5,712 there were some typical trigger factors , such as startle reaction , hyperventilation , stress , and fatigue.3 paroxysmal dystonia in some patients may subside within a few weeks spontaneously or after treatment.7 the most commonly prescribed medication was carbamazepine , but some patients did not have a satisfying response.2,4,12 a few cases reported trying to suppress symptoms with acetazolamide,911 phenytoin,12 adrenocorticotropic hormone,13 cannabis,14 and botulinum toxin.15 the therapeutic effects varied , and acetazolamide was considered to be the one with fewer side effects . in 1992 , sethi et al9 first adopted acetazolamide alone or in combination with carbamazepine in treating three patients with central demyelinating disease and paroxysmal dystonia . nardocci et al10 noted that paroxysmal dystonia completely receded with a 4-day treatment of acetazolamide ( 250 mg , three times a day ) , and paroxysmal tremor was also attenuated by a 1-month treatment in a patient . waubant et al11 reported that the symptoms stopped in a few hours post - acetazolamide treatment ( 250 mg , twice a day ) in their patients . waubant et al prolonged the treatment for 1 month , and the symptoms did not recur . in addition , acetazolamide was also proven to be effective in hereditary episodic ataxia type 2 , another kind of movement disorder.3,16 the exact mechanism of acetazolamide for paroxysmal dystonia in ms remains unclear . acetazolamide is a carbonic anhydrase inhibitor , which fosters metabolic acidosis across the blood brain barrier , thereby lowering the intracellular ph that reduces the potassium channels conductance and might alter the transmembranous potential and neuron excitability.1618 in addition , it could alternate the efflux of intracellular bicarbonate through -aminobutyric acid ( gaba)-activated channels.19 an animal study gave evidence that showed acetazolamide exerts antidystonic effects in the dt mutant hamster via inhibiting bicarbonate regeneration and reducing gaba - mediated excitation without affecting gaba - mediated inhibition.19 therefore , the neuronal hyperexcitability might be reduced , and the paroxysmal dystonia improved consequently . to the best of our knowledge , this type of ms with long - lasting paroxysmal dystonic posture of the hand is extremely rare in the previous literature . the common trigger factors of dystonia , such as startle reaction , hyperventilation , stress , and fatigue , did not induce the patient s dystonia . her dystonia did not respond to treatment with steroid , interferon , and baclofen . because of the good responses and few side effects of acetazolamide in previous case reports for typical short - lasting paroxysmal dystonia in ms,911 we treated our patient with this drug with a dosage of 250 mg , three times a day . there was no recurrence of dystonia in the next 17 months of follow - up . this dramatic effect of acetazolamide in our patient with ms and dystonia was an inspiring finding that would be of great interest for further study . paroxysmal dystonia is rare in patients with multiple sclerosis , but it significantly endangers the independence of patients in basic daily living activities . in addition to the policy of wait and see , medications with few side effects , such as acetazolamide , might be one of the treatment alternatives . we shared our clinical experience and advocate this effective and safe treatment option with acetazolamide for individuals with multiple sclerosis and dystonia .	dystonia is a rare manifestation of multiple sclerosis ( ms ) , but it always interferes with the functional performance and quality of life . we report a rare case of long - lasting paroxysmal dystonia associated with ms . the patient was a 40-year - old woman with relapsing- remitting ms for 6 years . during the latest attack of ms , she suffered from long - lasting paroxysmal dystonia in her left hand . despite treatment with pulse high - dose intravenous methylprednisolone , interferon , and baclofen , along with occupational therapy , the dystonia persisted and significantly bothered her daily activities . finally , she was treated with oral acetazolamide ( 250 mg , three times a day for 4 days ) , which was very effective for the control of her dystonia . the dystonic movement subsided without recurrence in a follow - up of 17 months . we advocate this effective and safe treatment for patients with paroxysmal dystonia associated with ms .
recent literature has refined the umbrella term malnutrition to include such conditions as starvation , sarcopenia , and cachexia [ 13 ] . in recent years , several consensus definitions for the diagnosis of sarcopenia [ 1 , 4 ] and cachexia [ 1 , 5 ] have been published , however , there is no indication that further work has been conducted to validate any of these definitions . consequently , there is still confusion over the diagnosis of these conditions as there are no universally shared criteria . however , indications are that simple starvation is the loss of fat and fat - free mass which occurs purely as a result of protein - energy deficiency ; sarcopenia is the loss of muscle mass and muscle strength which occurs with ageing ; cachexia is severe wasting , predominantly of fat - free mass , driven by inflammation [ 47 ] . guidelines used in practice , including the dietitians association of australia ( daa ) endorsed evidence - based practice guidelines for the nutritional management of malnutrition in adult patients across the continuum of care , invariably take a simplistic approach to malnutrition through focusing on protein - energy malnutrition , or starvation , alone [ 2 , 7 , 8 ] . the implications of the scarcity of recommendations for identification and treatment of alternate malnutrition conditions ( sarcopenia and cachexia ) are potential misdiagnosis and treatment , and ultimately suboptimal health outcomes . while increased energy and protein intake may be the principal , and most appropriate , intervention for treatment of starvation , it is becoming clear that conditions such as sarcopenia and cachexia can not be reversed by increased dietary intake alone . investigation into pharmacological therapies for sarcopenia has not provided strong evidence for efficacy , however , there is support for resistance training , in addition to adequate energy and protein intake , as a safe and effective therapy . considerable research has been conducted in an effort to find an effective therapy for cachexia . potential interventions include megestrol acetate , ghrelin , and fish oil , however , success has been limited and research is continuing . in the battle to combat malnutrition dietitians , are in the frontline yet the success of their practice could be limited unless they are armed with the correct knowledge in terms of identification and appropriate treatment strategies . the aim of this project was to determine whether dietitians understand and use the terms starvation , sarcopenia , and cachexia and provide targeted treatment strategies accordingly . an anonymous cross - sectional survey of members of the daa was undertaken to gain information on their attitudes and practices regarding the diagnosis and treatment of malnutrition in adults . all members are required to have graduated from a tertiary program designed to achieve entry level competencies as prescribed by the daa . members of the daa , including student members , were invited to participate via weekly email distribution from daa national office , in august 2010 . the email notified of the presence of a link to an online questionnaire and provided a brief introduction to the survey to enable prospective participants to gauge their level of interest in the project . upon entering the web - based survey , the daa website indicated a potential pool of approximately 4000 members , and all were eligible to participate in the survey . the protocol for this research project was approved by the social and behavioural research ethics committee of flinders university , adelaide , south australia , and conforms to the provisions of the declaration of helsinki ( as revised in edinburgh 2008 ) . this study was registered on the australian new zealand clinical trials registry ( actrn12610000403033 ) . following a review of the current literature on identification and classification of categories of malnutrition , including relevant consensus definitions [ 1 , 4 , 5 ] , a questionnaire was drafted by the authors using an online survey facility ( surveymonkey , palo alto , calif , usa ) . the tool was piloted on a small group of clinical dietitians and subsequently amended to reflect their comments where necessary . the final web - based questionnaire comprised a maximum of 42 questions ( some conditional release ) , depending on responses given ( see table 1 ) , and was available online from august - september 2010 . participants were asked to complete the survey by selecting appropriate answers from random - ordered lists provided and by giving further comment where necessary . a copy of the survey is available from the corresponding author on request . demographic information collected included age , gender , years of practice , and location . in order to ascertain current practice and use of terminology , respondents were asked to provide information on criteria for diagnosis , exposure to malnourished patients , use of the terms starvation , sarcopenia , and/or cachexia and criteria for diagnosis of any categories used . three case studies were provided ; one describing a patient in each of the three categories of malnutrition of interest , according to current literature [ 1 , 5 , 9 ] , and respondents were asked to provide a diagnosis based on information given . treatment regimes were investigated in a question asking respondents to rank ten possible interventions for malnutrition , from one to ten . further investigation was then conducted into the use of specific interventions ( fish oil , appetite stimulants , and resistance training ) , which have been investigated as potential therapies for sarcopenia and cachexia [ 10 , 14 , 15 ] . chi - squared tests were used to compare case study responses for selected subgroups of study participants . statistical significance was set at p < 0.05 . two hundred and twenty - one daa members , including ten student members , accessed the web - based survey during the data collection period , representing a response rate of approximately 5.5% . of those , 209 ( 95% ) provided full demographic details and 205 ( 93% ) continued to the main part of the survey . in total , 169 ( 76% ) responses were received from all states and territories in australia , with the majority from victoria ( 30% ) and new south wales ( 30% ) . almost half of respondents worked in the public hospital system ( 49% ) and one - quarter reported some work in private practice ( 25% ) . around 40% of survey participants reported seeing 25 malnourished adults each week , with some reporting that they consulted 10 , particularly in the aged care setting . with regard to terminology to describe categories of malnutrition , 81 respondents ( 43% ) indicated that they used at least one of the terms starvation , sarcopenia , and/or cachexia . of those , most used cachexia ( 89% ) , followed by starvation ( 31% ) and sarcopenia ( 19% ) . the most common criteria for diagnosis of malnutrition were reported to be muscle wasting ( 86% ) , loss of subcutaneous fat ( 81% ) , weight loss ( 77% ) , decreased appetite ( 76% ) , bmi < 18.5 kg / m ( < 22 kg / m for those aged 65 + years ) ( 67% ) , and anorexia ( 65% ) . for respondents reporting that they used the term starvation , the top three criteria used to diagnose this condition were muscle wasting ( 38% ) , anorexia ( 36% ) , and low bmi ( 36% ) . for respondents reporting that they used the term sarcopenia , the top three criteria used to diagnose this condition were muscle wasting ( 31% ) , loss of muscle strength ( 28% ) , and weight loss ( 16% ) . for respondents reporting that they used the term cachexia , the top three criteria used to diagnose this condition were muscle wasting ( 76% ) , loss of subcutaneous fat ( 74% ) , and anorexia ( 69% ) . when responding to the case study questions , the majority of respondents ( 77% , 63% ) used the term malnutrition to refer to what were clear cases of starvation and cachexia , respectively . the correct diagnosis of starvation was recorded by 6% of respondents ; 46% of respondents correctly identified the sarcopenia case ; 21% correctly identified cachexia . when case study responses were examined according to years of dietetic experience , more respondents with 5 or less years of experience correctly identified the condition in each case study , however these differences were not statistically significant ( see table 3 ) . when participants were asked to rank , from 1 to 10 , a range of possible interventions to treat malnutrition , according to their own criteria , the most frequently selected therapies were high - energy high - protein diet ( 75% of respondents ) , high - energy high - protein snacks ( 48% ) , oral nutritional supplements ( 42% ) , and enteral feeds ( 32% ) . three selected therapies were further investigated : 21% of respondents have recommended appetite stimulants to patients , 45% have recommended fish oil , and 57% have recommended resistance training . fifty percent of those recommending appetite stimulants indicated that this recommendation was for less than 5% of patients . similarly , 64% of those recommending fish oil , and 46% of those recommending resistance training , did so to less than 5% of their patients . most commonly , respondents referred patients for prescription appetite stimulants ( 56% ) and referred to a physiotherapist for resistance training ( 71% ) but made their own recommendations for fish oil . fish oil was proposed most often for high cholesterol ( 75% ) and rheumatoid arthritis ( 71% ) , with most respondents advising a daily dosage of 1 g of omega-3 fatty acids per day . the results from this cross - sectional survey provide insight into the inconsistency in the understanding and use of the terms starvation , sarcopenia , and cachexia amongst australian dietitians . furthermore , it appears that treatment strategies are generally focused on improving energy and protein intake , a strategy that alone is known to be largely ineffective in the treatment of cachexia and sarcopenia [ 7 , 10 ] . alternative terminology for different categories of malnutrition was not widely used amongst australian dietitians , with less than half of respondents using any of the terms starvation , sarcopenia , and/or cachexia . while the recently released daa endorsed evidence based practice guidelines for the nutritional management of malnutrition in adult patients across the continuum of care are explicit in their reference to protein - energy under nutrition only ; other commonly used guidelines and tools do not differentiate between categories of malnutrition [ 1719 ] , therefore they do not provide dietitians with the necessary guidance to correctly diagnose different malnutrition conditions . those who use the terms starvation , sarcopenia and/or cachexia do not appear to be consistent in using the most accepted criteria as part of their diagnosis . simple starvation is unintentional weight loss , with predominant loss of fat mass , purely as a result of inadequate protein and energy intake , yet 38% of respondents indicated that muscle wasting was the key criterion for diagnosis , more than reduced bmi or weight loss . the key characteristics of cachexia are elevated inflammatory markers and reduced body cell mass , so while 76% of respondents indicated that muscle wasting was their key criterion for diagnosis , only 24% would consider elevated inflammatory markers . sarcopenia is characterised by reduced muscle mass , strength , and function [ 1 , 4 ] , and 76% of survey participants indicated that muscle wasting was again the key criterion , but 50% of respondents indicated that muscle strength was a consideration hence this appears to indicate a clearer understanding of sarcopenia amongst australian dietitians . ability to identify the three conditions from case study information was poor , with 6% of respondents able to correctly diagnose starvation , 46% sarcopenia , and 21% cachexia . this gives further indication that dietitians are more familiar with sarcopenia than either starvation or cachexia and suggests that there is a need to increase the awareness among dietitians of the existence of separate categories of malnutrition and to educate on alternate diagnoses . however , for each case study , more newer graduates ( 5 or less years of experience ) than those with 6 or more years of experience , correctly identified the different categories of malnutrition . although this did not reach statistical significance it does suggest that these categories may receive more attention in current university curriculums . it is noteworthy that 63% of respondents to the cachexia case study indicated a diagnosis of malnutrition . while this is technically not incorrect as all cachectic patients are malnourished , the case study provided information on inflammatory status which should have alerted dietitians to the presence of cachexia . the inconsistency identified in the understanding and application of the terms sarcopenia , starvation , and cachexia extended to the interventions used to treat patients / clients . therapy for malnutrition was predominantly centred on increased energy and protein intake with most dietitians prescribing high - energy high - protein diet , snacks and/or enteral feeds . however , the literature indicates that the defining characteristic of starvation is that it is reversed by increased protein and energy intake , unlike sarcopenia or cachexia , therefore , provision of standard interventions , regardless of the aetiology of malnutrition is likely to address starvation alone . there are some limitations which should be considered in the interpretation of the data from this survey . participants were from australia and it should be acknowledged that the findings may differ elsewhere and therefore a repeat survey of dietitians from other countries would be valuable . the sample size may be considered small , however the response rate was similar to the expected level of response to surveys previously delivered by the organisation ( daa national office , personal communication , 2010 ) . it is also noteworthy that the majority of respondents were young and had been practicing for a short time and worked in metropolitan public hospitals . this profile reflects the workforce statistics for dietitians in australia ( daa national office , personal communication , 2010 ) . finally , the diagnostic criteria used in the questionnaire were based on consensus definitions which have not yet been validated however they were developed by leading experts in the field and are therefore likely to be proved to be valid and reliable in future work . further investigation into three interventions previously researched for use in different categories of malnutrition revealed that appetite stimulants , fish oil , and resistance training are not widely recommended to malnourished patients . recent research has shown that these interventions can be helpful in conjunction with adequate energy and protein intake to address sarcopenia and cachexia , which are unlikely to resolve purely with increased nutritional intake [ 10 , 14 , 15 ] . however , currently used guidelines for treatment of malnutrition do not advocate targeted therapies for malnutrition [ 8 , 17 , 19 ] thus do not provide dietitians with the correct advice to deliver optimal health outcomes to patients . in conclusion , the results of this study indicate a need for increased awareness of starvation , sarcopenia , cachexia , and appropriate methods of identification and treatment . more work is warranted to explore how dietitians might expand existing or collaborate to develop new screening and/or assessment tools to assist in differentiating the three distinct conditions , contribute to the evidence base for appropriate treatment strategies , and determine the cost effectiveness of this change in practice .	malnutrition is an umbrella term that includes starvation , sarcopenia , and cachexia ; however , differentiating between these terms is infrequent in clinical practice . given that the effectiveness of treatment depends on the aetiology of unintentional weight loss , it is important that clinicians are aware of the defining characteristics . the aim of this study was to determine whether australian dietitians understand and use the terms starvation , sarcopenia , and cachexia and provide targeted treatment strategies accordingly . members of the dietitians association of australia were surveyed to gain information on practices and attitudes to diagnosis and treatment of adult malnutrition . in addition , three case studies were provided to examine understanding of starvation , sarcopenia , and cachexia . 221 dietitians accessed the survey . 81 respondents ( 43% ) indicated the use of at least one alternate term ( starvation , sarcopenia , and/or cachexia ) . muscle wasting was the most commonly used diagnostic criterion . high - energy high - protein diet was the most common therapy prescribed . correct diagnoses for case studies were recorded by 6% of respondents for starvation , 46% for sarcopenia , and 21% for cachexia . there is a need for increased awareness of the existence of starvation , sarcopenia , and cachexia amongst australian dietitians and research into appropriate methods of identification and treatment for each condition .
the proposed criteria for the diagnostic and statistical manual of mental disorders , fifth edition ( dsm-5 , american psychiatric association , 2013 ) characterize hypersexual disorder ( hd ) as a repetitive and intense preoccupation with sexual fantasies , urges , and behaviors , leading to adverse consequences and clinically significant distress or impairment in social , occupational , or other important areas of functioning . one defining feature of the proposed disorder includes multiple unsuccessful attempts to control or diminish the amount of time an individual engages in sexual fantasies , urges , and behavior in response to dysphoric mood states or stressful life events ( kafka , 2010 ) . while some have suggested hd should be considered a behavioral addiction the empirical literature appears to fall short of supporting this conceptualization at the present time ( kor , fogel , reid & potenza , 2013 ) . nevertheless , there are many parallels concerning hd and addictive - related disorders and this appears especially true for shared commonalities between hd and a gambling disorder ( farre et al . , 2015 ) . however , it is unclear how severity for hypersexual behavior should be conceptualized and this is the first paper in the literature to explore how hd severity might be operationalized . this article is intended to be a catalyst to advancing some perspectives and discussion about assessing severity related to hd . as a caveat , the current article uses the term hd or hypersexual behavior for purposes of referring to the hd criteria proposed for dsm-5 while recognizing that ultimately the proposed disorder was excluded in the final publication for a number of reasons discussed elsewhere ( reid & kafka , 2014 ) . however , given the results of the dsm-5 field trial ( reid et al . , 2012 ) , the hd criteria are commonly used as the standard for measuring this phenomenon in clinical and research applications . the cambridge online dictionary defines severity as something serious or causing great pain , difficulty , or damage . severity in the dsm-5 has typically been operationalized from mild to severe and assigned to a diagnosis based on the number of symptom criteria endorsed . in the section on substance - related and addictive disorders , severity across time is also measured by reductions or increases in the frequency and/or doses of substances used . likewise , severity for gambling disorder is assigned by the number of symptoms endorsed at the time of evaluation . however , the dsm-5 operationalization of severity for addictive - related disorders is debatable and this is certainly true for hd for a vast array of reasons articulated below . symptom count is a gross measure of severity and makes an assumption that all symptoms for a given disorder are equal . during the dsm-5 field trial for hd , some patients barely met the diagnostic threshold for hd that would have likely been considered more serious or severe cases but such impressions were based on an examination of the broader scope of their overall clinical presentation that extended beyond the dsm-5 proposed criteria . this observation has also been noted elsewhere in substance - related disorders ( moss , 2011 ) . complicating matters , symptom endorsement for hd for example , several substance - use disorders can be diagnosed with as few as 2 of 10 or more symptoms . an hd diagnosis , however , required 4 out of 5 of the a criteria , evidence of impairment , and symptoms had to occur independent of a substance - related disorder , medical condition , or manic episode ( see figure 1 ) . collectively , the current structure of the hd proposed criteria makes severity by symptom endorsement difficult insofar as someone meeting the threshold for the diagnosis endorses 80% of the symptoms ( e.g. , a patient meeting criteria for hd would have either endorsed 4 or 5 of the a criteria , which does not allow for the current tri - categorized dsm-5 severity designations mild , moderate , and severe ) . the threshold for meeting the hd criteria would need to be lowered to 3 out of 5 of the a criteria to accommodate severity categorizations of mild , moderate , and severe , however , such a change would potentially introduce the problem of false - positive hd diagnosis . finally , symptom count as an index of severity also ignores the magnitude of how someone might experience a given symptom . for example patients may report significant clinical distress but the magnitude of the distress may vary across individuals . this is why someone with a greater magnitude across a few symptoms might be considered a more severe case than someone with more symptoms but of a lesser magnitude . given the limitations of the symptom - count approach to operationalizing severity , studies are needed to compare this method with alternative approaches . the field of hypersexuality research might consider some of the work challenging substance - use disorder severity designations as a model for research that could be applied to the hd criteria ( fazzino , rose , burt & helzer , 2014 ) . dsm-5 proposed criteria for hypersexual disorder one approach to assess severity might consider how much time is spent engaging in sexual fantasies , urges , and behavior ( e.g. time spent planning for and recovering from sexual activities ) . generally time as measured by the frequency and duration of sexual fantasies , urges , and behaviors can vary and may influence whether an activity is considered excessive or even problematic . for example , masturbation once a week for 15 minutes might not be considered excessive or problematic whereas sex with an extra - dyadic partner outside a monogamous committed relationship once a week for 15 minutes is likely both problematic and excessive . these examples raise questions about whether severity should consider the specific manifestation of sexual behavior . while no attempt was made to pathologize the expression of sexual behaviors with the dsm-5 proposal for hd , it is important to ask how the type of sexual behavior linked to hypersexuality might play a role in assessing levels of severity . for example , do solo - sex behaviors carry more or less potential to contribute to a severity index than relational sexual activities ? as evident above , considering severity based exclusively on level of excessiveness or frequency of a sexual activity has several limitations . another approach might consider severity based on the extent that hypersexual behavior causes significant impairment to one s social , occupational or other important areas of functioning . contributes to repetitive hypersexual behaviors that create consequences which impair one s ability to function . for example , multiple unsuccessful attempts to control or significantly reduce sexual fantasies , urges and behaviors might suggest a greater level of severity . perhaps diminished control in one context might have greater consequences than others ( e.g. inability to refrain from pornography on a work vs. a home computer ) . of course , the notion of diminished control makes an assumption that attempts to control the frequency or manifestations of hypersexual behavior have occurred . in cases where a patient may be pre - contemplative about change , it is possible attempts to control or change their behavior have never been made , and thus patients would deny experiencing multiple unsuccessful attempts to control their sexual behavior . further , the presence or absence of such efforts may occur , independent of the severity of their condition . consequences however , vary depending on several other factors and some consequences might be considered more severe based on the implications , frequency , or subjective values . in attempt to understand consequences , researchers developed the hypersexual behavior consequences scale which offered several insights about the types of consequences encountered by hypersexual patients ( reid , garos & fong , 2012 ) . for example , consequences leading to interference with friendships typically have different ramifications than those leading to divorce . a consequence such as job loss might also vary in severity depending on other variables . for example , job loss might be associated with greater severity if one is financially struggling for money or if it is highly publicized in the media as with some political scandals , celebrities , or well - known sports figures . the related legal problems that can be associated with hypersexual behavior can also heighten the seriousness of a case presentation and by extension , how severity is conceptualized . a patient s moral values might also influence whether a specific consequence is considered severe . one patient marginalized the consequence of an unintended pregnancy resulting from hypersexual behavior stating it was nt an issue , she just got an abortion . however , upon further investigation , it was discovered the woman felt pressured into having the abortion and afterward experienced significant depression , shame , and guilt as it contradicted her religious values . subsequently , it might be important to consider the extent to which others may be physically or emotionally harmed by hypersexual behavior and such factors weighted in assessing severity . further , a way of quantifying consequences is necessary to determine what types of consequences should be given more attention in assessing severity . an extension of the vast array of consequences encountered by hypersexual patients might consider whether hypersexual behavior contributes to comorbid psychological conditions such as depression , anxiety , substance abuse , suicidality or personality traits such as shame ( raymond , coleman & minor , 2003 ; reid , stein & carpenter , 2011 ) . such cases require special consideration and their complexity can influence the seriousness , pain , or suffering leading to a greater level of severity . repetitively engaging in sexual behavior while disregarding the risk for physical or emotional harm to self or others . arguably , not all risk taking behavior is equal and can vastly influence how severity might be operationalized . the word severe can also imply damage and some risk - taking behavior has significantly higher potential for greater damage . some examples might include risks associated with sexually transmitted infections , unintended pregnancies , physical harm to self or others , legal problems , job loss , divorce , and so forth . during the dsm-5 field trial , patients reported masturbating to the extent of incurring genital lesions requiring medical attention . some described watching pornography on their digital devices while driving placing them at risk for automobile accidents . a few patients meeting criteria for hd also had a comorbid paraphilic disorder of autoerotic asphyxiation , which has also been linked to unintended deaths . several patients described pursuing sex in neighborhoods that were known to have high crime rates that compromised their physical safety . while not part of the dsm-5 criteria for a gambling disorder , several studies show individuals with a gambling disorder and co - occurring illegal activities associated with gambling exhibited greater levels of gambling severity in comparison to gamblers without criminal histories ( ledgerwood , weinstock , morasco & petry , 2007 ; potenza , steinberg , mclaughlin , rounsaville & omalley , 2000 ; strong & kahler , 2007 ) . subsequently , if gambling research generalized to hypersexual patients , those who commit illegal acts in order to engage in sexual activities may represent more severe cases . collectively , the level of risk a patient is willing to disregard to pursue sexual fantasies , urges , and behaviors should be considered in assessing levels of severity . understanding severity for hd based on sexual cravings and urges creates several challenges . first , unlike substance - use disorders where abstinence is often a goal , clinicians try to help hypersexual patients reorganize their relationship with sexual cravings in order to cultivate healthy expressions of sexuality . thus , a sexual craving leading to sexual behavior may not always be problematic whereas a craving to use cocaine is likely always considered problematic . another challenge is the lack of research specifically focused on the role of cravings in the etiology or maintenance of hypersexual behavior . the field is in its infancy and only recently began developing measures of craving ( kraus & rosenberg , 2014 ) . however , more investigations are needed to understand the role of craving and its relationship to the concept of severity . for example , ecological momentary assessment could be used to further understand how cravings manifest among hypersexual individuals ( shiffman , stone & hufford , 2008 ) . such research might help resolve existing controversy where some have argued hd falls short of a pathological condition and is merely a manifestation of high sexual desire ( e.g. craving ) of behaviors and not an addictive process per se ( carvalho , stulhofer , vieira & jurin , 2015 ; steele , staley , fong & prause , 2013 ; winters , christoff & gorzalka , 2010 ) . as the field evolves , researchers might consider drawing on the work of those in the field of gambling disorders who have found some evidence between self - reported cravings and relapse ( oei & gordon , 2008 ) , risk - taking propensity in wagering ( ashrafioun , kostek & ziegelmeyer , 2013 ) , and gambling severity as measured by the problem - gambling severity index ( ferris & wynne , 2001 ) among gambling populations ( young & wohl , 2009 ) . however , other researchers in the field of behavioral addictions have considered alternative models to help explain the construct of cravings such as those often reported in food addiction ( rogers & hendrik , 2000 ) . collectively , further research is needed to understand the construct of cravings among hypersexual populations in order to consider how sexual cravings might be linked ( if at all ) in characterizing the severity of hd . regardless , cravings , as an index of severity , relapse , treatment response , or etiology , is likely to pose a difficult and challenging area of scientific inquiry as noted by other prominent craving researchers in the field of substance - use disorders ( tiffany & wray , 2009 ) . during the dsm-5 field trial for hd , the onset and clinical course of hd among participants was explored ( reid , carpenter et al . , 2012 ) . while the proposal limited an hd diagnosis to adults over the age of 18 , surprisingly , 54% of participants stated they began to experience difficulties regulating their sexual behavior as adolescents and 30% during college - age years . most ( 82.6% ) reported a gradual onset lasting several months or years with 17.4% indicating a rapid - acute onset > 90 days . the clinical course was split with 48.6% reporting a continuous pattern of hypersexual behavior and 51.4% stating episodic patterns . the substantial number of participants reported patterns of escalation including time ( 83.5% ) , frequency or intensity ( 81.7% ) , types of manifestations ( 62.4% ) , and increased risk ( 60.6% ) . there is no consensus about how these data might be associated with severity of hd , but similar data have been considered as surrogates for other indicators of severity in alcohol use disorders ( e.g. hingson , heeren & winter , 2006 ) . at present , further research is needed to investigate what implications the onset and clinical course of hd symptoms may have on attrition in therapy , treatment adherence , relapse , refractoriness to treatment all which might be markers of severity and the overall prognosis for hypersexual patients . such research might consider naturalistic longitudinal studies assessing the trajectory of sexual behaviors and hd across the lifespan in relation to different conceptualizations of severity . recently , item response theory ( irt ) has been used to evaluate severity ( saha , chou & grant , 2006 ) based on the assumption that symptoms less commonly endorsed reflect outliers with greater problems or severity . a key advantage of irt is the ability to select items based on the information they contribute which can inform the development of a severity index ( conway et al . , 2010 ) . for example , an irt approach has been used to assess the continuum of gambling problems using dsm criteria in exploring an index of problem severity ( strong & kahler , 2007 ) . alternatively , cluster analysis could be used to examine latent dimensions of hd and used to assess how best to threshold cases among populations of patients seeking help for hypersexual behavior . survival analysis is a plausible solution to evaluate the relationship between time and symptom remission in hypersexual patients allowing insights about treatment profiles and resistance to change . this resistance could subsequently be utilized as part of classifying a severity index . for example , if a particular patient profile is predictive of greater resistance to change and requires more treatment resources , such a profile might be classified as having a greater level of severity . the cambridge online dictionary defines severity as something serious or causing great pain , difficulty , or damage . severity in the dsm-5 has typically been operationalized from mild to severe and assigned to a diagnosis based on the number of symptom criteria endorsed . in the section on substance - related and addictive disorders , severity across time is also measured by reductions or increases in the frequency and/or doses of substances used . likewise , severity for gambling disorder is assigned by the number of symptoms endorsed at the time of evaluation . however , the dsm-5 operationalization of severity for addictive - related disorders is debatable and this is certainly true for hd for a vast array of reasons articulated below . symptom count is a gross measure of severity and makes an assumption that all symptoms for a given disorder are equal . during the dsm-5 field trial for hd , some patients barely met the diagnostic threshold for hd that would have likely been considered more serious or severe cases but such impressions were based on an examination of the broader scope of their overall clinical presentation that extended beyond the dsm-5 proposed criteria . this observation has also been noted elsewhere in substance - related disorders ( moss , 2011 ) . complicating matters , symptom endorsement for hd has a much higher threshold required compared to substance - related and gambling disorders . for example , several substance - use disorders can be diagnosed with as few as 2 of 10 or more symptoms . an hd diagnosis , however , required 4 out of 5 of the a criteria , evidence of impairment , and symptoms had to occur independent of a substance - related disorder , medical condition , or manic episode ( see figure 1 ) . collectively , the current structure of the hd proposed criteria makes severity by symptom endorsement difficult insofar as someone meeting the threshold for the diagnosis endorses 80% of the symptoms ( e.g. , a patient meeting criteria for hd would have either endorsed 4 or 5 of the a criteria , which does not allow for the current tri - categorized dsm-5 severity designations mild , moderate , and severe ) . the threshold for meeting the hd criteria would need to be lowered to 3 out of 5 of the a criteria to accommodate severity categorizations of mild , moderate , and severe , however , such a change would potentially introduce the problem of false - positive hd diagnosis . finally , symptom count as an index of severity also ignores the magnitude of how someone might experience a given symptom . for example patients may report significant clinical distress but the magnitude of the distress may vary across individuals . this is why someone with a greater magnitude across a few symptoms might be considered a more severe case than someone with more symptoms but of a lesser magnitude . given the limitations of the symptom - count approach to operationalizing severity , studies are needed to compare this method with alternative approaches . the field of hypersexuality research might consider some of the work challenging substance - use disorder severity designations as a model for research that could be applied to the hd criteria ( fazzino , rose , burt & helzer , 2014 ) . one approach to assess severity might consider how much time is spent engaging in sexual fantasies , urges , and behavior ( e.g. time spent planning for and recovering from sexual activities ) . generally time as measured by the frequency and duration of sexual fantasies , urges , and behaviors can vary and may influence whether an activity is considered excessive or even problematic . for example , masturbation once a week for 15 minutes might not be considered excessive or problematic whereas sex with an extra - dyadic partner outside a monogamous committed relationship once a week for 15 minutes is likely both problematic and excessive . these examples raise questions about whether severity should consider the specific manifestation of sexual behavior . while no attempt was made to pathologize the expression of sexual behaviors with the dsm-5 proposal for hd , it is important to ask how the type of sexual behavior linked to hypersexuality might play a role in assessing levels of severity . for example , do solo - sex behaviors carry more or less potential to contribute to a severity index than relational sexual activities ? as evident above , considering severity based exclusively on level of excessiveness or frequency of a sexual activity has several limitations . another approach might consider severity based on the extent that hypersexual behavior causes significant impairment to one s social , occupational or other important areas of functioning . closely related to impairment are the concepts of diminished control and consequences . certainly , these three concepts have some overlap and interaction . diminished control contributes to repetitive hypersexual behaviors that create consequences which impair one s ability to function . for example , multiple unsuccessful attempts to control or significantly reduce sexual fantasies , urges and behaviors might suggest a greater level of severity . perhaps diminished control in one context might have greater consequences than others ( e.g. inability to refrain from pornography on a work vs. a home computer ) . of course , the notion of diminished control makes an assumption that attempts to control the frequency or manifestations of hypersexual behavior have occurred . in cases where a patient may be pre - contemplative about change , it is possible attempts to control or change their behavior have never been made , and thus patients would deny experiencing multiple unsuccessful attempts to control their sexual behavior . further , the presence or absence of such efforts may occur , independent of the severity of their condition . consequences however , vary depending on several other factors and some consequences might be considered more severe based on the implications , frequency , or subjective values . in attempt to understand consequences , researchers developed the hypersexual behavior consequences scale which offered several insights about the types of consequences encountered by hypersexual patients ( reid , garos & fong , 2012 ) . for example , consequences leading to interference with friendships typically have different ramifications than those leading to divorce . a consequence such as job loss might also vary in severity depending on other variables . for example , job loss might be associated with greater severity if one is financially struggling for money or if it is highly publicized in the media as with some political scandals , celebrities , or well - known sports figures . the related legal problems that can be associated with hypersexual behavior can also heighten the seriousness of a case presentation and by extension , how severity is conceptualized . a patient s moral values might also influence whether a specific consequence is considered severe . one patient marginalized the consequence of an unintended pregnancy resulting from hypersexual behavior stating it was nt an issue , she just got an abortion . however , upon further investigation , it was discovered the woman felt pressured into having the abortion and afterward experienced significant depression , shame , and guilt as it contradicted her religious values . subsequently , it might be important to consider the extent to which others may be physically or emotionally harmed by hypersexual behavior and such factors weighted in assessing severity . further , a way of quantifying consequences is necessary to determine what types of consequences should be given more attention in assessing severity . an extension of the vast array of consequences encountered by hypersexual patients might consider whether hypersexual behavior contributes to comorbid psychological conditions such as depression , anxiety , substance abuse , suicidality or personality traits such as shame ( raymond , coleman & minor , 2003 ; reid , stein & carpenter , 2011 ) . such cases require special consideration and their complexity can influence the seriousness , pain , or suffering leading to a greater level of severity . one symptom of the hd proposal involves risk taking : repetitively engaging in sexual behavior while disregarding the risk for physical or emotional harm to self or others . arguably , not all risk taking behavior is equal and can vastly influence how severity might be operationalized . the word severe can also imply damage and some risk - taking behavior has significantly higher potential for greater damage . some examples might include risks associated with sexually transmitted infections , unintended pregnancies , physical harm to self or others , legal problems , job loss , divorce , and so forth . during the dsm-5 field trial , patients reported masturbating to the extent of incurring genital lesions requiring medical attention . some described watching pornography on their digital devices while driving placing them at risk for automobile accidents . a few patients meeting criteria for hd also had a comorbid paraphilic disorder of autoerotic asphyxiation , which has also been linked to unintended deaths . several patients described pursuing sex in neighborhoods that were known to have high crime rates that compromised their physical safety . while not part of the dsm-5 criteria for a gambling disorder , several studies show individuals with a gambling disorder and co - occurring illegal activities associated with gambling exhibited greater levels of gambling severity in comparison to gamblers without criminal histories ( ledgerwood , weinstock , morasco & petry , 2007 ; potenza , steinberg , mclaughlin , rounsaville & omalley , 2000 ; strong & kahler , 2007 ) . subsequently , if gambling research generalized to hypersexual patients , those who commit illegal acts in order to engage in sexual activities may represent more severe cases . collectively , the level of risk a patient is willing to disregard to pursue sexual fantasies , urges , and behaviors should be considered in assessing levels of severity . first , unlike substance - use disorders where abstinence is often a goal , clinicians try to help hypersexual patients reorganize their relationship with sexual cravings in order to cultivate healthy expressions of sexuality . thus , a sexual craving leading to sexual behavior may not always be problematic whereas a craving to use cocaine is likely always considered problematic . another challenge is the lack of research specifically focused on the role of cravings in the etiology or maintenance of hypersexual behavior . the field is in its infancy and only recently began developing measures of craving ( kraus & rosenberg , 2014 ) . however , more investigations are needed to understand the role of craving and its relationship to the concept of severity . for example , ecological momentary assessment could be used to further understand how cravings manifest among hypersexual individuals ( shiffman , stone & hufford , 2008 ) . such research might help resolve existing controversy where some have argued hd falls short of a pathological condition and is merely a manifestation of high sexual desire ( e.g. craving ) of behaviors and not an addictive process per se ( carvalho , stulhofer , vieira & jurin , 2015 ; steele , staley , fong & prause , 2013 ; winters , christoff & gorzalka , 2010 ) . as the field evolves , researchers might consider drawing on the work of those in the field of gambling disorders who have found some evidence between self - reported cravings and relapse ( oei & gordon , 2008 ) , risk - taking propensity in wagering ( ashrafioun , kostek & ziegelmeyer , 2013 ) , and gambling severity as measured by the problem - gambling severity index ( ferris & wynne , 2001 ) among gambling populations ( young & wohl , 2009 ) . however , other researchers in the field of behavioral addictions have considered alternative models to help explain the construct of cravings such as those often reported in food addiction ( rogers & hendrik , 2000 ) . collectively , further research is needed to understand the construct of cravings among hypersexual populations in order to consider how sexual cravings might be linked ( if at all ) in characterizing the severity of hd . regardless , cravings , as an index of severity , relapse , treatment response , or etiology , is likely to pose a difficult and challenging area of scientific inquiry as noted by other prominent craving researchers in the field of substance - use disorders ( tiffany & wray , 2009 ) . during the dsm-5 field trial for hd , the onset and clinical course of hd among participants was explored ( reid , carpenter et al . , 2012 ) . while the proposal limited an hd diagnosis to adults over the age of 18 , surprisingly , 54% of participants stated they began to experience difficulties regulating their sexual behavior as adolescents and 30% during college - age years . most ( 82.6% ) reported a gradual onset lasting several months or years with 17.4% indicating a rapid - acute onset > 90 days . the clinical course was split with 48.6% reporting a continuous pattern of hypersexual behavior and 51.4% stating episodic patterns . the substantial number of participants reported patterns of escalation including time ( 83.5% ) , frequency or intensity ( 81.7% ) , types of manifestations ( 62.4% ) , and increased risk ( 60.6% ) . there is no consensus about how these data might be associated with severity of hd , but similar data have been considered as surrogates for other indicators of severity in alcohol use disorders ( e.g. hingson , heeren & winter , 2006 ) . at present , further research is needed to investigate what implications the onset and clinical course of hd symptoms may have on attrition in therapy , treatment adherence , relapse , refractoriness to treatment all which might be markers of severity and the overall prognosis for hypersexual patients . such research might consider naturalistic longitudinal studies assessing the trajectory of sexual behaviors and hd across the lifespan in relation to different conceptualizations of severity . recently , item response theory ( irt ) has been used to evaluate severity ( saha , chou & grant , 2006 ) based on the assumption that symptoms less commonly endorsed reflect outliers with greater problems or severity . a key advantage of irt is the ability to select items based on the information they contribute which can inform the development of a severity index ( conway et al . , 2010 ) . for example , an irt approach has been used to assess the continuum of gambling problems using dsm criteria in exploring an index of problem severity ( strong & kahler , 2007 ) . alternatively , cluster analysis could be used to examine latent dimensions of hd and used to assess how best to threshold cases among populations of patients seeking help for hypersexual behavior . survival analysis is a plausible solution to evaluate the relationship between time and symptom remission in hypersexual patients allowing insights about treatment profiles and resistance to change . this resistance could subsequently be utilized as part of classifying a severity index . for example , if a particular patient profile is predictive of greater resistance to change and requires more treatment resources , such a profile might be classified as having a greater level of severity . the concept of severity among providers working with hypersexual behavior is frequently used , despite any lack of consensus about how severity should be operationalized . this article has highlighted several challenges to operationalizing severity for the dsm-5 proposed criteria for hd . regardless of what approach to classifying severity is ultimately adopted , the field should start dialogue about this concept given its relevance to determining level of patient care , risk , allocation of resources , and measuring treatment outcomes in clinical trials . the solo - author is responsible for all of the content and contributions to this manuscript .	background and aimsthe concept of severity among providers working with hypersexual behavior is frequently used despite a lack of consensus about how severity should be operationalized . the paucity of dialogue about severity for hypersexual behavior is disconcerting given its relevance in determining level of care , risk , allocation of resources , and measuring treatment outcomes in clinical practice and research trials . the aim of the current article is to highlight several considerations for assessing severity based on the proposed dsm-5 criteria for hypersexual disorder.methodsa review of current conceptualizations for severity among substance - use disorders and gambling disorder in the dsm-5 were considered and challenged as lacking applicability or clinical utility for hypersexual behavior.results and conclusionsthe current research in the field of hypersexual behavior is in its infancy . no concrete approach currently exists to assess severity in hypersexual populations . several factors in operationalizing severity are discussed and alternative approaches to defining severity are offered for readers to consider .
neurofibromatosis type 1 ( nf1 ) , known as von recklinghausen disease , is one of the most common gene disorders to human . pharmacopuncture is a type of neo - acupuncture that treats diseases by injecting herbal fluids at acupoints to gain the simultaneous effects of acupuncture and herbal medicine . guseonwangdo - go was prescribed as a tonifying medicine for treating internal damage in dongeui bogam . it can be consumed as a functional food because it is usually made into rice cakes . ( 2000 ) conducted the only study on guseonwangdo - go in korea . in this experimental study on rats and cells , guseonwangdo - go was reported to contribute to keeping homeostasis and to have an effect on anti - obesity and immune - system reinforcement . from these results , guseonwangdo - go pharmacopuncture was devised for the treatment of obesity and a weakened immune system . the previous experimental study , however , only proved the anti - obesity effect by administrating dried samples of guseonwangdo - go through an oral route to animals . therefore , this study was designed to evaluate the safety of administering a single - dose of guseonwangdo - go glucose through an intravenous route . the guseonwangdo - go glucose pharmacopuncture used in the experimental groups was prepared in the facility at the korean pharmacopuncture institute under the good manufacturing practice guidelines . according to bangyak happyeon , guseonwangdo - go consists of 160 g each of lotus seed ( nelumbinis semen ) , yam rhizome ( dioscoreae rhizoma , stir - baked ) , poria ( hoelen ) , and job s tears seed ( coicis semen ) , 80 g each of malt ( hordei fructus germinatus , stir - baked ) , dolichos bean seed ( dolichoris semen , stir - baked ) , fox nut seed ( euryales semen ) , and 40 g of persimmon frost ( kaki mannosum ) . because glucose is the main ingredient of persimmon frost , forty six - week - old sprague - dawley ( sd ) rats were used in this experiment : 20 male rats ( body weights : 193.9 210.5 g ) and 20 female rats ( body weights : 144.3 178.0 g ) . visual inspections and measurements of body weight by using electronic scales ( cp3202s , sartorius , germany ) were conducted on all animals when they were brought into the experiment . the general symptoms were observed once a day prior to the start of the experiments , with males and females within each groups being weighed and examined for general symptoms and body - weight changes on the last day of acclimatization . all animals were randomly distributed into four different groups with five individuals of each sex per group . the four different groups were labeled as follows : group 1 ( g1 , control group ) : normal saline solution , group 2 ( g2 ) : low - dose group , group 3 ( g3 ) : mid - dose group , and group 4 ( g4 ) : high - dose group ( table 1 ) . in a pilot test ( biotoxtech study no . : b12880p ) , 1.0 ml/ animal was administered through an intravenous route to one male and one female rat , which resulted in no deaths . from this result , the doses for guseonwangdo - go glucose pharmacopuncture in this study were set as follows : 1.0 ml / animal as the high dose ( g4 ) , 0.5 ml / animal as the mid - dose ( g3 ) , and 0.1 ml / animal as the low dose ( g2 ) . , korea ) as that of guseonwangdo - go glucose pharmacopuncture for the high - dose group was injected into the animals in the g1 , and the results were observed and compared with those of the experimental groups . all injections were administered at a caudal vein at a rate of 2 ml / min ( table 1 ) . this experiment was conducted at biotoxtech , an authorized institution for non - clinical studies , under the regulation of good laboratory practice ( glp ) of kfda notification no . 2012 - 61 ( guidelines for non - clinical laboratory studies , aug 24 , 2012 ) . the general symptoms and mortality were observed after 30 minutes , 1 , 2 , 4 , and 6 hours on the day of injection ( day 0 ) . from the next day to the 14 day after the injection , the general symptoms were examined once a day . * normal saline solution . the body weights were measured on the day of the injection and on the 3 , 7 , and 14 day after the injection . all animals were fasted during 18 hours before autopsy , the 15 day after the injection . they were then anesthetized with isoflurane , after which blood was collected from the abdominal aorta . for the hematological test , about 1 ml of the collected blood was placed in an ethylene - diamine - tetra - acetic acid tube and was analyzed with a hematology analyzer ( advia 120 , siemens , germany ) . for the coagulation test , about 2 ml of the collected blood was placed in a tube with 3.2% sodium citrate and centrifuged at 3,000 rpm for 10 minutes . different laboratory tests were conducted using a coagulation time analyzer ( coapresta 2000 , sekisui , japan ) . for the biochemical test , the blood remaining after carrying out the hematological tests was centrifuged at 3,000 rpm for 10 minutes , and the serum was collected . tests were done using a biochemistry analyzer ( 7180 , hitachi , japan ) and an electrolyte analyzer ( avl9181 , roche , germany ) . during the necropsy performed on all the animals , organs and tissues from the entire body tissues at the injection sites for all the animals were sampled and fixed in 10% neutral buffered formalin . routine histological methods , such as trimming , dehydration , and paraffin embedding , were conducted on the fixed organs and tissues . these were then sliced using a microtome and stained with hematoxylin & eosin ( h&e ) . all the results obtained were analyzed by using stata / se 9.2 for windows ( statacorp lp , college station , tx , usa ) . if the sample variances were equal , significant results were obtained using the one - way analysis of variance . if the sample variances were not equal , a kruskal - wallis test was performed . the number of animals in all of each group is 5 . * significantly different from control by dunnett s t - test ( p < 0.05 ) . during the observation , no deaths or abnormalities were observed in either the control or the experimental groups . however , crust formations were observed in the left ear , left neck , and left shoulder of one female of the control group . the body weights in both the control and the experimental groups continuously increased during the observation period , but no significant changes in body weight were observed . in the hematological test , the wbc counts were significantly increased in the female rats of g4 compared to g1 . however , no significant changes were found between the experimental groups and the control group ( table 2 ) . no significant biochemical changes were observed when the results of the experimental groups were compared with those of the control group . on necropsy , crust formation was observed in the posterior neck and the left posterior cranium of one female rat of the control group . however , no abnormalities were observed when visual inspections were conducted on both the control group and the experimental groups ( table 3 ) . in the tolerance test on the injection sites , the crust found in the one female rat of the control group was confirmed , in the autopsy , to be an ulcer / erosion with crust . granulation tissues were observed around the injection site - perivascular area of the lateral vein - of one female rat of g4 . however , no abnormal histopathological signs were observed in either the control or the experimental groups ( table 4 ) . a toxicant refers to any substance that may harm the body in a sufficient concentration . swiss scientist paracelsus said , all substances are poisons ; there is none which is not a poison . therefore , a toxicity test should be conducted to prove the safety of a drug before its clinical use because any drug with minimum side effects can be toxic , depending on the dose . there are three different types of toxicity tests : an acute toxicity test injecting a single - dose , a sub - acute toxicity test injecting a dose repeatedly for one month , and a chronic toxicity test injecting a dose repeatedly for over three months . the toxicity test has recently been perceived as being better for focusing on finding the relationship between the dose and toxicity changes before and after the administration than for simply acquiring ld50 values . the safety of a pharmacopuncture with respect to its toxicity needs to be proven because a pharmacopuncture uses an injection route , which is different from the oral administration route used in previous treatments . guseonwangdo - go was first prescribed in manbyeonghwechun . in the japbyeong - pyeon chapter of dongui bogam , guseonwangdo - go , as one of tonifying remedies for internal damage , was recorded to nourish essence - spirit , reinforce source - qi , fortify spleen and stomach , digest diets , recover depletion , produce muscles , and eliminate dampness - heat . it is usually made in the form of rice cakes due to its starch - rich and mild drug properties . also , it can be used as functional food in modern society as it can be eaten with pleasure as gruel . ( 2000 ) reported that guseonwangdo - go suppressed the proliferation of preadipocytes in the adipocytes - formation process and caused white blood cells , monocytes , and hemoglobin to increase and plasma glutamic - oxaloacetic transaminase ( got ) and cholesterol to decrease . in addition , guseonwangdo - go was reported to be more efficient in the activation of a complement as the glucose content was higher , and it showed no or marginal cytotoxicity , with an ic50 value of more than 5000 g / ml . therefore , it can be utilized for clinical purposes because it is expected to have effects on immunity reinforcement , and obesity prevention and to have minimum side effects . however , toxicity tests in animals have not been reported in korea yet . in china , injectable formulations of chinese medicine such as houttuyniae herba , bupleuri radix , astragali radix , and radix salviae miltiorrhizae composita were made and administered through intravenous injection to animals . many studies have been conducted in various areas such as clinical treatment and side - effect management . however , pharmacopuncture studies in korea are mainly conducted via subcutaneous or intramuscular injection through an acupoint or a tender point . studies of intravascular pharmacopuncture injection have rarely been conducted , the only exception being a study on wild ginseng radix pharmacopuncture [ 9 , 14 ] , because of the limited use of intramuscular injection in medical practice in korea . therefore , a toxicity test for guseonwangdo - go iv injection was conducted in this experiment . sd rats have been widely used in safety tests of drugs and related materials because of the system of their supply is stable and because they have consistent reactions to drugs . no deaths from toxicity occurred as a result of guseonwangdo - go glucose injection into rats of both sexes , and abnormal symptoms were not observed . in regards to changes in weight , no significant differences were observed between the control group and the experimental group . in the hematological test , female rats of g4 showed a significant increase in wbc counts compared to the control group . however , the result demonstrated a small variance and showed no dose - dependency or consistent variance by sex . no significant changes were observed in the biochemical test when the results from the experimental groups and the control group were compared . on autopsy , a crust was observed with the naked eyes on the posterior neck and the cranium of one female rat of the control group . it was confirmed to be an ulcer or erosion , but it was concluded to be a one - time or accidental change . in the local tolerance test , granulation tissue was observed around the injection site in one female rat of g4 . however , the formation of granulation tissue is a general mechanism of wound healing , as are angiogenesis and increased vascular permeability . the formation of granulation tissue was observed in only one individual , so it was concluded to be due to changes caused by insertion of the needle into the vein . no other results were found in the autopsy and the local tolerance test on the injected sites . a single - dose of guseonwangdo - go glucose was injected through an iv route into sd rats , and no toxicity was observed in the 0.1- , 0.5- , and 1.0-ml injection groups . thus , we consider its approximate lethal dose to be over 1.0 ml/ animal in both sexes , and we can conclude that it is relatively safe to use in an acute toxicity test . as no toxicity was observed in a maximum 1.0 ml injection for sd rats with body weights of 144.3 210.5 g , about 238 347 ml of guseonwangdo - go glucose 20% intravenous injection may be a safe dosage in humans weighting 50 kg . also , toxicity tests for non - rodents and sub - acute or chronic toxicity tests with repeated administration should be conducted . a single - dose of guseonwangdo - go glucose was injected through an iv route into sd rats , and no toxicity was observed in the 0.1- , 0.5- , and 1.0-ml injection groups . thus , the approximate lethal dose of guseonwangdo - go glucose is considered to be over 1.0-ml / animal .	objectives : this study was performed to evaluate the single - dose intravenous toxicity of guseonwangdo - go glucose 20% pharmacopuncture.methods:forty sprague - dawley rats were divided into four groups of five males and five females per group : an intravenous ( iv ) injection of 1.0 ml of normal saline solution per animal was administered to group 1 ( g1 , control group ) ; an iv injections of 0.1 , 0.5 , and 1.0 ml of guseonwangdo - go glucose pharmacopuncture per animal were administered to experimental groups 2 , 3 , and 4 ( g2 , g3 , and g4 ) , respectively . general symptoms , body weights , hematological and biochemical test results , and necropsy histopathological observation were recorded in all groups . in the statistical analyses , significance was determined by using the one - way analysis of variance ( anova ) . the significance level was 0.05 in all comparisons.results:for 14 days , no deaths or abnormalities were observed in any of the 4 groups . the body weights of all groups continuously increased during the observation period . in the hematological test , the wbc count was significantly increased in female rats of g4 compared to the control group , but this difference was considered not to be statistically meaningful . no significant biochemical changes were observed . on necropsy , crust formation was observed in one rat of the control group , and granulation tissues were observed around the injection site in one rat of g4 ; these changes were concluded to have been caused by injection of the needle into a vein.conclusion:the findings suggest that the lethal dose of guseonwangdo - go glucose pharmacopuncture is more than 1.0 ml per animal in both male and female rats . thus , we can conclude that guseonwangdo - go glucose pharmacopuncture injection is relatively safe to use in acute toxicity tests . further studies are needed to establish more detailed evidences of its toxicity .
a 75-year - old right - handed woman was admitted because of the abrupt development of mental confusion , left hemiparesis , and dysarthria . one day before admission , she developed weakness in her left arm and disorientation , followed 20 hours later by inappropriate speech , agitation , and dysarthria . she had a history of hypertension and diabetes , but no history of psychiatric illness . , she showed partially impaired orientation to time , but had well - preserved orientation to place and person . the neurological examination revealed left homonymous hemianopsia , but no obvious motor or sensory deficits . she showed no deficits in the line bisection and cancellation tasks , but was unable to copy an interlocked pentagon . three days later , she developed motor weakness ( grade 4 ) and hypesthesia in her left arm and face . the motor deficits and dysarthria were improved the next day , but the sensory deficit was unchanged . initial brain magnetic resonance imaging ( mri ) with diffusion - weighted images ( dwi ) was performed on admission , which revealed an acute infarction in the right parietal lobe with subtle involvement of the posterior temporal area ( figure 1a ) . magnetic resonance angiography ( mra ) revealed an obstruction of portion m1 of the right middle cerebral artery ( figure 1b ) . repeat mri on the fourth hospital day after the motor deficits developed revealed that the infarction had increased in size ( figure 1c ) . brain 2-deoxy-2-[f]fluoro - d - glucose positron emission tomography ( f - fdg pet ) performed 13 days after admission showed reduced fdg uptake in the right hemisphere , which was most severe in the right parietal lobe and left cerebellum ( figure 2a ) . when she was discharged 2 weeks after admission after 13 months , her neurological deficits and manic symptoms had resolved completely , except for the left - sided visual field defect . repeated brain f - fdg pet showed that the previous reduction in fdg uptake in the right hemisphere had improved ( figure 2b ) . this patient had sudden - onset restlessness with manic symptoms , including agitated mood , irritability , euphoria , talkativeness , pressured speech , and decreased need for sleep . the close temporal relationship between the onset of her symptoms and ischemic stroke , along with the absence of a history of affective disorders , supports the hypothesis that her restlessness with mania was caused by acute infarction of the right parietal lobe . an extensive literature search revealed 17 reports that described 29 patients who had post - stroke manic episodes . of these , eleven patients developed manic symptoms concomitantly with their strokes . in the other cases , the temporal relationship between the stroke and mania was obscure , either with a fairly long latency or due to insufficient information . the reported anatomical locations of the strokes were the temporal and frontal cortex ( 13 patients ) , 1,2,5,6,1517 thalamus ( 9 patients),3,5,8,9,11,13 caudate nucleus ( 2 patients),6 and ventral pontine region ( 2 patients).7 some patients had infarctions in widespread regions involving the frontal , temporal , and parietal lobes . our patient had an infarction that was mainly confined to the right parietal region on the initial brain mri , although there was subtle involvement in the posterior temporal area . post - stroke mania due to a localized infarction in the right parietal lobe has not previously been reported . one report described a case affecting the right parieto - occipital area involving the geniculocalcarine tract with agitation , hallucinations , and delusion , but the computed tomography result was not reported.18 widespread reduction in right hemispheric activity was seen on the brain fdg pet . although there was no structural damage ( i.e. , infarction ) , this may have been responsible for the restlessness and manic symptoms in this patient . however , the persistence of reduced activity in the right hemisphere , as shown on the repeated brain pet , along with a complete resolution of the manic symptoms , supports the possible relationship between the manic symptoms and the right parietal infarction . disinhibition syndrome , including restlessness and manic features , is usually caused by lesions in the orbitofrontal and basotemporal cortices of the right hemisphere.5,19 these areas are thought to inhibit the motor , instinctive , affective , and intellectual behaviors elaborated in the dorsal cortex selectively.19 lesions in these areas or in their connections to the dorsal cortex could produce disinhibition syndrome . this may range from mildly inappropriate social behavior to full - blown mania.19 caplan et al.20 observed that about half of the patients with infarcts of the inferior division of the right middle cerebral artery were in an agitated , confused state , showing hyperactivity , restlessness , and easy distractibility . et al.20 suggested , the inferior parietal lesions in our patient may interrupt the connections between the medial limbic cortex and superior parietal and frontal lobes , altering the emotional tone and affective behavior .	mood disorders following acute stroke are relatively common . however , restlessness with manic episodes has rarely been reported . lesions responsible for post - stroke mania can be located in the thalamus , caudate nucleus , and temporal and frontal lobes . we present a patient who exhibited restlessness with manic episodes after an acute infarction in the right parietal lobe , and summarize the case reports involving post - stroke mania . the right parietal stroke causing mania in our case is a novel observation that may help us to understand the mechanisms underlying restlessness with mania following acute stroke .
there is an urgent need for new , effective drug delivery systems . in the last few decades , nanotechnology has been increasingly employed in searching for methods to transport drugs to certain organs within the human body . transporting systems able to transmit cell - insoluble drugs into cells ; non - toxic for the surroundings ; and with time - prolonged releasing of active substances , have remarkable potential for improving cancer therapy efficacy.1 several bacteria isolates may be applied for possible new drug delivery mechanisms . their unique features including : potential to move ; intracellular survival ; and ability to settle in precisely programmed points of the body , allow the use of bacteria as specific medicine carriers.2 however , the following has still to be elucidated : how can bacteria be enforced to ingest medicines ; how can uncontrolled proliferation and distribution of bacteria be avoided within a host organism ; and can nanoparticles be used as drugs , or links between drugs and bacteria ? nanoparticles derived from noble metals are recognized as being non - genotoxic for the organism.3 moreover , gold and silver nanoparticles did not reveal proinflammatory activity4 and even antiangiogenic properties of au nanoparticles ( nano - au ) were observed.5 nano - au have been identified as promising candidates for many biological and biomedical applications.6 they have been widely used as an inert agent for cell imaging , targeted drug delivery , cancer diagnostics , and various therapeutic applications , due to their small sizes , convenience in bioconjugation , and strong absorbing and scattering properties.710 they entrap small drugs , or biopharmaceutical agents such as therapeutic proteins and dna , and can be designed to release these agents at the target site . platinum nanoparticles ( nano - pt ) are of great interest due to their exceptional catalytic activity , which is affected by the ligands at their external surface . nano - pt may display multiple functional groups at the surface , which can be hydrophilic , lipophilic , and chemically reactive.11 some of the pt compounds are used as very effective anticancer agents . this property is associated with the inhibition of dna replication and mitosis by the addition of nano - pt to dna strand.12 on the other side , research of hikosaka et al13 has revealed that nano - pt have a nicotinamide adenine dinucleotide ( nadh ) : ubiquinone oxidoreductase - like activity . this finding suggests that nano - pt can be used as a remedy for oxidative stress diseases with suppressed mitochondrial complex i.13 affinity of nano - pt to dna strand can also support their activity as carriers locating medicine within bacteria . salmonella enteritidis is a motile , gram - negative ( g ) , rod - shaped bacterium belonging to the enterobacteriaceae family . recent work has shown that salmonella enteritidis reside within macrophages.14 listeria monocytogenes is a gram - positive ( g+ ) , food - borne pathogenic bacterium , which possesses the ability to be internalized by macrophages and non - phagocytic cells , including epithelial cells , endothelial cells , and hepatocytes . it enters the cell using advanced mechanisms , proliferates intracellularly and spreads without contact with the extracellular medium , using elongated protrusions.15 methods to deliver functional nano - molecules to specific targets within the body still remain to be investigated . in addition , the impact of noble metals nanoparticles on bacteria s cells is still unclear . considering physical and chemical properties of gold ( au ) and platinum ( pt ) nanoparticles and biological properties of salmonella enteritidis and listeria monocytogenes , consequently , the objective of the present investigation was to evaluate morphological characteristics of the assembles of nano - au and nano - pt with g+ and g bacteria , to reveal possibilities of constructing bacteria- nanoparticle vehicles . hydrocolloids of nano - au and nano - pt were obtained from nano - tech polska ( warsaw , poland ) . they were produced by a patented electric non - explosive method ( polish patent 380649 ) from high purity metals ( 99.9999% ) and high purity demineralized water . shape and size of nanoparticles were inspected using a jem-1220 transmission electron microscope ( tem ) at 80 kev ( jeol , tokyo , japan ) , with a morada 11 mega - pixel camera ( olympus soft imaging solutions gmbh , mnster , germany ) ( figure 1 ) . diameter of particles ranged from 229 nm for au and from 219 nm for pt . samples of au and pt for tem were prepared by placing droplets of hydrocolloids on to formvar - coated copper grids ( agar scientific ltd . , stansted , uk ) . immediately after drying of the droplets in dry air , grids were inserted into the tem . bacteria strains ( salmonella enterica subspecies enterica serovar enteritidis atcc 13076 and listeria monocytogenes atcc 19111 ) were obtained from lgc standards ( lomianki , poland ) . salmonella enteritidis were grown on nutritive 2.8% agar ( bio - rad , hercules , ca , usa ) . listeria monocytogenes were grown on brain heart infusion agar ( bio - rad , hercules , ca , usa ) for 24 hours at 37c . bacteria were gently washed out from the agar using sterile distilled water . to remove liquid remains of the medium , bacteria were centrifuged at 4000 rpm for 5 min on an eppendorf minispin centrifuge ( eppendorf , hamburg , germany ) and the sediment ( pellet ) was re - suspended in milli - q water ( millipore , billerica , ma , usa ) . to calculate the exact number of bacteria in the suspension , the optical density at 600 nm ( od600 ) was estimated using a spectrophotometer helios epsilon unicam ( usa ) and serial dilutions of the suspension were prepared . based on the results of measurements carried out in triplicate , the density of bacteria suspension od600 = 0.130 corresponded to 2.2 10 cfu.ml ; and od600 = 0.133 corresponded to 4 10 cfu.ml , for salmonella enteritidis and listeria monocytogenes respectively . hydrocolloids of nano - au or nano - pt ( 200 l ) were added to two bacteria suspensions ( 200 l ) in the following order : nano - au + salmonella enteritidis ; nano - au + listeria monocytogenes ; nano - pt + salmonella enteritidis ; nano - pt + listeria monocytogenes . the samples were gently mixed for 15 min and then droplets of the samples were put on formvar - coated 300 mesh copper grids ( agar scientific ltd . the samples were dried at room temperature , under sterile conditions , and inspected using a jem-2000ex tem ( jeol , tokyo , japan ) . hydrocolloids of nano - pt ( 200 l ) were added to salmonella enteritidis suspension ( 200 l ) . to remove unattached nanoparticles , the samples were gently mixed for 15 min and then centrifuged at 4000 rpm for 5 min , washed with ultrapure milli - q water , centrifuged at 4000 rpm for 5 min , and re - suspended in milli - q water . control samples of bacteria not treated with nanoparticles were prepared using the same methods . in the third experiment , we wanted to confirm that the substance realized from salmonella enteritidis , after nano - pt treatment , was dna . furthermore , the viability of bacteria after incubation with nano - pt was determined . bacteria suspended in phosphate buffering solution ( pbs ) were divided into the control sample , to which 0.5 ml of milli - q water was added , and four samples , to which 0.5 ml of colloidal nano - pt was added . samples were incubated for 45 , 60 , and 90 min and then centrifuged at 5000 rpm for 5 min . the supernatant was discarded and the remaining pellet containing bacteria was suspended in 0.5 ml solution with the fluorescent marker dapi ( 4,6-diamidyno-2-fenyloindol ) and pbs in the proportion 1 l dapi to 1 ml pbs . bacteria with the fluorescent dye were incubated for 15 min . after this time , to remove overdose of the dye , samples were centrifuged ( 5000 rpm for 5 min ) and the obtained pellet was suspended in 200 l pbs . the control was a suspension of bacteria in 200 l of pbs , subjected to all stages of the experiment , except the incubation with nano - pt . the resulting suspension was thoroughly stirred and one drop was placed on a microscope slide and immediately covered with a coverslip . the resulting preparations were observed at 300x magnification with an olympus fv1000 confocal microscope , with fv10-asw software version 1.4 ( olympus corporation , tokyo , japan ) . the preparations were observed at the excitation wave length of 405 nm and in the nomarski contrast . the viability of salmonella enteritidis was also measured . samples of salmonella enteritidis , prepared as in experiment 1 , were incubated with hydrocolloids of nano - pt ( 1:1 ) and with milli - q water as the control , for 45 , 60 , and 90 min . samples of salmonella enteritidis were then spread and grown on nutritive 2.8% agar ( bio - rad , hercules , ca , usa ) , for 24 hours , at 37c and bacterial colonies were counted . hydrocolloids of nano - au and nano - pt were obtained from nano - tech polska ( warsaw , poland ) . they were produced by a patented electric non - explosive method ( polish patent 380649 ) from high purity metals ( 99.9999% ) and high purity demineralized water . shape and size of nanoparticles were inspected using a jem-1220 transmission electron microscope ( tem ) at 80 kev ( jeol , tokyo , japan ) , with a morada 11 mega - pixel camera ( olympus soft imaging solutions gmbh , mnster , germany ) ( figure 1 ) . diameter of particles ranged from 229 nm for au and from 219 nm for pt . samples of au and pt for tem were prepared by placing droplets of hydrocolloids on to formvar - coated copper grids ( agar scientific ltd . , stansted , uk ) . immediately after drying of the droplets in dry air , grids were inserted into the tem . bacteria strains ( salmonella enterica subspecies enterica serovar enteritidis atcc 13076 and listeria monocytogenes atcc 19111 ) were obtained from lgc standards ( lomianki , poland ) . salmonella enteritidis were grown on nutritive 2.8% agar ( bio - rad , hercules , ca , usa ) . listeria monocytogenes were grown on brain heart infusion agar ( bio - rad , hercules , ca , usa ) for 24 hours at 37c . bacteria were gently washed out from the agar using sterile distilled water . to remove liquid remains of the medium , bacteria were centrifuged at 4000 rpm for 5 min on an eppendorf minispin centrifuge ( eppendorf , hamburg , germany ) and the sediment ( pellet ) was re - suspended in milli - q water ( millipore , billerica , ma , usa ) . to calculate the exact number of bacteria in the suspension , the optical density at 600 nm ( od600 ) was estimated using a spectrophotometer helios epsilon unicam ( usa ) and serial dilutions of the suspension were prepared . based on the results of measurements carried out in triplicate , the density of bacteria suspension od600 = 0.130 corresponded to 2.2 10 cfu.ml ; and od600 = 0.133 corresponded to 4 10 cfu.ml , for salmonella enteritidis and listeria monocytogenes respectively . hydrocolloids of nano - au or nano - pt ( 200 l ) were added to two bacteria suspensions ( 200 l ) in the following order : nano - au + salmonella enteritidis ; nano - au + listeria monocytogenes ; nano - pt + salmonella enteritidis ; nano - pt + listeria monocytogenes . the samples were gently mixed for 15 min and then droplets of the samples were put on formvar - coated 300 mesh copper grids ( agar scientific ltd . , stansted , uk ) . the samples were dried at room temperature , under sterile conditions , and inspected using a jem-2000ex tem ( jeol , tokyo , japan ) . hydrocolloids of nano - pt ( 200 l ) were added to salmonella enteritidis suspension ( 200 l ) . to remove unattached nanoparticles , the samples were gently mixed for 15 min and then centrifuged at 4000 rpm for 5 min , washed with ultrapure milli - q water , centrifuged at 4000 rpm for 5 min , and re - suspended in milli - q water . control samples of bacteria not treated with nanoparticles were prepared using the same methods . in the third experiment , we wanted to confirm that the substance realized from salmonella enteritidis , after nano - pt treatment , was dna . furthermore , the viability of bacteria after incubation with nano - pt was determined . bacteria suspended in phosphate buffering solution ( pbs ) were divided into the control sample , to which 0.5 ml of milli - q water was added , and four samples , to which 0.5 ml of colloidal nano - pt was added . samples were incubated for 45 , 60 , and 90 min and then centrifuged at 5000 rpm for 5 min . the supernatant was discarded and the remaining pellet containing bacteria was suspended in 0.5 ml solution with the fluorescent marker dapi ( 4,6-diamidyno-2-fenyloindol ) and pbs in the proportion 1 l dapi to 1 ml pbs . bacteria with the fluorescent dye were incubated for 15 min . after this time , to remove overdose of the dye , samples were centrifuged ( 5000 rpm for 5 min ) and the obtained pellet was suspended in 200 l pbs . the control was a suspension of bacteria in 200 l of pbs , subjected to all stages of the experiment , except the incubation with nano - pt . the resulting suspension was thoroughly stirred and one drop was placed on a microscope slide and immediately covered with a coverslip . the resulting preparations were observed at 300x magnification with an olympus fv1000 confocal microscope , with fv10-asw software version 1.4 ( olympus corporation , tokyo , japan ) . the preparations were observed at the excitation wave length of 405 nm and in the nomarski contrast . samples of salmonella enteritidis , prepared as in experiment 1 , were incubated with hydrocolloids of nano - pt ( 1:1 ) and with milli - q water as the control , for 45 , 60 , and 90 min . samples of salmonella enteritidis were then spread and grown on nutritive 2.8% agar ( bio - rad , hercules , ca , usa ) , for 24 hours , at 37c and bacterial colonies were counted . hydrocolloids of nano - au or nano - pt ( 200 l ) were added to two bacteria suspensions ( 200 l ) in the following order : nano - au + salmonella enteritidis ; nano - au + listeria monocytogenes ; nano - pt + salmonella enteritidis ; nano - pt + listeria monocytogenes . the samples were gently mixed for 15 min and then droplets of the samples were put on formvar - coated 300 mesh copper grids ( agar scientific ltd . , stansted , uk ) . the samples were dried at room temperature , under sterile conditions , and inspected using a jem-2000ex tem ( jeol , tokyo , japan ) . hydrocolloids of nano - pt ( 200 l ) were added to salmonella enteritidis suspension ( 200 l ) . to remove unattached nanoparticles , the samples were gently mixed for 15 min and then centrifuged at 4000 rpm for 5 min , washed with ultrapure milli - q water , centrifuged at 4000 rpm for 5 min , and re - suspended in milli - q water . in the third experiment , we wanted to confirm that the substance realized from salmonella enteritidis , after nano - pt treatment , was dna . furthermore , the viability of bacteria after incubation with nano - pt was determined . bacteria suspended in phosphate buffering solution ( pbs ) were divided into the control sample , to which 0.5 ml of milli - q water was added , and four samples , to which 0.5 ml of colloidal nano - pt was added . samples were incubated for 45 , 60 , and 90 min and then centrifuged at 5000 rpm for 5 min . the supernatant was discarded and the remaining pellet containing bacteria was suspended in 0.5 ml solution with the fluorescent marker dapi ( 4,6-diamidyno-2-fenyloindol ) and pbs in the proportion 1 l dapi to 1 ml pbs . bacteria with the fluorescent dye were incubated for 15 min . after this time , to remove overdose of the dye , samples were centrifuged ( 5000 rpm for 5 min ) and the obtained pellet was suspended in 200 l pbs . the control was a suspension of bacteria in 200 l of pbs , subjected to all stages of the experiment , except the incubation with nano - pt . the resulting suspension was thoroughly stirred and one drop was placed on a microscope slide and immediately covered with a coverslip . the resulting preparations were observed at 300x magnification with an olympus fv1000 confocal microscope , with fv10-asw software version 1.4 ( olympus corporation , tokyo , japan ) . the preparations were observed at the excitation wave length of 405 nm and in the nomarski contrast . the viability of salmonella enteritidis was also measured . samples of salmonella enteritidis , prepared as in experiment 1 , were incubated with hydrocolloids of nano - pt ( 1:1 ) and with milli - q water as the control , for 45 , 60 , and 90 min . samples of salmonella enteritidis were then spread and grown on nutritive 2.8% agar ( bio - rad , hercules , ca , usa ) , for 24 hours , at 37c and bacterial colonies were counted . examination of the control bacteria indicated some changes caused by the used methods ( centrifugation , milli - q water , drying ) , but did not show any toxicological or destructive alteration of bacteria . morphology of interaction between nano - au and salmonella enteritidis is presented in figure 2 . the cells were malformed , had shrunk , and a loss of cytoplasm was observed . interestingly , nano - au was trapped within the flagella - biofilm network and kept at some distance from bacteria . the results of the morphological interaction of gold nanoparticles with listeria monocytogenes also showed that nano - au were aggregated and non - specifically placed on the cell wall ( figure 3 ) . the visualization of the interaction between nano - pt and salmonella enteritidis is shown in figure 4 . the picture shows that nano - pt disintegrated bacteria cell walls and cytoplasmatic membranes ; moreover , they entered bacteria and were seen as intensively black spots within the cells . nano - pt were located specifically , probably attached to some intracellular compounds ( dna ) , but under microscope electron beam , these black spots evaporated becoming white ( figure 4c ) . the image of interaction between nano - pt and listeria monocytogenes , shown in figure 5 , indicated that nano - pt are destructive to these bacteria . the influence of nano - pt on listeria monocytogenes clearly pointed out that nano - pt were seen within bacteria , moreover , they were located close to dna and probably bound to it . interestingly , these new molecules ( nano - pt with dna ) were removed or escaped from the bacteria cells . as in the case of salmonella enteritidis , nano - pt evaporated under the electron beam . this experiment was performed to elucidate to what extent nanoparticles , identified from morphological pictures in experiment one , as attached or being inside bacteria , were truly attached or were stably anchored bacteria cells . to remove unattached particles , mixtures with bacteria it was revealed that platinum complexes seen as intensively black spots were still within the bacteria ( figure 6 ) . this experiment was performed to confirm that nano - pt is actually removing dna from salmonella enteritidis , and whether this process is time - dependent . in this experiment , the average number of bacteria observed by the confocal microscope in the nomar - ski contrast and the average number of bacteria labelled with dapi ( dna - binding fluorochrome ) were determined . the average number of bacteria cells ( table 1 ) seen as a cell wall image observed with the nomar- ski contrast ( figure 7 ) , after incubation for 45 , 60 , and 90 min with nano - pt , did not change . however , the number of cells containing dna ( seen as a blue ) was reduced after 60 and 90 min . viability of salmonella enteritidis was also decreased and after 60 and 90 min of incubation , was about one quarter of that measured in the control condition ( table 1 ) . examination of the control bacteria indicated some changes caused by the used methods ( centrifugation , milli - q water , drying ) , but did not show any toxicological or destructive alteration of bacteria . morphology of interaction between nano - au and salmonella enteritidis is presented in figure 2 . the cells were malformed , had shrunk , and a loss of cytoplasm was observed . interestingly , nano - au was trapped within the flagella - biofilm network and kept at some distance from bacteria . the results of the morphological interaction of gold nanoparticles with listeria monocytogenes also showed that nano - au were aggregated and non - specifically placed on the cell wall ( figure 3 ) . the visualization of the interaction between nano - pt and salmonella enteritidis is shown in figure 4 . the picture shows that nano - pt disintegrated bacteria cell walls and cytoplasmatic membranes ; moreover , they entered bacteria and were seen as intensively black spots within the cells . nano - pt were located specifically , probably attached to some intracellular compounds ( dna ) , but under microscope electron beam , these black spots evaporated becoming white ( figure 4c ) . the image of interaction between nano - pt and listeria monocytogenes , shown in figure 5 , indicated that nano - pt are destructive to these bacteria . the influence of nano - pt on listeria monocytogenes clearly pointed out that nano - pt were seen within bacteria , moreover , they were located close to dna and probably bound to it . interestingly , these new molecules ( nano - pt with dna ) were removed or escaped from the bacteria cells . as in the case of salmonella enteritidis , nano - pt evaporated under the electron beam . this experiment was performed to elucidate to what extent nanoparticles , identified from morphological pictures in experiment one , as attached or being inside bacteria , were truly attached or were stably anchored bacteria cells . to remove unattached particles , mixtures with bacteria it was revealed that platinum complexes seen as intensively black spots were still within the bacteria ( figure 6 ) . this experiment was performed to confirm that nano - pt is actually removing dna from salmonella enteritidis , and whether this process is time - dependent . in this experiment , the average number of bacteria observed by the confocal microscope in the nomar - ski contrast and the average number of bacteria labelled with dapi ( dna - binding fluorochrome ) were determined . the average number of bacteria cells ( table 1 ) seen as a cell wall image observed with the nomar- ski contrast ( figure 7 ) , after incubation for 45 , 60 , and 90 min with nano - pt , did not change . however , the number of cells containing dna ( seen as a blue ) was reduced after 60 and 90 min . viability of salmonella enteritidis was also decreased and after 60 and 90 min of incubation , was about one quarter of that measured in the control condition ( table 1 ) . the purpose of the present study was to identify possibilities of using bacteria as a vehicle for transporting nanoparticles and/or nanoparticle - drug molecules . we focused on possibilities of placing nanoparticles of au and pt within bacteria , using a simple mechanism of self - organization , but without destroying cell walls of bacteria . the biological action of nanoparticles on mammalian and bacterial cells is highly dependent on their functional groups present on the surface of nanoparticles , which may determine their toxicity.16,17 to avoid influence of the additional chemical groups which surround nanoparticles and to determine chemical properties of nanoparticles , we used nano - au and nano - pt produced in high purity water by electric methods ( the same for au and pt ) . consequently , only hydrogen ion ( h ) or hydroxide ion ( oh ) groups could be present on the nanoparticle surface . the structure of the cell wall of g listeria monocytogenes and g- salmonella enteritidis is quite different . listeria monocytogenes has a thick ( 2080 nm ) , continuous cell wall composed of highly cross - linked layers of peptidoglycan , covalently bound to teichoic acids , while the salmonella enteritidis cell wall is thin ( 510 nm ) but more complex . peptidoglycan is surrounded by an outer membrane containing lipolysaccharide and also by some non - specific porins , which can potentially transport smaller nanoparticles ( around 1 nm ) . interestingly , in spite of significant differences in cell walls structure , the self - organization between nanoparticles and bacteria was affected by the type of bacteria to a lesser degree than by the type of nanoparticles . in our studies , nano - au formed aggregates trapped within flagella network , when examined with salmonella enteritidis ( figure 2 ) . the pictures might indicate that this process is initiated by fimbria network creation , which probably promotes the development of biofilm.18 formations of biofilm by salmonella enteritidis increase colonization , but also protect bacteria cells,19 which in this case could be a system of protection against nanoparticles . the formation of nano - au aggregates kept the nanoparticles away from the bacteria cells , which might be more safe for the bacteria but unsuitable for constructing a bacteria - nanoparticle drug delivery system . listeria monocytogenes also produced biofilm , but mainly showed abilities to form colonies , making it impossible to observe single cells as it was in the case of salmonella enteritidis ( figure 3 ) . probably , building a multi - cells structure was , for listeria monocytogenes , a form of protection against nano - au , but make it difficult to use listeria monocytogenes as a drug delivery vehicle . nanoparticles of au were mainly placed outside bacteria ( figure 2 and 3 ) , however , some aggregates , containing small ( about 510 nm ) particles of nano - au , were non - specifically placed on the surface of both bacteria . bacterial cell walls and cytoplasm membrane in both bacteria were disintegrated , but nano - au were not seen inside the cells . pan et al20 observed that particles of 12 nm were highly toxic and that nanoparticles larger than 15 nm were not toxic . consequently , the possibility that the smallest ( about 1 nm ) nanoparticles could enter the cells could be considered . however , in the present experiment , it was not possible to observe such small nanoparticles . in the present experiment , the visualization of the association between nano - pt and bacteria indicated significant effects of nano - pt on both salmonella enteritidis and listeria monocytogenes . cell wall and cytoplasmic membrane of both bacteria were disintegrated , however , listeria monocytogenes , like in the case of nano - au , created colonies , probably as a defence against nano - pt . interestingly , salmonella enteritidis did not demonstrate the mechanism of fimbria - biofilm production , which was seen as a remedy against nano - au . moreover , we noticed that the cell wall and cytoplasmic membrane of both microorganisms were permeable for nano - pt , which could be seen as intensively black spots , being black even when microscopic pictures were lightened and not existing in the control bacteria . moreover , the present pictures ( figure 4 , 5 ) showed that nano - pt was bound to specific intracellular compounds ; probably dna . our results are in agreement with previous studies,21 revealing that platinum demonstrates effective affinity to dna strand . these properties may be utilized for using nano - pt conjugates to inhibit dna replication , by binding to its strand and therefore acting as potential anticancer agents . the antioxidant activity of platinum complexes , which can penetrate the nucleus membrane and enter the nucleus , has recently gained attention due to its potential for the treatment of a variety of cancers.22 for example , cisplatin and carboplatin are used as chemotherapeutic agents for ovarian and testicular cancer.23 it is well known that in cancer therapy , tissue - unspecific drug application is a key problem . the possibility to locate nano - pt inside bacteria ghost , being a locomotive force , could provide target - specific drug delivery . previously , bacterial cells as ghosts were used as a very promising drug delivery system in a study with escherichia coli.2 in the case of listeria monocytogenes , we observed that nano - pt - dna molecules ( as intensively black spots ) were seen inside the cells , but the majority was placed outside the cells . it is therefore likely that nano - pt , after binding with dna , was removed from listeria monocytogenes ( figure 5 ) . in the case of salmonella enteritidis , the association of nano - pt and dna was also visualized as dark spots ( figure 4 ) , but seen rather within bacteria cells . interestingly , these spots were disappearing under the microscope electron beam , especially when magnification was increasing . in salmonella enteritidis , in contrast to listeria monocytogenes , the spots were mostly inside the bacterial cells and only a few were removed from bacteria cells . considering the results from the first experiment , only salmonella enteritidis and nano - pt were chosen in the second experiment , because only nano - pt was undoubtedly seen within salmonella enteritidis , and did not flow out from the cells . in this experiment , salmonella enteritidis were mixed with nano - pt and then centrifuged and washed to remove nanoparticles being unbound or unattached to bacteria s structures . as we supposed , dark spots placed within bacteria could be observed ( figure 6 ) . however , only a part of bacterial cells still had the spots while the other bacterial bodies were empty . in experiment 3 , we confirmed our hypothesis that after treatment with nano - pt , dna is removed from cells of salmonella enteritidis . however , after 60 min of incubation , only about half of the cells lost their dna , moreover , this process did not increase in the following 30 min . abilities of nano - pt entering salmonella cells and binding with dna seem to be very promising for a drug delivery system , even when only part of bacterial cells contain nano - pt - dna molecules . a fundamental problem seems to be determination of optimal time and other conditions of incubation allowing location of nano - pt within bacteria and binding with dna . we have also examined viability of salmonella enteritidis after nano - pt treatment , showing that after 90 min of incubation , about 25% of bacteria was still able to replicate . it can be suspected that if about half of bacteria after incubation for 6090 min with nano - pt still contained dna , and about a quarter of bacteria was still alive , the other quarter of bacteria contains nano - pt - dna molecule , but it is not able to replicate . visualization of morphological interaction between nano - au and salmonella enteritidis and listeria monocytogenes , showed that nano - au were aggregated within flagella or biofilm network , but were not specifically attached . in addition , nano - au visible under the microscope did not penetrate inside the bacterial cell . the analysis of morphological effects of interaction between nano - pt and bacteria revealed that nano - pt entered cells of listeria monocytogenes but were removed from the cells . in the case of salmonella enteritidis , nano - pt were seen inside bacteria cells , indicating that the bacteria cells could be used as a vehicle to deliver nano - pt , and active substances attached to pt , to specific points in the body .	purposerapid development of nanotechnology has recently brought significant attention to the extraordinary biological features of nanomaterials . the objective of the present investigation was to evaluate morphological characteristics of the assembles of gold and platinum nanoparticles ( nano - au and nano - pt respectively ) , with salmonella enteritidis ( gram - negative ) and listeria monocytogenes ( gram - positive ) , to reveal possibilities of constructing bacteria - nanoparticle vehicles.methodshydrocolloids of nano - au or nano - pt were added to two bacteria suspensions in the following order : nano - au + salmonella enteritidis ; nano - au + listeria monocytogenes ; nano - pt + salmonella enteritidis ; nano - pt + listeria monocytogenes . samples were inspected by transmission electron microscope.resultsvisualization of morphological interaction between nano - au and salmonella enteritidis and listeria monocytogenes , showed that nano - au were aggregated within flagella or biofilm network and did not penetrate the bacterial cell . the analysis of morphological effects of interaction of nano - pt with bacteria revealed that nano - pt entered cells of listeria monocytogenes and were removed from the cells . in the case of salmonella enteritidis , nano - pt were seen inside bacteria cells , probably bound to dna and partly left bacterial cells . after washing and centrifugation , some of the nano - pt - dna complexes were observed within salmonella enteritidis.conclusionthe results indicate that the bacteria could be used as a vehicle to deliver nano - pt to specific points in the body .
thyroglobulin ( tg ) is excreted exclusively by thyroid cells and is one of the key factors in the follow - up of patients with follicular differentiated thyroid carcinoma ( fdtc ) . while lymph node metastasis may be associated with low tg levels , the highest level of tg is seen in bone metastasis . the sensitivity of tg is very high for detection of bone metastasis especially after thyroid stimulating hormone ( tsh ) stimulation . low tg associated with normal neck ultrasonography is reported to have a very high negative predictive value for persistent disease and is used in many clinical trials as a proof of patient response to treatment . we are reporting a patient with diffuse skeletal metastasis associated with low serum tg level bringing a point of caution in evaluation of patients with low tg . a 23-year - old man presented to our thyroid cancer clinic on may 2006 with a history of papillary thyroid carcinoma ( ptc ) . he was operated about 10 years ago with no follow - up and no radio - iodine therapy . primary pathology was ptc in a 4.5 cm 4.0 cm 3.0 cm nodule in the right thyroid lobe . the stage of the disease at the time of diagnosis was considered pt3nxmx . on presentation , he had no symptoms and neck examination revealed a small 1 cm mass in the thyroid bed in midline . he underwent repeat neck exploration , and pathology examination revealed adenomatous nodule . following discontinuation of levothyroxine for 4 weeks , thyroid values were measured in the serum and tsh was 150 miu / l , tg level was 3.5 ng / ml and anti - tg antibody level was 11 iu / ml . the biocode ( s. a , liege , belgium ) kit was used for determination of tg level with a sensitivity of 0.35 ng / ml and an inter assay variation of 4.8% and between assay variation of 6.1% . after 2007 , tg measurements were done using elecsys_electrochemiluminescent immunoassay from roche diagnostics ( meylan , france ) . anti - tg antibody was measured using elisa method ( trinity - biotech ) before 2007 and by elecsys electrochemiluminescent immunoassay from roche diagnostics ( meylan , france ) after 2007 . diagnostic whole body iodine scan was performed 2 days after administration of 74 mbq i-131 and widespread bone metastasis , regional lymph node metastasis , and thyroid bed uptake was detected [ figure 1 ] . patient had no complaint and after asking him about any bone pain , remembered just occasional mild low back pain . postablation whole body iodine scan ( anterior and posterior views ) performed 7 days after 7400 mbq of i-131 shows multiple areas of iodine uptake in the skull , sternum , thyroid bed , cervical spine , left clavicle , thoracic spine , lumbar spine , pelvis and left femoral diaphysis the patient was treated with 7400 mbq of 131-iodine . suppressive therapy was started , and measurement of tg was done after discontinuation of the drugs 8 months later . tsh level was 380 miu / ml with simultaneous tg level of 6 ng / ml and anti - tg antibody level of 11 iu / ml . treatment was repeated with another dose of 7400 mbq of i-131 . postablation whole body iodine scan again showed multiple foci of metastases with no significant change compared with the previous scan . radiography of the pelvis showed a small lytic lesion in the iliac bone with a rim of sclerosis [ figure 2a ] . ( a ) pelvic radiography shows lytic lesion in the right iliac bone with a rim of sclerosis . ( b ) computed tomography scan image from 2 lumbar spine shows a lytic lesion in vertebral body with sclerotic rim and internal septa . these findings strongly suggest low grade malignant lesion since that time , the patient has been treated 5 more times with i ( total accumulated dose 51.8 gbq i-131 ) . tg level was always below 6 ng / ml in spite of multiple bone metastases . thoraco - lumbar computed tomography scan was done for more confirmation and indicated the presence of skeletal involvement corresponding to the lytic lesions detected in the radiographies [ figure 2b ] . the patient underwent surgical biopsy from the lumbar spine ( l2 ) and the pathology result was positive for metastatic carcinoma . immunohistochemistry of the removed tissue was positive for thyroid transcription factor-1 and tg markers in tumoral cells , confirming thyroid origin of the lesions [ figure 3 ] . ( a ) carcinoma cells with follicle - like structure formation ( h and e , 10 ) . a low stimulated tg level ( < 1 ng / ml ) associated with normal neck ultrasonography is considered as the most reliable criteria for complete remission in low risk patients . also stimulated tg between 1 and 10 ng / ml concomitant with normal neck ultrasonography is considered as acceptable response . however false negative tg in the presence of metastasis is reported in 6.8% of patients , although most of the patients in this report had only lymph node metastasis and only one patient had bone metastasis . another study in 194 patients with differentiated thyroid carcinoma ( dtc ) , with a mean follow - up of 7.7 years found persistent disease in 1.5% of the patients . in that study , there was no patient with low serum tg level concomitant with distant metastasis . falsely low or high tg level is also reported in the presence of heterophile antibodies in serum of the patient . in our study tg was measured with two different techniques and showed persistently low tg and anti - tg level throughout the years of follow - up . in the present patient , inappropriate low serum tg level in the presence of widespread skeletal metastases was noted in spite of measurement of tg with two different kits and controlling for heterophile antibodies . only few cases of skeletal metastases associated with low tg level was reported in the literature , and only one of the reported cases had diffuse skeletal metastases . our patient brings more caution in definition of response as low tg and normal neck ultrasonography . our patient is a case of pediatric thyroid cancer , operated at the age of 13 years old . as distant metastases are more commonly seen in pediatric patients , diagnostic wbis may be useful in these patients irrespective of the result of tg or neck ultrasonography . early recognition and radio - iodine therapy are mandatory in the majority of these patients to prevent long term morbidity and possibly to improve survival . the other teaching point in our patient this finding again emphasis on careful scrutiny in patients with dtc and not relying only on tg level and symptoms . low tg level and absence of symptom does not exclude widespread skeletal metastases in patients with ptc .	thyroglobulin ( tg ) is frequently measured in follow - up of patients with differentiated thyroid carcinoma . a low stimulated tg level ( < 1 ng / ml ) associated with normal neck ultrasonography is considered as the most reliable criteria for complete remission in low risk patients . we are reporting a case of pediatric thyroid cancer with diffuse skeletal metastasis associated with low serum tg level bringing a point of caution in evaluation of patients with low tg .
this survey was conducted during january february 2009 at phc facilities ( health centers and polyclinics ) located in various geographical zones of benghazi . the survey included nine primary health centers and seven polyclinics selected through a lottery method and giving geographical ( cluster ) representation ( table 1 ) . this survey was carried out by face - to - face interviews with the patients . the charleston psychiatric outpatient satisfaction scale ( likert - type scale ) was translated into arabic . this 15-item e5 response format ( excellent , very good , good , fair , and poor ) is intended to minimize positive response bias and optimize variability and predictive validity ( 9 ) . the 15th item assesses behavioral intent to recommend the facility by using a 4-point scale ( yes definitely , yes probably , no probably not , and no definitely not ) . some are predictor items , such as overall quality and recommendation intent , and the other items assess administrative and environmental factors unique to the outpatient setting : helpfulness of secretariat , waiting time , amount of information received about the problem , respect for opinions , matching treatment plan with individual needs , helpfulness of services , appearance of waiting room , appearance of office , working hours , location , and parking . the survey process was standardized by allotting three man - days in each facility during the morning shift . it was decided to conduct exit interviews with as many beneficiaries as possible within these three man days by adopting a random sampling method . survey monitoring and data quality assurance was carried out by regular discussions with researchers , editing at field level , and centralized editing . a total patient satisfaction score was calculated by adding the scores of all the items except items 2 and 14 ( payment and billing , which are not applicable because the services are offered free ) , item 8 ( overall quality , an anchor item ) , and item 15 ( recommendation intent ) . with each of the 11 included items scored as 5 ( excellent ) , 4 ( very good ) , 3 ( good ) , 2 ( fair ) , or 1 ( poor ) , the total score varies between 11 and 55 . the data were analyzed by anova ( one - way and two - way ) , t - test , and chi - square test . data collection was carried out with approval from faculty of public health , al arab medical university and also after obtaining permission from the secretariat of health and the concerned centers . the patients were mostly females ( 64.2% ) , had low levels of education , were married , and worked in service - oriented jobs ( table 2 ) . characteristics of patients twenty patients in al birka did not indicate educational level and are excluded from the calculation of percentage . the female male ratio was highest at al birka and city centre ( 7:3 each ) than in al slawy ( 3:2 ) . women have been reported to be frequent users of phc in other populations as well ( 10 ) . female - oriented issues and childhood , likely take a higher importance at phc facilities . a higher proportion of females ( 20.6% as against 9.0% in males ) were in the age group of 1524 years , indicating that there was a high demand for adolescent health and reproductive health care services at these facilities . the pattern of educational status was similar in al slawy and city centre , but with a slight trend to a lower level in the former . in al birka , 20 patients ( 25% ) did not answer this question , which makes it unadvisable to draw conclusions regarding the seemingly higher educational level in this area . this might not relate to the national population characteristics , as the mean age at marriage remains high in libya ( 33 years for males and 29 years for females ) ( 1 ) . more patients from al birka were married ( 78.8% ) compared to the city centre ( 64.7% ) and al slawy ( 45.0% ) . among patients from al slawy there was a higher proportion of divorcees ( 33.8% ) . two - thirds ( 66.8% ) of those who were married were females and over half ( 56.8% ) were males . a larger proportion of divorced persons were males ( 30.6% ) compared to females ( 15.2% ) . though family bonds were strong in libya , families were generally nuclear , and this was observed among the patients . the proportion of nuclear families was highest in al birka ( 85.0% ) , followed by city centre ( 75.3% ) and al slawy ( 72.5% ) . the frequency of small families ( up to six members ) was higher in al birka ( 56.3% ) than in the city centre ( 45.3% ) and al slawy ( 43.8% ) . the total satisfaction score was calculated by summing up items 113 of the charleston scale , but excluding items 2 and 8 . two - way anova , one - way anova , t - test , and chi - square test were used to identify variables affecting satisfaction . geographic zone was found to be an important factor in creating satisfaction ( f - value = 13.09 ; p = 0.00 ) . thus , the location of the health facility was important in determining satisfaction ( table 3 ) . mean satisfaction level was higher in the city centre ( 44.4 ) than in al slawy ( a mean of 41.5 ) and al birka ( 40.7 ) . mean satisfaction and f values of variables used in one - way anova twenty persons did not respond . there was no significant difference in patient satisfaction between the married and unmarried persons ( f = 1.99 ; p = 0.14 ) . education , occupation , and family size had no relevance in creating differences in satisfaction . the effect of the type of facility , gender , and type of family on satisfaction was analyzed by an independent sample t - test ( table 4 ) . only the type of facility was found to produce a significant difference in satisfaction ( t = 2.04 ; p = 0.04 ) , with patients expressing greater satisfaction with health centers than with polyclinics . mean satisfaction scores mean satisfaction among males and females were not significantly different ( p = 0.12 ) . there was also no significant difference in satisfaction level across the three localities ( table 5 ) . satisfaction levels and variables used in chi - square test comparison of satisfaction level in age groups showed that patients who were 25 years or older were significantly more satisfied ( p = 0.06 ) than those who were younger ( table 5 ) . these results confirm the findings of a similar study conducted in united arab emirates ( 12 ) . . a higher proportion of persons from al birka and al slawy sought facilities outside their residential zone ( =24.01 ; p<0.01 ) . half of those residing in al birka and over one - third of those from al slawy went outside their residential area ( table 6a ) . by contrast , only 13.6% of the city centre residents went to another zone . looked at from the other perspective , 49.9% of those attending facilities in the city centre came from other zones , whereas only 8.8% of those attending al slawy facilities were from other zones ( table 6b ) . health - seeking behavior seeking facilities by location of residence seeking facilities by the location of facility these results indicate that in general there was more satisfaction with the facilities in the city centre . this was supported by analysis of satisfaction among the 100 persons who attended facilities outside their zones . those attending facilities in the city centre were more satisfied ( mean 44.6 ) than those going to al birka and al slawy ( means 39.6 and 34.6 , respectively ) ( table 7 ) . the differences in satisfaction scores are statistically significant ( f - value = 18.0 ; p = 0.00 ) . this suggests that persons seek services in facilities with which they were more satisfied even if they were outside their residential zone . mean satisfaction and variables in one - way anova for those who seek facilities outside two patients gave no response . family size was also related to satisfaction ( f - value = 4.8 ; p = 0.10 ) . mean satisfaction was higher among those who reside in small families as compared to medium and large families ( 44.8 , 41.4 , and 41.2 ) . more satisfaction was expressed by those attending health centers ( mean 43.9 ) than polyclinics ( 41.0 ) ( t - value = 2.4 ; p = 0.02 ) . these statistics indicate that those who access health centers located at zones outside their own residential zone were more satisfied . on the other hand , health centers were more satisfactory than polyclinics for those who seek care away from their homes . mean satisfaction and variables in t - test for those who seek facilities outside in creating differences in satisfaction , there was an interaction effect between the two types of facilities on the one hand , and the three zones in which the facilities were located , on the other hand . while both of these variables create differences in satisfaction independently , differences were also created through interaction of these two sets of variables ( table 9 ) . the difference in patient satisfaction due to the main effect of facilities was found to be insignificant ( f = 2.31 ; p = 0.128 ) . the main effect of the zone was found to be highly significant ( f = 14.3 ; p = 0.000 ) , meaning that satisfaction varies widely across zones . the interaction of these variables in patient satisfaction was highly significant ( f = 31.6 ; p = 0.000 ) , which means that variations in patient satisfaction across zones were created through the type of facilities . interaction of facilities and zones ( two - way anova ) on satisfaction note : r = 0.985 . this interaction was evident from table 8 , which shows that health centers create a high level of satisfaction ( mean of 43.2 ) compared to polyclinics ( mean of 41.7 ) . health centers in city centre create a satisfaction score of 45.3 , which was followed by al birka ( mean of 42.9 ) and al slawy ( mean of 39.4 ) ( table 10 ) . polyclinics , on the other hand , were highly satisfactory at al slawy ( mean of 47.8 ) , followed by city centre ( mean of 41.9 ) and al birka ( mean of 38.5 ) . thus , there was a disparity in the health center versus polyclinic issue in that polyclinics at al slawy were more satisfactory in offering quality services , whereas health centers in city centre and al birka were more satisfactory . the patients were mostly females ( 64.2% ) , had low levels of education , were married , and worked in service - oriented jobs ( table 2 ) . characteristics of patients twenty patients in al birka did not indicate educational level and are excluded from the calculation of percentage . the female male ratio was highest at al birka and city centre ( 7:3 each ) than in al slawy ( 3:2 ) . women have been reported to be frequent users of phc in other populations as well ( 10 ) . female - oriented issues and childhood , likely take a higher importance at phc facilities . a higher proportion of females ( 20.6% as against 9.0% in males ) were in the age group of 1524 years , indicating that there was a high demand for adolescent health and reproductive health care services at these facilities . the pattern of educational status was similar in al slawy and city centre , but with a slight trend to a lower level in the former . in al birka , 20 patients ( 25% ) did not answer this question , which makes it unadvisable to draw conclusions regarding the seemingly higher educational level in this area . this might not relate to the national population characteristics , as the mean age at marriage remains high in libya ( 33 years for males and 29 years for females ) ( 1 ) . more patients from al birka were married ( 78.8% ) compared to the city centre ( 64.7% ) and al slawy ( 45.0% ) . among patients from al slawy there was a higher proportion of divorcees ( 33.8% ) . two - thirds ( 66.8% ) of those who were married were females and over half ( 56.8% ) were males . a larger proportion of divorced persons were males ( 30.6% ) compared to females ( 15.2% ) . though family bonds were strong in libya , families were generally nuclear , and this was observed among the patients . the proportion of nuclear families was highest in al birka ( 85.0% ) , followed by city centre ( 75.3% ) and al slawy ( 72.5% ) . the frequency of small families ( up to six members ) was higher in al birka ( 56.3% ) than in the city centre ( 45.3% ) and al slawy ( 43.8% ) . the total satisfaction score was calculated by summing up items 113 of the charleston scale , but excluding items 2 and 8 . two - way anova , one - way anova , t - test , and chi - square test were used to identify variables affecting satisfaction . geographic zone was found to be an important factor in creating satisfaction ( f - value = 13.09 ; p = 0.00 ) . thus , the location of the health facility was important in determining satisfaction ( table 3 ) . mean satisfaction level was higher in the city centre ( 44.4 ) than in al slawy ( a mean of 41.5 ) and al birka ( 40.7 ) . mean satisfaction and f values of variables used in one - way anova twenty persons did not respond . there was no significant difference in patient satisfaction between the married and unmarried persons ( f = 1.99 ; p = 0.14 ) . education , occupation , and family size had no relevance in creating differences in satisfaction . the effect of the type of facility , gender , and type of family on satisfaction was analyzed by an independent sample t - test ( table 4 ) . only the type of facility was found to produce a significant difference in satisfaction ( t = 2.04 ; p = 0.04 ) , with patients expressing greater satisfaction with health centers than with polyclinics . mean satisfaction scores mean satisfaction among males and females were not significantly different ( p = 0.12 ) . there was also no significant difference in satisfaction level across the three localities ( table 5 ) . satisfaction levels and variables used in chi - square test comparison of satisfaction level in age groups showed that patients who were 25 years or older were significantly more satisfied ( p = 0.06 ) than those who were younger ( table 5 ) . these results confirm the findings of a similar study conducted in united arab emirates ( 12 ) . this choice was significantly associated with the zone of residence . a higher proportion of persons from al birka and al slawy sought facilities outside their residential zone ( =24.01 ; p<0.01 ) . half of those residing in al birka and over one - third of those from al slawy went outside their residential area ( table 6a ) . by contrast , only 13.6% of the city centre residents went to another zone . looked at from the other perspective , 49.9% of those attending facilities in the city centre came from other zones , whereas only 8.8% of those attending al slawy facilities were from other zones ( table 6b ) . health - seeking behavior seeking facilities by location of residence seeking facilities by the location of facility these results indicate that in general there was more satisfaction with the facilities in the city centre . this was supported by analysis of satisfaction among the 100 persons who attended facilities outside their zones . those attending facilities in the city centre were more satisfied ( mean 44.6 ) than those going to al birka and al slawy ( means 39.6 and 34.6 , respectively ) ( table 7 ) . the differences in satisfaction scores are statistically significant ( f - value = 18.0 ; p = 0.00 ) . this suggests that persons seek services in facilities with which they were more satisfied even if they were outside their residential zone . mean satisfaction and variables in one - way anova for those who seek facilities outside two patients gave no response . family size was also related to satisfaction ( f - value = 4.8 ; p = 0.10 ) . mean satisfaction was higher among those who reside in small families as compared to medium and large families ( 44.8 , 41.4 , and 41.2 ) . more satisfaction was expressed by those attending health centers ( mean 43.9 ) than polyclinics ( 41.0 ) ( t - value = 2.4 ; p = 0.02 ) . these statistics indicate that those who access health centers located at zones outside their own residential zone were more satisfied . on the other hand , health centers were more satisfactory than polyclinics for those who seek care away from their homes . in creating differences in satisfaction , there was an interaction effect between the two types of facilities on the one hand , and the three zones in which the facilities were located , on the other hand . while both of these variables create differences in satisfaction independently , differences were also created through interaction of these two sets of variables ( table 9 ) . the difference in patient satisfaction due to the main effect of facilities was found to be insignificant ( f = 2.31 ; p = 0.128 ) . the main effect of the zone was found to be highly significant ( f = 14.3 ; p = 0.000 ) , meaning that satisfaction varies widely across zones . the interaction of these variables in patient satisfaction was highly significant ( f = 31.6 ; p = 0.000 ) , which means that variations in patient satisfaction across zones were created through the type of facilities . interaction of facilities and zones ( two - way anova ) on satisfaction note : r = 0.985 . this interaction was evident from table 8 , which shows that health centers create a high level of satisfaction ( mean of 43.2 ) compared to polyclinics ( mean of 41.7 ) . health centers in city centre create a satisfaction score of 45.3 , which was followed by al birka ( mean of 42.9 ) and al slawy ( mean of 39.4 ) ( table 10 ) . polyclinics , on the other hand , were highly satisfactory at al slawy ( mean of 47.8 ) , followed by city centre ( mean of 41.9 ) and al birka ( mean of 38.5 ) . thus , there was a disparity in the health center versus polyclinic issue in that polyclinics at al slawy were more satisfactory in offering quality services , whereas health centers in city centre and al birka were more satisfactory . factors related to differences in satisfaction were type and location of facility and age of customer . more satisfaction was expressed for health centers than for polyclinics , and there was an interaction of satisfaction between the geographical location and type of facility . the authors have not received any funding or benefits from industry or elsewhere to conduct this study .	introductionthe libyan national health system ( lnhs ) is debated for the paradox of its performance versus impact . it has poor performance , but the national health statistics are good and competitive . there are concerted efforts to manage health care services and to regain the lost trust . a primary health care ( phc ) system that focuses on preventive and promotive care is the core focus of lnhs efforts.objectivesto assess patient satisfaction with quality of phc assessed in terms of ( a ) customer profile , ( b ) patient satisfaction , and ( c ) health care - seeking behavior.methodologya sample of nine health centers and seven polyclinics from various locations in benghazi , libya were selected for gathering information by structured face - to - face interviews . a total of 310 beneficiaries were interviewed by using an arabic translation of the charleston psychiatric outpatient satisfaction scale.resultsthe beneficiaries appear to be quite satisfied with the quality of services . geographical zone , marital status of beneficiary , and type of facility are satisfaction - related factors . there are preferences for facilities located within the city centre over those located elsewhere . there is also an interaction effect of the geographical zone and the type of facility in creating differences in satisfaction.conclusionsa customer - friendly facility concept that emphasizes reception , physician interaction , and cordiality shall add value . polyclinics require more attention as does the al slawy area . a few utility services might also be considered .
stroke is a medical emergency in neurology , and is one of the leading causes of death nowadays . at a recent time , a therapeutic method used in adequate conditions is thrombolysis , a treatment of an emerging clot in the brain vascular system by alteplase . this is a brief report of a case with thrombolysis complication which manifested as a spleen rupture . despite significant improvement in therapy in the past two decades , stroke is the leading cause of death and disability worldwide ( 1 ) . the use of recombinant tissue plasminogen activator ( iv - tpa ) is a well - established standard of treatment for patients presenting with acute ischemic stroke ( ais ) within the first 4.5 h from the symptom onset ( 2 - 5 ) . in order to minimize the unwanted side effects ( 6 ) , there are clear and strictly defined criteria for appropriate patient selection for the systemic treatment with rt - pa . intravenous application of alteplase is the only therapy approved by the us food and drug administration ( fda ) for the treatment of patients with ischemic stroke . its use is associated with improved outcomes for a wide group of patients ( 7 ) and its earlier treatment is associated with better outcomes ( 8) . during thrombolytic therapy , there is a risk of intracerebral hemorrhage with a reported incidence of about 6% ( 9 , 10 ) . other , less common complications of thrombolytic therapy include systemic hemorrhage , angioedema and allergic reactions ( 11 ) . we report a case of a female patient with ais who developed symptoms of internal bleeding shortly after administration of thrombolytic therapy . a diagnosis of spontaneous splenic rupture was made by the following clinical and radiological examination . a 45-year old female patient was admitted to hospital with weakness of the left extremities , dizziness , nausea and vomiting . in the past medical history , she asserted frequent urinary infections and hyperventilation as a result of panic attacks , and therefore she was taking oxazepam temporarily . on admission , dysarthria , eyeballs deviation to the right , moderate paresis of the left hand , plegia of the left leg , diminished ipsilateral reflexes , and diminished left plantar reflex were observed in the neurological status . laboratory findings showed increased blood glucose level ( 7.4 mmol / l ) and low potassium level ( 3.5 mmol / l ) . the patient satisfied the criteria for thrombolytic therapy and intravenous alteplase was administered at the dose of 0.9 mg / kg . mild dysarthria , incomplete left homonymous hemianopsia , left - sided central facial palsy , mild to moderate paresis of the left hand , severe paresis of the left leg and left hemihypesthesia were observed in neurological status . control ct of the brain performed six hours after admission showed no signs of intracerebral hemorrhage or ischemia . the patient developed tachycardia with a rate of 125 bpm , her blood pressure dropped to 75/55 mmhg and hemoglobin level decreased from 139 g / l to 99g / l , which indicated the bleeding occurrence and patient was moved to the intensive care unit . an urgent ct scan of abdomen and pelvis was performed and it showed a splenic rupture with massive intraperitoneal bleeding ( figure 1 ) . the patient underwent splenectomy . on control neurological examination , left homonymous hemianopsia , unreactive left pupil , moderate paresis of the left hand , plegia of the left leg , heightened reflexes and positive babinski sign left were observed . control ct scan of the brain performed a week after admission showed demarcation of an extensive hypodense lesion in the irrigation area of the right posterior cerebral artery . the patient was discharged with a recommendation for physical therapy , appropriate for her condition . ct scan of abdomen and pelvis showed a splenic rupture with massive intraperitoneal bleeding in the area of ruptured spleen , perihepatic space , in both paracolic gutters and in the pelvis , there was a history of significant trauma prior to thrombolysis ( 12 , 13 ) . friedrich and colleagues described a case of a patient with polycythemia rubra vera in which spontaneous spleen rupture followed thrombolytic therapy for myocardial infarction ( 14 ) , while in the case described by jankowski et al , spontaneous spleen rupture after application of thrombolytic therapy for ais was preceded by splenomegaly secondary to infection ( 15 ) . a few cases in which spleen rupture followed the streptokinase therapy have been reported ( 16 - 19 ) . in one of these cases , thrombolytic therapy was used for deep venous thrombosis ( 16 ) , while in other cases it was used for acute myocardial infarction ( 17 - 19 ) . in a case described by cheung and colleagues , 3 hours after administration of the t - pa for acute myocardial infarction , shock and acute abdomen developed due to spleen rupture ( 20 ) . revesz et al reported a case in which spontaneous spleen rupture followed t - pa for peripheral arterial occlusion ( 21 ) . harries and gomez reported a case of a patient with acute myocardial infarction , in which spleen rupture occurred as a complication of the thrombolytic therapy with tenecteplase ( 22 ) . spontaneous spleen rupture following alteplase therapy for stroke was reported only once ( 23 ) . the aim of current therapy for ais is to improve the patients long - term functional outcome . to date , the only proven therapy for ais is early recanalization ( 24 ) . the fda approved the use of alteplase in treatment of ischemic stroke based on the results of the ninds ( national institute of neurological disorders and stroke ) study in 1996 ( 2 ) . in this study , the time window was 3 hours and it showed that alteplase - treated patients had a 30 % higher probability of recovering with little or no deficit after 3 months . this clinical benefit of alteplase treatment overcame the risk of symptomatic intracerebral hemorrhage that occurred in 5.8% of cases , compared with placebo in which intracerebral hemorrhage was reported in 0.6% of cases ( 26 ) . several years later therapeutic time window was extended up to 4.5 hours , based on results of the ecass iii ( european cooperative acute stroke study ) study ( 26 ) . however , this study excluded patients over 80 years of age , those with severe stroke ( clinically with nihss > 25 , or radiologically with more than one - third of the middle cerebral artery territory involved ) and patients with a combination of diabetes and a prior stroke . alteplase promotes thrombolysis by hydrolyzing the arginine - valine peptide bond in plasminogen to form the active proteolytic enzyme plasmin , leading to an effective fibrin clot dissolution . in contrast to streptokinase and urokinase , alteplase is relatively fibrin - selective plasminogen activator , and therefore it is relatively inactive in the systemic circulation ( 27 ) . beside the risk of intra cranial hemorrhage and allergic reactions as adverse effects of alteplase , note should be taken of angioedema ( 11 ) , that occurs in 1 - 2 % of cases and it is more common in patients taking ace inhibitors ) ( 28 ) . the benefit of thrombolytic therapy is undeniable , but each patient receiving it should be observed carefully . although rare , splenic rupture should be considered in any patient receiving thrombolytic therapy , with signs of systemic bleeding , especially in those in which there is a history of trauma or of spleen disease , which is a predisposing factor for rupture . in our case , there was no history of trauma , myeloproliferative diseases or splenomegaly . signs of systemic bleeding developed shortly after administration of alteplase and ct scan of abdomen and pelvis confirmed the splenic rupture .	introduction : stroke is a medical emergency in neurology , and is one of the leading causes of death nowadays . at a recent time , a therapeutic method used in adequate conditions is thrombolysis , a treatment of an emerging clot in the brain vascular system by alteplase . the application of alteplase also has a high risk of life threatening conditions.case report : this is a brief report of a case with thrombolysis complication which manifested as a spleen rupture .
children with physical disabilities have lower bone mineral density ( bmd ) and a higher risk of osteoporosis than healthy children . contributing factors include decreased functional level , nutritional status , reduced calcium intake , intake of antiepileptic drugs , limited sunlight exposure related to decreased outdoor activities , and immobilization . childhood is a critically important period in the achievement of healthy bone mass . as the results of failure in achievement of optimal bone health , children with developmental disabilities are at a high risk of lifetime osteoporosis and are prone to traumatic fractures , even during simple activities such as dressing and undressing . nearly 20% of nonambulatory children and young adults had sustained a femur fracture at some time in their life . the incidence of fractures in cerebral palsy ( cp ) children with moderate to severe motor impairment has been reported up to 9.7% per year . reduced physical activity and abnormal muscle tone can also negatively influence bone growth in length . weight bearing standing exercise is widely used in rehabilitation programs and may enhance functional abilities , increase bmd , and prevent or minimize musculoskeletal problems . however , relatively little is known about the effect of standing or weight bearing exercise on bone health during vigorous growth period . because of the limited clinical evidence of its effects on bmd and bony length growth , the frequency and duration of weight bearing standing exercise are usually decided based on the experience of the clinician . the present study was performed to determine the effect of regular programmed weight bearing exercise on bmd and bone growth in children with cp . we included only the spastic cp children who were unable to stand without support , with a functional level of gross motor functional classification scale ( gmfcs ) v , for homogeneity of participants , because muscle spasticity or different functional level may influence the bmd . exclusion criteria were as follows : previous fracture of the lower limb , severe spinal deformity , hip dislocation , surgery on the spine , lower limbs , or nerve block within the previous 3 months , oral anticonvulsant intake , and current vitamin d supplementation or history of vitamin d supplementation within 6 months . between december 1 , 2011 and june 30 , 2012 , 44 children with cp were recruited from the physical medicine and rehabilitation department of a tertiary hospital . of the 44 children with cp who underwent an eligibility assessment , 15 were deemed eligible for the randomization procedure . participants were randomly assigned by the study biostatistician , who was not involved in the outcome measure . this randomization was achieved by assigning participants to either the programmed standing exercise group ( group a ) or the standing as usual group according to conventional rehabilitation treatment ( group b ) with equal probability within randomly permuted blocks of size 2 , 4 , and 8 . due to the preliminary nature of the study , stratification in grouping was not performed . one child in group a and 2 children in group b discontinued participation in the study . finally data from 6 healthy children ( control group ) and 12 children with cp ( group a = 7 children ; group b = 5 children ) were analyzed . participants in the control group were healthy and free of any diseases that affect growth or development . those with a history of leg fractures , scoliosis , and current or previous pharmacotherapy within 6 months that may affect growth were excluded . written informed consent was obtained from primary caregivers of all study participants , and approval for the study was obtained from the institutional review board . all participants in group a and group b were enrolled in each program for 6 months : group a with programmed assisted standing for more than 2 hours a day , at least 5 days a week ; and group b with assisted standing for 20 minutes a day , 2 to 3 days a week . with exception of assisted standing program , other conventional rehabilitation program including neurodevelopmental treatment , gross motor task training , functional electrical stimulation was undertaken in the same manner in both groups . all participants underwent a dual - energy x - ray absorptiometry ( dexa ) scan and full - length anteroposterior radiography to measure femur and tibia bone lengths at baseline and after 6 months . standard scanning procedures were used for the femur neck . to minimize motion - related variability , subjects with severe involuntary muscle contractions or uncontrolled movement were supported by a frame or sedated with 0.7 mg / kg body weight midazolam 15 to 30 minutes before radiologic procedures . leg length was measured by supine long - leg radiographs , and 2 measurements were made by 1 physiatrist . first , the length between the highest point on the femoral head and the projection of the center of the intercondylar notch on a line touching the femoral condyles was measured . second , the distance from the same point on the line between the femoral condyles up to the lowest point on the tibial articular surface in the ankle was measured . the wilcoxon signed - rank test was used to analyze the change of bmd and bone length at baseline and at 6 months . we included only the spastic cp children who were unable to stand without support , with a functional level of gross motor functional classification scale ( gmfcs ) v , for homogeneity of participants , because muscle spasticity or different functional level may influence the bmd . exclusion criteria were as follows : previous fracture of the lower limb , severe spinal deformity , hip dislocation , surgery on the spine , lower limbs , or nerve block within the previous 3 months , oral anticonvulsant intake , and current vitamin d supplementation or history of vitamin d supplementation within 6 months . between december 1 , 2011 and june 30 , 2012 , 44 children with cp were recruited from the physical medicine and rehabilitation department of a tertiary hospital . of the 44 children with cp who underwent an eligibility assessment , 15 were deemed eligible for the randomization procedure . participants were randomly assigned by the study biostatistician , who was not involved in the outcome measure . this randomization was achieved by assigning participants to either the programmed standing exercise group ( group a ) or the standing as usual group according to conventional rehabilitation treatment ( group b ) with equal probability within randomly permuted blocks of size 2 , 4 , and 8 . due to the preliminary nature of the study , stratification in grouping was not performed . one child in group a and 2 children in group b discontinued participation in the study . finally data from 6 healthy children ( control group ) and 12 children with cp ( group a = 7 children ; group b = 5 children ) were analyzed . participants in the control group were healthy and free of any diseases that affect growth or development . those with a history of leg fractures , scoliosis , and current or previous pharmacotherapy within 6 months that may affect growth were excluded . written informed consent was obtained from primary caregivers of all study participants , and approval for the study was obtained from the institutional review board . all participants in group a and group b were enrolled in each program for 6 months : group a with programmed assisted standing for more than 2 hours a day , at least 5 days a week ; and group b with assisted standing for 20 minutes a day , 2 to 3 days a week . with exception of assisted standing program , other conventional rehabilitation program including neurodevelopmental treatment , gross motor task training , functional electrical stimulation was undertaken in the same manner in both groups . all participants underwent a dual - energy x - ray absorptiometry ( dexa ) scan and full - length anteroposterior radiography to measure femur and tibia bone lengths at baseline and after 6 months . standard scanning procedures were used for the femur neck . to minimize motion - related variability , subjects with severe involuntary muscle contractions or uncontrolled movement were supported by a frame or sedated with 0.7 mg / kg body weight midazolam 15 to 30 minutes before radiologic procedures . leg length was measured by supine long - leg radiographs , and 2 measurements were made by 1 physiatrist . first , the length between the highest point on the femoral head and the projection of the center of the intercondylar notch on a line touching the femoral condyles was measured . second , the distance from the same point on the line between the femoral condyles up to the lowest point on the tibial articular surface in the ankle was measured . the wilcoxon signed - rank test was used to analyze the change of bmd and bone length at baseline and at 6 months . the healthy control group ( 3 girls and 3 boys ; age range , 2777 months ; mean age , 40.33 19.14 months ) , group a ( 4 girls and 3 boys ; age range , 2262 months ; mean age , 34.43 13.91 months ) , and group b ( 4 girls and 1 boy ; age range , 2777 months ; mean age , 38.80 21.61 months ) showed no statistical differences in age and sex . at 6-month follow - up , there was no change in the level of gmfcs for all participants . bmd changes for each group are demonstrated in table 1 . at baseline , the femoral neck bmd in group a and group there was no difference in the femoral neck bmd between group a and group b at baseline . femoral neck bmd was significantly lower in groups a and b after 6 months than in the control group . six - month follow - up showed a significant increase in femoral neck bmd in the control group . although bmd changes in groups a and b were not significant , group a demonstrated an increasing trend for bmd , whereas a decreasing trend was observed for group b. table 2 demonstrates changes in bone length of the lower limb in each group . changes in bone length were not statistically significant ; however , the greatest change was observed in the control group and the smallest change was found in group b. changes in bone length of the lower limbs . in the general population , regular vigorous weight bearing exercise of 1 hour or more per week is associated with an increase in bmd . moderate but important increases in skeletal mass are also associated with increased physical activity levels in children . several intervention studies with weight bearing activity have identified improvements in bmd in cp ; however , there are inadequate data ( level u ) to support a recommendation for weight bearing activities as an effective intervention for improving bmd . this preliminary study was performed to reveal the effect of weight bearing exercise on changes in bmd and bone growth . although no statistical significance was found in this study , the increased trend of bmd in the regular programmed exercise group ( group a ) and the decreased trend of bmd in the conventional treatment group ( group b ) indicate a positive effect of weight bearing exercise in increasing bmd in children with cp . therefore , if available , weight bearing exercise , even with equipment , should be considered as a prerequisite in rehabilitation programs for children with cp at a high risk of osteoporosis . the limited ability to maintain antigravity head and trunk postures and control leg and arm movements may negatively impact quantitative and qualitative bone growth . quadriplegic cp children are 5% shorter than healthy children at 2 years of age and more than 10% shorter at 8 years of age . in the present study , bone length of children in the control group was highest at baseline and after 6 months , although there was no significant difference between the groups . changes in bone growth after 6 months were highest in the control group and lowest in group b. from this result , we can assume that weight bearing exercise may help children with cp to catch up in normal height growth . the key strength of this study is the homogeneity of experimental groups that we recruited only the cp children with same functional level , gmfcs level v , who are totally dependent during transportation . in addition , healthy children were recruited as a control group to compare patterns of changes in bmd and bone growth with those in children with cp . all children in the control group were siblings of children in the patient groups . from an ethical perspective , weight bearing exercise could not be completely deprived in group b , which would have reduced the statistical significance of bmd differences between patient groups . interestingly , conventional treatment including weight bearing exercise for 20 minutes a day , 2 to 3 days a week , resulted in decreased bmd after 6 months . on the basis of these results , there appears to be a minimum requirement of weight bearing activity for promoting an increase in bmd in children with cp . in the present study , a regular standing program was designed as standing for more than 2 hours a day , more than 5 days a week . furthermore , standing for only 2 hours a day is very little compared with standing in healthy children . however , this program positively affected children with cp who are unable to walk , because the mechanical loading set point ( mechanostat ) , as theorized by frost , may be lowered in children with disabilities compared with healthy children . in adults , a deficit of 1 sd in bone mass is associated with a 1.5- to 3-fold increase in the incidence of fractures . accrual of bone mass occurs throughout childhood and early adulthood , and peak bone mass is a key determinant of the lifetime risk of osteoporosis . according to the pathophysiology of osteoporosis in children with cp , bone remodeling is a life - long process where bone resorption and formation is damaged in young adults and middle - aged adults . children with cp and gmfcs levels iv v have an increased risk of fractures compared with normally developing children . the femur is subjected to mechanical loading only during standing and is vulnerable to osteoporosis . furthermore , bone density of the vertebrae may be affected by both standing and sitting . therefore , measurement of femur bmd may have a greater clinical significance , which is why femur bmd was selected as a parameter to monitor bmd change after weight bearing exercise in the present study . the results presented in this study may provide the basis of an effective means of increasing peak bone mass in children who are unable to stand . however , further studies are required to reveal the implications of increased bmd on pathological situations . during the course of the study , no complications were observed in any of the participants . as weight bearing exercise using a standing frame was demonstrated to be safe in the present study , we suggest that children with cp use a standing frame for more than 2 hours a day , at least 5 days a week . the sample size was determined without considering statistical significance due to the limited number of volunteers . it was only conducted in a small number of patients and healthy controls . because of the difficulty in recruiting a sufficient number of healthy children to calculate the z score , the t score was used in bmd analysis . although the t score is of little use in the diagnosis of osteoporosis in children , it may be used to monitor the relative change in bmd . weight bearing exercise may play an important role in increasing or maintaining bmd in children with cp and is expected to promote bone growth . programmed standing exercise ( at least 2 hours a day , more than 5 days a week ) could be used as an effective treatment to increase bmd and to catch up in normal bone growth in children with cp . however , further studies with a larger patient cohort and longer follow - up periods are required to reveal the benefit of weight bearing exercise and to establish a detailed program . further considerations are also required on increasing bmd in children with disabilities who are unable to tolerate standing posture .	abstractbackground : the present study aims to explore the effect of weight bearing exercise on bone mineral density ( bmd ) and bone growth in children with cerebral palsy ( cp).methods : twelve children with cp of functional level of gross motor functional classification scale ( gmfcs ) v and 6 healthy children ( control group ) were included in the study . participants underwent a dual - energy x - ray absorptiometry scan to measure the bmd of the femur and full - length anteroposterior radiography to measure the bone length of the femur and tibia at baseline and after 6 months . patients were randomly divided into 2 groups : group a with programmed standing exercises and assisted standing for more than 2 hours a day , more than 5 days a week ; and group b with conventional physiotherapy with a standing program for 20 minutes a day , 2 to 3 days a week.results:a 6-month follow - up showed significantly increased bmd on the femur neck in the control group . although the changes in bmd were not significant in both groups , group a demonstrated an increased trend of bmd , whereas group b showed a decreased trend . bone length was significantly increased in all 3 groups at the 6-month follow - up . although this increase was not significant , the change in bone length was greatest in the control group . the smallest changes were observed in group b.conclusions:weight bearing exercise may play an important role in increasing or maintaining bmd in children with cp and is also expected to promote bone growth . programmed standing may be used as an effective treatment method to increase bmd in children with cp . however , further studies with a larger cohort and longer follow - up period are required to reveal further information on the benefit of weight bearing exercise and to develop a detailed program .
it has been reported that mild dilatation of saphenous vein grafts ( svg ) occurs in 14% of the cases at 57 years after surgery 1 . however , giant aneurysm of the svg is a rare complication of coronary artery bypass surgery , with an overall incidence of < 1% 2 , 3 . although svg aneurysms are often incidentally identified on any type of imaging modality , there are some reports about cases of rupture 4 , fistula formation with neighboring anatomy 5 , and hemodynamic compromise resulting from compression of adjacent cardiac and vascular structures 6 . moreover , inhospital mortality associated with giant svg aneurysms was reported to be 15.7% 7 . svg aneurysms have been generally treated by surgical repair , such as resection with or without bypass of the affected territory . although the use of percutaneous treatment has been increasing recently 3 , 8 , 9 , 10 , 11 , there is no report about svg aneurysms treated with percutaneous revascularization followed by transcatheter coil embolization . here , we report the case of a giant svg aneurysm that was successfully treated with catheter intervention . the patient was a 67yearold man who had undergone coronary artery bypass graft surgery ( cabg ) using saphenous vein graft ( svg ) 22 years before and had not presented chest symptoms during follow up . transthoracic echocardiography demonstrated that the svg aneurysm had been progressively enlarging at a rate of 5 mm within 6 months from july 2012 ( fig . besides , chest xray showed a masslike shadow on the left side of mediastinum ( fig . 1b ) . enhanced computed tomography ( ct ) revealed a large aneurysm of the svg ( diameter of aneurysm was 38 42 mm and its length was 80 mm ) ( fig . the cardiac surgeon considered that it was necessary to perform surgical resection with revascularization to avoid fatal complications such as rupture of the aneurysm which could lead to sudden death . however , because the patient rejected rethoracotomy , he was referred to our division to treat this svg aneurysm with catheter intervention . ct revealed a large aneurysm of the svg ( 38 42 mm in diameter ; 80mm long ) and graft patency . d : coronary angiogram of the left coronary artery at the left anterior oblique with caudal view . the white arrow indicates the leakage of contrast medium from the proximal portion of the svg . rv , right ventricle ; pa , pulmonary artery ; svg , saphenous vein graft ; ct , computed tomography ; lad , left anterior descending artery . coronary angiogram ( cag ) demonstrated that the native left anterior descending artery ( lad ) was totally occluded from the origin to its mid portion ( fig . in addition , there was a large leakage of contrast medium from the proximal portion of the svg ( fig . first , we performed percutaneous coronary intervention ( pci ) for chronic total occlusion ( cto ) of the native lad with bidirectional approach via the svg . an 8fr xblad3.5 guiding catheter ( britetip , cordis , nj ) was engaged into the left coronary artery and an 8fr amplatz left 1.0 with a shorttip guiding catheter ( britetip , cordis ) was engaged into the svg . we introduced a floppy guidewire ( sion blue , asahi intecc , aichi , japan ) and an intravascular ultrasound ( ivus ) catheter ( volcano platinum , volcano , san diego , ca ) into the high lateral branch and identified the orifice of the lad . after that , we advanced a crusade catheter ( double lumen microcatheter ) ( kaneka medix , osaka , japan ) along this floppy guidewire and penetrated the proximal cap of the lad cto using a guidewire with an intermediate tip load ( 3.5 g ) and a tapered tip ( gaia second , asahi intecc ) via the side hole of the crusade catheter . after confirming the position of the gaia second guidewire using ivus , we exchanged the microcatheter from the crusade to a corsair microcatheter ( asahi intecc ) and advanced this guidewire to the more distal portion of the cto . however , because the gaia second guidewire progressed into the subintimal space , we switched to the retrograde approach via the svg . we penetrated the distal cap of the cto using the gaia first guidewire ( tip load of 1.5 g and tapered tip ) ( asahi intecc ) and advanced this guidewire toward the antegrade guidewire with the support of the corsair microcatheter . finally , we could successfully recanalize the cto lesion under ivusguided reverse controlled antegrade and retrograde subintimal tracking ( cart ) technique using a balloon catheter of 2.5 mm in diameter . we implanted two biolimuseluting stents ( nobori , terumo , tokyo , japan ) from the mid portion of the lad to the orifice of the left main trunk and performed simultaneously kissing balloon technique between the lad and the left circumflex artery ( lcx ) . the final cag showed a wellexpanded cto lesion without disappearance of any branches ( fig . one month after the pci for the cto lesion of the lad , we performed transcatheter embolization ( tce ) for the svg . at first , we deployed a drugeluting stent ( xience prime , abbott vascular , santa clara , ca ) for the distal portion of the lad at the anastomosis of the svg to avoid migration of coils from the svg to the lad . then , we engaged a 7fr judkins right 4.0 guiding catheter of short length ( 85 cm ) ( camino , goodman , aichi , japan ) to the svg and inserted a 5.2fr angiocatheter ( selecon mp catheter ii , terumo clinical supply , gifu , japan ) , which had a balloon of 9.0 mm in diameter at the tip of the catheter , to the svg via the 7fr guiding catheter ( fig . 2a ) . during the procedure , we inflated the balloon at the orifice of the svg to shut off the blood flow from the aorta and to avoid drift of coils and distal embolism of the native lad ( fig . a microcatheter ( renegade18 , boston scientific , natick , ma ) was introduced into the svg through the 5.2fr angiocatheter and 4 coils ( gdc18 8 300 mm , gdc18 5 200 mm , idcsoft 3 100 mm , vortex diamond18 4 41 mm , stryker , kalamazoo , mi ) were deployed at the distal portion of the svg via this microcatheter . we then deployed another 4 coils ( gdc18 12 300 mm , gdc18 10 300 mm , idc 8 200 mm , idcsoft 5 120 mm , stryker ) at the proximal portion of the svg . transcatheter embolization systems and its fluoroscopic image . a : picture of transcatheter embolization systems . a 5.2fr and 100cm length angiocatheter was introduced into a 7fr jr4.0 shaped with 85cm length guiding catheter via the unidirectional valve . moreover , b : fluoroscopic image . the white circle indicates the inflated balloon of the 5.2fr angiocatheter . during transcatheter embolization , we inflated this balloon at the orifice of the svg to shut off the blood flow from the aorta . enhanced ct revealed that the svg remained occluded and that the size of the svg aneurysm had decreased ( diameter was 30 32 mm ) . c : cag of the left coronary artery at the left anterior oblique with caudal view . just after the percutaneous coronary intervention for chronic total occlusion of the lad . d : cag of the left coronary artery at the left anterior oblique with caudal view . ct , computed tomography ; svg , saphenous vein graft ; cag , coronary angiogram ; lad , left anterior descending artery ; tce , transcatheter embolization . three months after the tce , chest xray and ct demonstrated that the svg had not recanalized and the size of the aneurysm had decreased ( fig . one year after the tce , cag revealed that the lad maintained its patency ( fig . first , we performed percutaneous coronary intervention ( pci ) for chronic total occlusion ( cto ) of the native lad with bidirectional approach via the svg . an 8fr xblad3.5 guiding catheter ( britetip , cordis , nj ) was engaged into the left coronary artery and an 8fr amplatz left 1.0 with a shorttip guiding catheter ( britetip , cordis ) was engaged into the svg . we introduced a floppy guidewire ( sion blue , asahi intecc , aichi , japan ) and an intravascular ultrasound ( ivus ) catheter ( volcano platinum , volcano , san diego , ca ) into the high lateral branch and identified the orifice of the lad . after that , we advanced a crusade catheter ( double lumen microcatheter ) ( kaneka medix , osaka , japan ) along this floppy guidewire and penetrated the proximal cap of the lad cto using a guidewire with an intermediate tip load ( 3.5 g ) and a tapered tip ( gaia second , asahi intecc ) via the side hole of the crusade catheter . after confirming the position of the gaia second guidewire using ivus , we exchanged the microcatheter from the crusade to a corsair microcatheter ( asahi intecc ) and advanced this guidewire to the more distal portion of the cto . however , because the gaia second guidewire progressed into the subintimal space , we switched to the retrograde approach via the svg . we penetrated the distal cap of the cto using the gaia first guidewire ( tip load of 1.5 g and tapered tip ) ( asahi intecc ) and advanced this guidewire toward the antegrade guidewire with the support of the corsair microcatheter . finally , we could successfully recanalize the cto lesion under ivusguided reverse controlled antegrade and retrograde subintimal tracking ( cart ) technique using a balloon catheter of 2.5 mm in diameter . we implanted two biolimuseluting stents ( nobori , terumo , tokyo , japan ) from the mid portion of the lad to the orifice of the left main trunk and performed simultaneously kissing balloon technique between the lad and the left circumflex artery ( lcx ) . the final cag showed a wellexpanded cto lesion without disappearance of any branches ( fig . one month after the pci for the cto lesion of the lad , we performed transcatheter embolization ( tce ) for the svg . at first , we deployed a drugeluting stent ( xience prime , abbott vascular , santa clara , ca ) for the distal portion of the lad at the anastomosis of the svg to avoid migration of coils from the svg to the lad . then , we engaged a 7fr judkins right 4.0 guiding catheter of short length ( 85 cm ) ( camino , goodman , aichi , japan ) to the svg and inserted a 5.2fr angiocatheter ( selecon mp catheter ii , terumo clinical supply , gifu , japan ) , which had a balloon of 9.0 mm in diameter at the tip of the catheter , to the svg via the 7fr guiding catheter ( fig . 2a ) . during the procedure , we inflated the balloon at the orifice of the svg to shut off the blood flow from the aorta and to avoid drift of coils and distal embolism of the native lad ( fig . a microcatheter ( renegade18 , boston scientific , natick , ma ) was introduced into the svg through the 5.2fr angiocatheter and 4 coils ( gdc18 8 300 mm , gdc18 5 200 mm , idcsoft 3 100 mm , vortex diamond18 4 41 mm , stryker , kalamazoo , mi ) were deployed at the distal portion of the svg via this microcatheter . we then deployed another 4 coils ( gdc18 12 300 mm , gdc18 10 300 mm , idc 8 200 mm , idcsoft 5 120 mm , stryker ) at the proximal portion of the svg . was introduced into a 7fr jr4.0 shaped with 85cm length guiding catheter via the unidirectional valve . moreover , , we inflated this balloon at the orifice of the svg to shut off the blood flow from the aorta . enhanced ct revealed that the svg remained occluded and that the size of the svg aneurysm had decreased ( diameter was 30 32 mm ) . c : cag of the left coronary artery at the left anterior oblique with caudal view . just after the percutaneous coronary intervention for chronic total occlusion of the lad . d : cag of the left coronary artery at the left anterior oblique with caudal view . ct , computed tomography ; svg , saphenous vein graft ; cag , coronary angiogram ; lad , left anterior descending artery ; tce , transcatheter embolization . three months after the tce , chest xray and ct demonstrated that the svg had not recanalized and the size of the aneurysm had decreased ( fig . one year after the tce , cag revealed that the lad maintained its patency ( fig . a giant svg aneurysm is a rare complication of cabg . nevertheless , because of its high potential for morbidity and mortality , we should diagnose and treat this complication adequately . in our case , we could detect a progressively enlarging svg aneurysm by multiple imaging modalities ( echocardiography , ct , and cag ) and successfully treat it with catheter intervention . a review article reported that only 4.2% of patients develop an svg aneurysm within the first year after cabg , with 6.1% identified between 1 and 5 years , 21.2% between 5 and 10 years , and 68.5% at over 10 years after surgery 3 . clinical symptoms associated with svg aneurysms vary greatly among patients , and include chest pain ( 46.4% ) , shortness of breath ( 12.9% ) , myocardial infarction ( 7.7% ) and so on . nevertheless , one third of svg aneurysms were discovered incidentally 3 . in the present case , we identified the svg aneurysm coincidentally 22 years after cabg during follow up using echocardiography , ct , and cag . adverse events associated with svg aneurysms are mechanical complications such as compression , fistula formation to adjacent structures , or aneurysm rupture 3 , 4 , 5 , 6 . moreover , it has been reported that an event rate of mechanical complications was 33.3% even in 20 mm diameter of small svg aneurysms 3 . thus , to avoid these serious complications we should carefully follow the patients after cabg using svgs , especially when several years have passed after the surgery , and it is desirable to perform some kind of treatment once svg aneurysm is identified . as for the management of svg aneurysms , not only catheterization but also crosssectional imaging modalities such as ct or magnetic resonance imaging are necessary because angiography can not identify an intraluminal thrombus and the size of the aneurysm or the interaction with adjacent structures can not be evaluated . strategy of treatment for svg aneurysm has not been fully established because of small number of case reports about svg aneurysm . patients with other surgical indications ( i.e. concomitant valvular disease or multiple territories requiring revascularization ) or mechanical complications are recommended to undergo surgical treatment including aneurysm resection with subsequent bypass grafting 12 . in addition , for patients with patent affected grafts in whom the graft anatomy is not favorable for implantation of covered stents , surgical treatment should be selected . on the other hand , the number of patients subjected to percutaneous management for svg aneurysm has been increasing recently 3 , 8 , 9 , 10 , 11 . in patients whose svg remains patent and in whom continued antegrade blood flow through the svg is necessary , percutaneous management with covered stents implantation is suitable 3 . however , fusiform aneurysms can pose a problem due to the limited length of coronary covered stents or veincovered stents 8 . also , in case of percutaneous treatment with peripheral covered stents 9 , it is quite difficult to deliver the treatment system into the svg and it involves many risks such as vessel rupture , distal embolism , and so on . the svg aneurysm can be occluded by coil embolization 1 , 3 , 11 or vascular plugs 10 in patients whose svg is occluded but shows unilateral patency and persistent flow into the aneurysmal segment or in whom the affected graft supplies a small territory . in our patient , the svg remained patent and the territory of myocardium that was supplied by this svg was broad . however , the svg aneurysm was too large to be treated with the covered stents available in japan . furthermore , because cabg had been performed 22 years previously and his svg became degenerated , the risk of thromboembolism or new aneurysm formation had to be considered even if the svg could have been treated using covered stents . moreover , he was still not so old , 67yearold , and did not complicate other comorbidities . thus , at first , we considered that a surgical repair combined aneurysm resection and bypass grafting was feasible for this patient . on the other hand , there was no severe stenotic lesion at his right coronary artery and lcx , and he rejected rethoracotomy . in addition , redo cabg has greater risks of operative mortality compared with primary cabg 13 . in consequence of a discussion among a heart team including cardiologists and cardiac surgeons of our institution , we decided to treat this patient with catheter intervention , which meant that we primarily recanalize the cto of the native lad with bidirectional approach via the svg and subsequently perform tce using endovascular coils . nevertheless , if we could not recanalize the cto of the native lad , we adopted a surgical treatment as a backup plan and the patient consented to this strategy . although we performed the tce 1 month after the pci with a view to thrombotic occlusion of the svg after recanalization of the native lad , it is feasible to perform recanalization and tce consecutively within several days because the svg maintained the patency even 1 month after the recanalization of the native lad and this situation had a risk of occlusion of the native coronary artery due to competition of coronary blood flow . moreover , simultaneous procedure of recanalization and tce is unfavorable because acute stent thrombosis may occur . this is the first report of svg aneurysm successfully treated by catheter intervention including pci for the cto of native coronary artery and tce for the svg aneurysm . in case of an svg aneurysm that remains patent , percutaneous revascularization of native coronary artery followed by tce is considered a useful strategy to treat an svg aneurysm because the introduction of drugeluting stent has resulted in improved patency of cto in the chronic phase 14 and the initial success rate of pci for cto lesions has been improved with advancing of pci techniques 15 . we successfully treated a patient with giant svg aneurysm with two times of catheter intervention . tce after revascularization with pci for native coronary artery is one of the useful options of the treatment of svg aneurysm .	the patient was a 67yearold man who had undergone coronary artery bypass graft surgery using a saphenous vein graft ( svg ) 22 years before . computed tomography angiogram revealed a large aneurysm of the svg ( 38 42 mm in diameter ; 80mm long ) and total occlusion of the left anterior descending artery ( lad ) . we first performed percutaneous coronary intervention for chronic total occlusion of the native lad with bidirectional approach via the svg . one month later , we performed the transcatheter embolization of the svg and occluded the svg using multiple coils . this case demonstrates that transcatheter embolization after recanalization of native coronary artery is an effective strategy to treat an svg aneurysm . 2015 wiley periodicals , inc .
since strabismus surgery manipulates extraocular muscles ( eoms ) mechanically to correct binocular misalignment , accurate determination of quasistatic biomechanical properties of eoms may be important . more recently , comprehensive biomechanical methods have been employed to characterize constitutive models for eoms , representing their viscoelastic , or history - dependent relationship between stress and strain [ 6 , 7 ] . several important mechanical behaviors reflect viscoelasticity : hysteresis , relaxation , attenuation of acoustic waves , and creep . hysteresis is history dependent variation in mechanical behavior , resulting in differences in stress between loading and unloading phases . hysteresis implies work , energy dissipation due to loading and unloading [ 5 , 8 , 9 ] . relaxation describes how a material deformed by external perturbation returns to equilibrium . in stress relaxation testing , a material is rapidly subjected to displacement and then maintained over an extended time interval at the same displacement by external feedback while a decline in required external force is observed [ 6 , 1013 ] . creep is the tendency of a material to deform permanently under constant force . to characterize creep , tensile loading is rapidly imposed and maintained at a constant level while specimen elongation is observed over an extended time [ 13 , 14 ] . creep is likely to be particularly significant in binocular alignment and strabismus , where agonist and antagonist eoms remain under loading for extended time periods , and where such loading is purposely altered by , for example , strabismus surgery that alters eom tension . while viscoelastic properties have often been neglected in interpretation of conventional eom length - tension data , realistic constitutive modeling is essential for accurate finite element models ( fems ) that graphically simulate interactions of eoms with orbital connective tissues . supporting this contention , quaia et al . and our previous investigation highlighted the need for valid constitutive models of eoms in application of quasilinear viscoelastic ( qlv ) theory to constitutive modeling . soft tissues generally , however , undergo larger physiological strains in the range of tens of percent where behavior becomes nonlinear . modeling of eoms should consider the hyperelastic nature of soft tissue , which means that it is highly nonlinearly elastic [ 12 , 1520 ] fung 's qlv theory [ 5 , 2123 ] is appropriate because it incorporates both linear viscoelastic and nonlinear elastic characteristics . we previously reported that a qlv model based upon in vitro relaxation data was effective in describing mechanical behavior of passive bovine eoms . quaia et al . found limitations in qlv describing constitutive properties of in vivo simian eoms at short - time scale and proposed a modification that they termed adaptive quasilinear viscoelasticity ( aqlv ) . although aqlv was superior to qlv in describing eom properties relevant to the most rapid eye movements ( saccades ) , the approach is computationally intensive and requires extensive data collection that would limit its practicality for fem . we supposed that the simpler qlv formulation might be adequate to quasistatic , passive behavior of eoms relevant in binocular alignment and strabismus . since the eoms are not contracting when under tensile loading , we emulated a previous study with the assumption that absence of perfusion and innervation would not strongly influence constitutive properties . by investigating eom creep , we aimed to extend understanding of their time - dependent stress - strain behavior and to validate the previously reported relaxation - based qlv model applicable to fem . the eom specimens prepared from heads of cattle freshly slaughtered for food . the total time from slaughter to mechanical testing ranged from 3 to 4 hours . during the preparation of specimens , the tissues were constantly irrigated with lactated ringer solution , which was kept at 37c , to prevent any dehydration . the eom specimens were cut into 23 cm long rectangular prisms ( length of actual bovine eoms ) having 2 mm 2 mm cross - section . actual test length of each specimen was 10 mm , with 5 mm on both ends that was clamped on the load cell , excluding the terminal tendon . as in the previous study , a tabletop microtensile load cell , instron model 4411 ( instron , norwood , mass , series ix software ) was enclosed in a plastic chamber where warm water vapor and radiant heat maintained physiologic 97% humidity and 37c temperature as indicated by a sensor ( fisher scientific , chino , calif ) . preconditioning was omitted because a previous investigation of passive eom relaxation properties demonstrated that preconditioning did not influence results . for both creep testing and preliminary experiments to optimize the testing parameters , total of 60 eom specimens from 8 different bovine orbits were used . , an initial tensile force should be imposed on the specimen instantaneously and then maintained at a constant level for an extended time [ 5 , 13 ] . since instantaneous imposition of tensile force is physically impossible , tensile force is rapidly increased at a constant ramp rate to a level that is subsequently maintained . based upon tensile - loading results , we selected 0.2 n , which produces 30% strain that is well within the linear range , as an appropriate ramp force . although the maximum loading rate of the load cell was 550 mm / min , it was necessary to limit the rate to 100 mm / min to maintain stable force feedback . therefore , all the creep tests were performed at the following four rates : 10 , 20 , 50 , and 100 mm / min , equivalent to 1.67 , 3.33 , 8.33 , and 16.67%/s , respectively . after the initial loading of 0.2 n was imposed , this force was then maintained by the load cell 's feedback servo for 1,500 s during recording of specimen length . , strain is held constant and the stress declines as a function of time alone : ( 1)(t)=0g(t ) , where g(t ) is the time dependent reduced relaxation function with g(0 ) = 1 , and 0 is initial stress . similarly , for static creep in which stress is held constant , strain varies as a function of time alone : ( 2)(t)=0j(t ) , where j(t ) is the time dependent reduced creep function with j(0 ) = 1 and 0 is the initial strain . for linear viscoelasticity , which is a special case of the more general qlv theory , time - varying stress relaxation function g(t ) and creep function j(t ) should be related as follows : ( 3)g(0)=1j(0),g()=1j( ) , or ( 4)g(s)j(s)=1s2 , where g(s ) and j(s ) are laplace transforms of the functions g(t ) and j(t ) , respectively . a nonlinear least square method was used in previous study fit the stress relaxation data , determining constants a , b , c , d , g , and h : ( 5)g(t)=ae(bt)+ce(dt)+ge(ht ) . using the laplace transform of the stress relaxation function and ( 4 ) , a predicted creep function for eom was determined using matlab ( the mathworks inc , natick , mass , usa ) . as is typical for creep testing generally [ 5 , 24 , 25 ] , a logarithmically increasing displacement was observed after initial ramp loading of each specimen , without asymptote . data were plotted as normalized displacement j(t ) , the ratio of instantaneous specimen length to initial length . although relaxation and tensile properties were similar among the four rectus and both oblique bovine eoms in the previous study , preliminary experiments were conducted on two specimens of each of the six eoms to determine if they exhibit similar creep at 3.33%/s strain rate . as can be seen from figure 1 , if eom properties depend on initial loading , experiments were conducted for five specimens each at four ramp - loading rates : 1.67 , 3.33 , 8.33 , and 16.67%/s . as an example , figures 2(a ) and 2(b ) show the force displacement and creep plots for 0.2 n force . creep at other ramp - loading rates was similar to figure 2 , with maximum standard deviations ( sds ) for five specimens each of 0.029 , 0.056 , and 0.036 for 3.33 , 8.33 , and 16.67%/s loading , respectively . as seen in figure 3 , for all four initial loading rates , creep coefficients at the end of 1,500 seconds were similar at 1.37 0.033 . despite the ultimate similarity of creep behavior , the linear part of each curve under initial loading steepened as loading rate increased , and the shape of each curve varied with loading rate ( figure 3 ) . as loading rate decreased , the curve showed slower elongation . since eom is a soft tissue , reaching a true asymptotic value during creep testing is neither practical nor theoretically necessary [ 5 , 25 ] . although the creeping behavior exhibited by eom after ramp loading is used in its viscoelastic characterization , the rate - dependent ramp behavior can also be used to understand the contribution of eom elasticity in its overall creep behavior . with behavior of the eom during the ramp phase being less affected by viscosity as the loading rate is decreased , one can optimize the loading rate for tensile testing to extract the tensile elasticity of the eom . all constants in the reduced relaxation function were derived from the published relaxation function and are shown in table 1 . using parameters extracted from the qlv relaxation model , ( 4 ) in the time domain was converted to a reduced creep function using the inverse laplace transform function and symbolic toolbox in matlab , 6 constant values from the laplace s domain ( 6 ) , and 7 constants from the time domain ( 7 ) of the resulting reduced creep function are listed in table 2 ( 6)j(s)=1s2(a1/(s+a2)+a3/(s+a4)+a5/(s+a6 ) ) , ( 7)j(t)=c1t+c2sinh(c3t)c4cosh(c3t)c5exp(c6t)+c7 . since ideal initial loading is assumed instantaneous , data obtained at higher rates of initial ramp loading should more closely reflect ideal creep . hence , the reduced creep function derived from the reduced relaxation function was compared with experimental data for the highest ramp - loading rate , showing close agreement ( figure 4 ) . experimental creep coefficient values fell within 2.7% of theoretical values for all time points tested . when an eom is innervated and perfused with oxygenated blood , it is constantly under tension . it is important to understand eom creep properties for simulation of the common situation where a physiologically relaxing eom is still experiencing tensile force exerted by its antagonist . in the current study , passive bovine eom creep was investigated within the framework of linear viscoelasticity by comparing converted creep function from qlv model based upon stress relaxation data . the current experiment varied initial elongation rate and demonstrated excellent agreement with the theoretical prediction based upon qlv relaxation model . it has been suggested that initial ramp loading for creep testing of articular cartilage should occur within 250 ms ; however , even over a longer time of 800 ms , the data for bovine eoms agreed closely with values derived from relaxation function . regardless of which anatomical rectus or oblique eom was tested , creep for initial ramp - loading rates from 1.67 to 16.7%/s showed similar asymptotic creep coefficients of 1.37 0.03 over 1,500 s. however , dynamic creep varied with initial strain rate . creep coefficient j(t ) reached a higher initial value when the loading rate was lower , but subsequently increased more slowly ( figure 3 ) . it is a well - accepted notion that when solid specimen is stretched slowly , dynamic contributions are reduced because at low initial loading speed , elasticity predominates over viscous effects [ 2628 ] . with predominantly elastic effects , since the present models were based upon data from low strain rates , we suggest that the qlv models derived from these investigations are most applicable to slow eye movements such as fixations and pursuit . since loading for both relaxation and creep tests closely approximates ideal step loading , the relaxation and creep functions are likely to accurately reflect eom viscosity . since a reduced creep function is the inverse laplace transform of a reduced relaxation function , agreement between experimental creep and the previously published reduced relaxation function constitutes a strong test of the relaxation - based qlv model for passive eoms , with experimental agreement to within 2.7% of theoretical values . hence , we can infer that a qlv model based upon relaxation effectively describes the constitutive properties of passive eoms . the present validation of the quantitative viscoelastic constitutive relationship for passive bovine eom provides better understanding of eom biomechanics , in a theoretical framework practical for graphical simulation of quasistatic ocular motility using fem .	this paper characterized bovine extraocular muscles ( eoms ) using creep , which represents long - term stretching induced by a constant force . after preliminary optimization of testing conditions , 20 fresh eom samples were subjected to four different loading rates of 1.67 , 3.33 , 8.33 , and 16.67%/s , after which creep was observed for 1,500 s. a published quasilinear viscoelastic ( qlv ) relaxation function was transformed to a creep function that was compared with data . repeatable creep was observed for each loading rate and was similar among all six anatomical eoms . the mean creep coefficient after 1,500 seconds for a wide range of initial loading rates was at 1.37 0.03 ( standard deviation , sd ) . the creep function derived from the relaxation - based qlv model agreed with observed creep to within 2.7% following 16.67%/s ramp loading . measured creep agrees closely with a derived qlv model of eom relaxation , validating a previous qlv model for characterization of eom biomechanics .
ischemic stroke caused by large vessel occlusion is associated with poor outcome with high rates of death and disability.1 recent studies have indicated safety and efficacy of mechanical thrombectomy ( mt ) in the treatment of acute ischemic stroke ( ais).26 baseline national institutes of health stroke scale ( nihss ) score , age , and degree of recanalization are independent predictors of clinical outcome in patients undergoing mt.7 time to recanalization is crucial for a good clinical outcome as well,811 and is the strongest modifiable factor beyond patient characteristics . factors influencing time to recanalization can be divided into pre - hospital factors ( eg , detection of symptoms , contact of the emergency medical service , hospital transfer ) and in - hospital ( mostly logistic ) factors . several studies have already indicated that a single or combined logistic measure can be implemented and may shorten time to recanalization by reducing door - to - puncture time and/or picture - to - puncture ( p2p ) time.9 1214 a further reduction in p2p time and time to recanalization may be achieved by merging stroke imaging and mt in a dedicated combined imaging and treatment suite . this would deem patient transfer from imaging suite to angiography suite unnecessary , decrease logistic effort , and save time . a combination of imaging and angiography could be achieved by angiographic or flat - panel ct in the angiography suite . flat - panel ct is already used in peri - interventional visualization of complications ( eg , intracranial hemorrhage ) and perfusion imaging,1518 but image quality is not yet sufficient to rule out small intracranial hemorrhage19 or to detect early signs of an ischemic stroke . these imaging findings , however , are essential for the indication of intravenous thrombolytics and mt and , therefore , flat - panel ct so far can not replace conventional ct or mri of ais . to combine both imaging modalities we aimed to perform diagnostic angiography and mt on the table of the ct scanner in a dedicated setting . this was achieved by a new mobile c - arm x - ray device ( cios alpha ; siemens ag , healthcare sector , erlangen , germany ) using full - view flat detector technology and adapted to the spiral ct system . here the cios alpha is a commercially available mobile c - arm x - ray device and a mobile monitor cart . the device is equipped with a 3030 cm ( 1212 inch ) , 1.51.5 k full - view flat detector and a 25 kw power generator . a motorization package and a remote control unit allow direct control of the c - arm in a sterile field during endovascular therapy . the monitor cart mounts two high - brightness high - contrast thin - film transistor ( tft ) monitors , the data interface , and storage unit . the ct imaging suite is equipped with a commercially available 64-slice somatom definition as ct scanner ( siemens ag , healthcare sector , forchheim , germany ) . the ct patient table is installed 95 cm ( 37.4 inch ) away from the gantry , in contrast to the standard setup ( 30 cm ( 11.8 inch ) ) , thus enabling the c - arm of the cios alpha to be positioned in the gap between the gantry and patient table during mt ( figure 1a ) . horizontal and orbital movements of the c - arm are unrestricted whereas angular movement of the c - arm is limited to 20 in the anterior or posterior direction to avoid collision of the c - arm with the patient , gantry or patient table . for direct remote control of the patient table , ct scanner and c - arm , two remote control units for both the c - arm and ct scanner ( i - control ; siemens ag , healthcare sector , forchheim , germany ) are mounted to the side rail of the patient table . these switches allow the neurointerventionalist to move the ct scanner , maneuver the c - arm , and move the patient table in almost the same mode as in the existing standard biplane angiographic setup ( figure 1b ) . two ceiling - mounted monitors ( part of the adaptive three - dimensional intervention suite ; siemens ag , healthcare sector , erlangen , germany ) were installed to replace the monitor cart within the ct imaging suite . the monitor cart is stationed in an adjacent room allowing concurrent viewing and , if necessary , data processing without exposure to x - rays . in case of a malfunction of the ceiling - mounted monitors , the monitor cart can be brought immediately to the ct imaging room . to protect the neurointerventionalist from radiation , an adjustable ceiling - mounted upper body protective shield and a lower body x - ray protective shield are in place . for the treating anesthesiologist or neurointensivist , a mobile height - adjustable full body x - ray protective shield is available for upper and lower body protection from radiation . ( a ) setup of the ct / c - arm intervention suite with the cios alpha in its operating position ( partially covered by the height - adjustable upper body and the mobile lower body x - ray protection shields ) . note : the remote controls for the c - arm and the ct ( center , bottom ) can be mounted in alternative order to the side rail of the ct table . on admission , a stroke neurologist performed physical neurologic examinations and detailed assessment of the nihss score in the emergency room . patients with suspected ais and an nihss score of 10 received a standardized stroke imaging protocol consisting of a non - contrast enhanced cranial ct ( cct ) , ct perfusion of the brain as well as ct angiography of the aortic arch , cervical , and intracranial arteries . during the intervention , patients received standardized peri - interventional management and monitoring of physiological target values according to in - house standard operating procedures and the discretion of the treating neurointensivist adapted to the patient and situation . to detect possible peri - procedural complications , cct and/or ct perfusion all patients were initially observed in a neurologic intensive care unit after the interventional procedure . transfer to the stroke unit occurred as soon as possible depending on the clinical condition of the patient . follow - up ct or mri was routinely performed at 2036 h after treatment , or earlier if neurologic deterioration occurred . post - interventional nihss and modified rankin scale scores were assessed by detailed physical examinations performed by an independent stroke neurologist at discharge . the cios alpha is a commercially available mobile c - arm x - ray device and a mobile monitor cart . the device is equipped with a 3030 cm ( 1212 inch ) , 1.51.5 k full - view flat detector and a 25 kw power generator . a motorization package and a remote control unit allow direct control of the c - arm in a sterile field during endovascular therapy . the monitor cart mounts two high - brightness high - contrast thin - film transistor ( tft ) monitors , the data interface , and storage unit . the ct imaging suite is equipped with a commercially available 64-slice somatom definition as ct scanner ( siemens ag , healthcare sector , forchheim , germany ) . the ct patient table is installed 95 cm ( 37.4 inch ) away from the gantry , in contrast to the standard setup ( 30 cm ( 11.8 inch ) ) , thus enabling the c - arm of the cios alpha to be positioned in the gap between the gantry and patient table during mt ( figure 1a ) . horizontal and orbital movements of the c - arm are unrestricted whereas angular movement of the c - arm is limited to 20 in the anterior or posterior direction to avoid collision of the c - arm with the patient , gantry or patient table . for direct remote control of the patient table , ct scanner and c - arm , two remote control units for both the c - arm and ct scanner ( i - control ; siemens ag , healthcare sector , forchheim , germany ) are mounted to the side rail of the patient table . these switches allow the neurointerventionalist to move the ct scanner , maneuver the c - arm , and move the patient table in almost the same mode as in the existing standard biplane angiographic setup ( figure 1b ) . two ceiling - mounted monitors ( part of the adaptive three - dimensional intervention suite ; siemens ag , healthcare sector , erlangen , germany ) were installed to replace the monitor cart within the ct imaging suite . the monitor cart is stationed in an adjacent room allowing concurrent viewing and , if necessary , data processing without exposure to x - rays . in case of a malfunction of the ceiling - mounted monitors , the monitor cart can be brought immediately to the ct imaging room . to protect the neurointerventionalist from radiation , an adjustable ceiling - mounted upper body protective shield and a lower body x - ray protective shield are in place . for the treating anesthesiologist or neurointensivist , a mobile height - adjustable full body x - ray protective shield is available for upper and lower body protection from radiation . ( a ) setup of the ct / c - arm intervention suite with the cios alpha in its operating position ( partially covered by the height - adjustable upper body and the mobile lower body x - ray protection shields ) . note : the remote controls for the c - arm and the ct ( center , bottom ) can be mounted in alternative order to the side rail of the ct table . on admission , a stroke neurologist performed physical neurologic examinations and detailed assessment of the nihss score in the emergency room . patients with suspected ais and an nihss score of 10 received a standardized stroke imaging protocol consisting of a non - contrast enhanced cranial ct ( cct ) , ct perfusion of the brain as well as ct angiography of the aortic arch , cervical , and intracranial arteries . during the intervention , patients received standardized peri - interventional management and monitoring of physiological target values according to in - house standard operating procedures and the discretion of the treating neurointensivist adapted to the patient and situation . to detect possible peri - procedural complications , cct and/or ct perfusion all patients were initially observed in a neurologic intensive care unit after the interventional procedure . transfer to the stroke unit occurred as soon as possible depending on the clinical condition of the patient . follow - up ct or mri was routinely performed at 2036 h after treatment , or earlier if neurologic deterioration occurred . post - interventional nihss and modified rankin scale scores were assessed by detailed physical examinations performed by an independent stroke neurologist at discharge . we report our preliminary clinical experience with the ct / c - arm stroke system in three patients starting in october 2014 . the decision to perform the procedure on the new system and the choice of the endovascular devices was made by the respective neurointerventionalist and neurologist . an 84-year - old man presented with low - grade right - sided weakness and reduced sense of sensation which had worsened until arrival at the hospital ( nihss 17 ) . he had a medical history of hypertension and diabetes mellitus type 2 , but was otherwise previously independent . ct showed a dense artery sign in the left middle cerebral artery ( mca ) and a significant increase in time to peak perfusion in the corresponding territory ( figure 2a , b ) . ct angiography revealed an occlusion of the left mca ( image not shown ) and the decision for mt was made . initiation of general anesthesia and intubation on the ct table was followed by sterile draping and skin disinfection of the right - sided groin for puncture and placement of an 8 f introducer into the femoral artery . with support of a sim2 angiographic catheter , a balloon guide catheter ( cello 8 f , covidien , dublin , ireland ) angiographic imaging showed a left - sided high - grade cervical internal carotid artery stenosis ( 80% , figure 2c ) . interventional therapy started with an angioplasty of the stenosis using a 320 mm pta balloon which was followed by placement of a carotid wallstent ( 740 mm ; boston scientific , marlborough , massachusetts , usa ) ( figure 2d ) . afterwards the internal carotid artery could be passed by a 5 f distal access catheter ( sofia , microvention , tustin , california , usa ) . angiographic imaging confirmed an occlusion of the mca ( m1 segment , figure 2e ) , which was subsequently passed by a guidewire and microcatheter ( traxcess , rebar-18 microcatheter , covidien ) to deploy a stent - retriever device ( solitaire 2 , 620 mm , covidien ) . after a single mechanical retrieval maneuver , the clot in the m1 segment was removed completely ( thrombolysis in cerebral infarction ( tici ) grade 3 ; figure 2f , g ) . post - interventional ct imaging showed no signs of intracranial hemorrhage and a symmetric perfusion ( figure 2h , i ) . ( a ) dense artery sign in the left middle cerebral artery ( mca ) . ( b ) increase of time - to - peak in ct perfusion in the left mca territory . ( c , d ) a high - grade stenosis of the left cervical internal carotid artery before and after stent - assisted angioplasty . ( e ) intracranial occlusion of the left mca ( m1 segment ) . ( h , i ) ct and ct perfusion after treatment without a sign of infarct demarcation or perfusion deficit . a 51-year - old man without relevant comorbidity was found unconscious at home with unknown time of onset of symptoms and intubated on site by the emergency medical service . stroke imaging revealed a dense artery sign at the tip of the basilar artery ( ba ) and a matching increase in time to peak perfusion ( figure 3a , b ) . after standardized preparation for vascular arterial access , a 7 f long sheath was placed in the left subclavian artery allowing a 5 f vertebral angiographic catheter to be positioned in the left vertebral artery . a distal access catheter ( sofia ) was placed into the root of the ba . the occlusion was passed by a guidewire and microcatheter ( traxcess , rebar-18 microcatheter ) which were placed into the left posterior cerebral artery . a stent - retriever device ( solitaire 2 , 620 mm ) was deployed and a single mechanical retrieval maneuver removed the clot completely ( tici grade 3 ; figure 3d , e ) . ( a ) non - contrast enhanced ct reveals a dense artery sign in the basilar artery . ( b ) ct perfusion shows a significant reduction of perfusion of the pons and the superior cerebellar artery territory on both sides . ( d , e ) posterior circulation after mechanical thrombectomy without evidence of a remaining occlusion . an 83-year - old man with moderate to severe ais ( nihss 17 ) was referred for further diagnostics and possible interventional treatment from a primary care hospital where he had already received intravenous recombinant tissue plasminogen activator . cct was repeated because of clinical deterioration during transfer to exclude intracranial hemorrhage ( figure 4a ) . ct perfusion and ct angiography showed a left - sided proximal m2 occlusion ( superior trunk ) with an equivalent decrease in brain perfusion ( figure 4b ) . while the patient was intubated on the ct table by the neurointensivist , the neurointerventionalist concurrently started the interventional procedure by prepping the placing of an 8 f guiding catheter . angiographic imaging confirmed a distal m2 occlusion with poor leptomeningeal collaterals ( figure 4c ) . a distal access catheter ( sofia ) was placed into the internal carotid artery and the m2 occlusion was subsequently passed by a guidewire and microcatheter ( traxcess ; rebar-18 ) . a stent - retriever device ( solitaire 2 , 620 mm ) was deployed in the occluded m2 segment . the clot was removed completely after three mechanical retrieval maneuvers leaving no perfusion deficit ( tici grade 3 ; figure 4d , f ) . ( a ) non - contrast enhanced ct shows hypodensity in the lenticular nucleus and the insular cortex and excludes intracranial hemorrhage . ( b ) ct perfusion demonstrates a fairly large deficit in almost two - thirds of the left middle cerebral artery territory . ( d , e ) the superior trunk of the left middle cerebral artery is reperfused after mechanical thrombectomy . since biplane angiographic systems are usually used in our center and other centers for mt , we provide preliminary technical and time relevant data from the first patients who received mt using the ct / c - arm system in comparison with a small historic patient cohort in august to october 2014 ( n=16 ) . median p2p time was 35 min ( iqr 3443 ) and median onset to recanalization ( tici 2b/3 ) time was 183 min ( iqr 175243 ) in the ct / c - arm system group . for patients who received mt using a biplane angiographic system , median p2p time aggregated to 57 min ( iqr 5071 ) and median onset to recanalization time totaled 250 min ( iqr 214319 ) . using the ct / c - arm system , meansd signal - to - noise ratio ( snr ) was 38 ( 8) and radiation dose was 4045 ( 834 ) gym compared with a mean snr of 45 ( 8) and a radiation dose of 18 797 ( 8357 ) gym using the biplane angiographic system in comparable procedures . we report our preliminary clinical experience with the ct / c - arm stroke system in three patients starting in october 2014 . the decision to perform the procedure on the new system and the choice of the endovascular devices was made by the respective neurointerventionalist and neurologist . an 84-year - old man presented with low - grade right - sided weakness and reduced sense of sensation which had worsened until arrival at the hospital ( nihss 17 ) . he had a medical history of hypertension and diabetes mellitus type 2 , but was otherwise previously independent . ct showed a dense artery sign in the left middle cerebral artery ( mca ) and a significant increase in time to peak perfusion in the corresponding territory ( figure 2a , b ) . ct angiography revealed an occlusion of the left mca ( image not shown ) and the decision for mt was made . initiation of general anesthesia and intubation on the ct table was followed by sterile draping and skin disinfection of the right - sided groin for puncture and placement of an 8 f introducer into the femoral artery . with support of a sim2 angiographic catheter , a balloon guide catheter ( cello 8 f , covidien , dublin , ireland ) angiographic imaging showed a left - sided high - grade cervical internal carotid artery stenosis ( 80% , figure 2c ) . interventional therapy started with an angioplasty of the stenosis using a 320 mm pta balloon which was followed by placement of a carotid wallstent ( 740 mm ; boston scientific , marlborough , massachusetts , usa ) ( figure 2d ) . afterwards the internal carotid artery could be passed by a 5 f distal access catheter ( sofia , microvention , tustin , california , usa ) . angiographic imaging confirmed an occlusion of the mca ( m1 segment , figure 2e ) , which was subsequently passed by a guidewire and microcatheter ( traxcess , rebar-18 microcatheter , covidien ) to deploy a stent - retriever device ( solitaire 2 , 620 mm , covidien ) . after a single mechanical retrieval maneuver , the clot in the m1 segment was removed completely ( thrombolysis in cerebral infarction ( tici ) grade 3 ; figure 2f , g ) . post - interventional ct imaging showed no signs of intracranial hemorrhage and a symmetric perfusion ( figure 2h , i ) . ( a ) dense artery sign in the left middle cerebral artery ( mca ) . ( b ) increase of time - to - peak in ct perfusion in the left mca territory . ( c , d ) a high - grade stenosis of the left cervical internal carotid artery before and after stent - assisted angioplasty . ( e ) intracranial occlusion of the left mca ( m1 segment ) . ( h , i ) ct and ct perfusion after treatment without a sign of infarct demarcation or perfusion deficit . a 51-year - old man without relevant comorbidity was found unconscious at home with unknown time of onset of symptoms and intubated on site by the emergency medical service . stroke imaging revealed a dense artery sign at the tip of the basilar artery ( ba ) and a matching increase in time to peak perfusion ( figure 3a , b ) . after standardized preparation for vascular arterial access , a 7 f long sheath was placed in the left subclavian artery allowing a 5 f vertebral angiographic catheter to be positioned in the left vertebral artery . a distal access catheter ( sofia ) was placed into the root of the ba . the occlusion was passed by a guidewire and microcatheter ( traxcess , rebar-18 microcatheter ) which were placed into the left posterior cerebral artery . a stent - retriever device ( solitaire 2 , 620 mm ) was deployed and a single mechanical retrieval maneuver removed the clot completely ( tici grade 3 ; figure 3d , e ) . ( a ) non - contrast enhanced ct reveals a dense artery sign in the basilar artery . ( b ) ct perfusion shows a significant reduction of perfusion of the pons and the superior cerebellar artery territory on both sides . ( d , e ) posterior circulation after mechanical thrombectomy without evidence of a remaining occlusion . an 83-year - old man with moderate to severe ais ( nihss 17 ) was referred for further diagnostics and possible interventional treatment from a primary care hospital where he had already received intravenous recombinant tissue plasminogen activator . cct was repeated because of clinical deterioration during transfer to exclude intracranial hemorrhage ( figure 4a ) . ct perfusion and ct angiography showed a left - sided proximal m2 occlusion ( superior trunk ) with an equivalent decrease in brain perfusion ( figure 4b ) . while the patient was intubated on the ct table by the neurointensivist , the neurointerventionalist concurrently started the interventional procedure by prepping the placing of an 8 f guiding catheter . angiographic imaging confirmed a distal m2 occlusion with poor leptomeningeal collaterals ( figure 4c ) . a distal access catheter ( sofia ) was placed into the internal carotid artery and the m2 occlusion was subsequently passed by a guidewire and microcatheter ( traxcess ; rebar-18 ) . a stent - retriever device ( solitaire 2 , 620 mm ) was deployed in the occluded m2 segment . the clot was removed completely after three mechanical retrieval maneuvers leaving no perfusion deficit ( tici grade 3 ; figure 4d , f ) . ( a ) non - contrast enhanced ct shows hypodensity in the lenticular nucleus and the insular cortex and excludes intracranial hemorrhage . ( b ) ct perfusion demonstrates a fairly large deficit in almost two - thirds of the left middle cerebral artery territory . ( d , e ) the superior trunk of the left middle cerebral artery is reperfused after mechanical thrombectomy . an 84-year - old man presented with low - grade right - sided weakness and reduced sense of sensation which had worsened until arrival at the hospital ( nihss 17 ) . he had a medical history of hypertension and diabetes mellitus type 2 , but was otherwise previously independent . ct showed a dense artery sign in the left middle cerebral artery ( mca ) and a significant increase in time to peak perfusion in the corresponding territory ( figure 2a , b ) . ct angiography revealed an occlusion of the left mca ( image not shown ) and the decision for mt was made . initiation of general anesthesia and intubation on the ct table was followed by sterile draping and skin disinfection of the right - sided groin for puncture and placement of an 8 f introducer into the femoral artery . with support of a sim2 angiographic catheter , a balloon guide catheter ( cello 8 f , covidien , dublin , ireland ) angiographic imaging showed a left - sided high - grade cervical internal carotid artery stenosis ( 80% , figure 2c ) . interventional therapy started with an angioplasty of the stenosis using a 320 mm pta balloon which was followed by placement of a carotid wallstent ( 740 mm ; boston scientific , marlborough , massachusetts , usa ) ( figure 2d ) . afterwards the internal carotid artery could be passed by a 5 f distal access catheter ( sofia , microvention , tustin , california , usa ) . angiographic imaging confirmed an occlusion of the mca ( m1 segment , figure 2e ) , which was subsequently passed by a guidewire and microcatheter ( traxcess , rebar-18 microcatheter , covidien ) to deploy a stent - retriever device ( solitaire 2 , 620 mm , covidien ) . after a single mechanical retrieval maneuver , the clot in the m1 segment was removed completely ( thrombolysis in cerebral infarction ( tici ) grade 3 ; figure 2f , g ) . post - interventional ct imaging showed no signs of intracranial hemorrhage and a symmetric perfusion ( figure 2h , i ) . ( a ) dense artery sign in the left middle cerebral artery ( mca ) . ( b ) increase of time - to - peak in ct perfusion in the left mca territory . ( c , d ) a high - grade stenosis of the left cervical internal carotid artery before and after stent - assisted angioplasty . ( e ) intracranial occlusion of the left mca ( m1 segment ) . ( h , i ) ct and ct perfusion after treatment without a sign of infarct demarcation or perfusion deficit . a 51-year - old man without relevant comorbidity was found unconscious at home with unknown time of onset of symptoms and intubated on site by the emergency medical service . stroke imaging revealed a dense artery sign at the tip of the basilar artery ( ba ) and a matching increase in time to peak perfusion ( figure 3a , b ) . after standardized preparation for vascular arterial access , a 7 f long sheath was placed in the left subclavian artery allowing a 5 f vertebral angiographic catheter to be positioned in the left vertebral artery . a distal access catheter ( sofia ) was placed into the root of the ba . the occlusion was passed by a guidewire and microcatheter ( traxcess , rebar-18 microcatheter ) which were placed into the left posterior cerebral artery . a stent - retriever device ( solitaire 2 , 620 mm ) was deployed and a single mechanical retrieval maneuver removed the clot completely ( tici grade 3 ; figure 3d , e ) . ( a ) non - contrast enhanced ct reveals a dense artery sign in the basilar artery . ( b ) ct perfusion shows a significant reduction of perfusion of the pons and the superior cerebellar artery territory on both sides . ( d , e ) posterior circulation after mechanical thrombectomy without evidence of a remaining occlusion . an 83-year - old man with moderate to severe ais ( nihss 17 ) was referred for further diagnostics and possible interventional treatment from a primary care hospital where he had already received intravenous recombinant tissue plasminogen activator . cct was repeated because of clinical deterioration during transfer to exclude intracranial hemorrhage ( figure 4a ) . ct perfusion and ct angiography showed a left - sided proximal m2 occlusion ( superior trunk ) with an equivalent decrease in brain perfusion ( figure 4b ) . while the patient was intubated on the ct table by the neurointensivist , the neurointerventionalist concurrently started the interventional procedure by prepping the placing of an 8 f guiding catheter . angiographic imaging confirmed a distal m2 occlusion with poor leptomeningeal collaterals ( figure 4c ) . a distal access catheter ( sofia ) was placed into the internal carotid artery and the m2 occlusion was subsequently passed by a guidewire and microcatheter ( traxcess ; rebar-18 ) . a stent - retriever device ( solitaire 2 , 620 mm ) was deployed in the occluded m2 segment . the clot was removed completely after three mechanical retrieval maneuvers leaving no perfusion deficit ( tici grade 3 ; figure 4d , f ) . ( a ) non - contrast enhanced ct shows hypodensity in the lenticular nucleus and the insular cortex and excludes intracranial hemorrhage . ( b ) ct perfusion demonstrates a fairly large deficit in almost two - thirds of the left middle cerebral artery territory . ( d , e ) the superior trunk of the left middle cerebral artery is reperfused after mechanical thrombectomy . since biplane angiographic systems are usually used in our center and other centers for mt , we provide preliminary technical and time relevant data from the first patients who received mt using the ct / c - arm system in comparison with a small historic patient cohort in august to october 2014 ( n=16 ) . median p2p time was 35 min ( iqr 3443 ) and median onset to recanalization ( tici 2b/3 ) time was 183 min ( iqr 175243 ) in the ct / c - arm system group . for patients who received mt using a biplane angiographic system , median p2p time aggregated to 57 min ( iqr 5071 ) and median onset to recanalization time totaled 250 min ( iqr 214319 ) . using the ct / c - arm system , meansd signal - to - noise ratio ( snr ) was 38 ( 8) and radiation dose was 4045 ( 834 ) gym compared with a mean snr of 45 ( 8) and a radiation dose of 18 797 ( 8357 ) gym using the biplane angiographic system in comparable procedures . mt using stent - retrievers has been proven to be safe and effective in stroke due to large vessel occlusions.24 2022 besides final recanalization , the time to recanalization after stroke onset is crucial for patient outcome.8 23 24 among all in - hospital factors , logistic measures are crucial to reduce time to treatment that is , recanalization in the endovascular setting . here we report the first use of a combined ct / c - arm system for interventional stroke treatment in order to decrease time from ct to recanalization , increase the safety of the procedure , and extend the application of endovascular stroke treatment to a more general setting , in particular in centers lacking a dedicated neurointerventional x - ray suite . using the combined ct / c - arm system , the time window between symptom onset and endovascular therapy may be reduced by accelerating initiation of endovascular therapy . this affects time metrics relevant for clinical outcome such as p2p time , onset to puncture time , and onset to recanalization time.9 1113 in these few cases , p2p time and onset to recanalization time were clearly reduced compared with large multicenter studies.3 4 since this is a series of only three patients , these time windows should be viewed cautiously and investigated in a larger patient cohort . while accelerating initiation of endovascular therapy is very important , the ct / c - arm system does not neglect the need for state of the art stroke imaging , which is a prerequisite for initiation of intravenous thrombolysis and mt . on the contrary , this setting allows precise pretherapeutic imaging as well as immediate , intra- or post - interventional detection and monitoring of peri-/intraprocedural complications via repeated ct scans with high imaging quality ( in contrast to flat - panel ct ) . furthermore , repeated ct perfusion after mt could demonstrate successful brain reperfusion , which complements angiographic findings and therefore allows a better prognostic estimation . performing endovascular therapy on the ct table using this combined ct / c - arm system eliminates further patient transfers . this is important because of possible time saving but , in addition , in these severely ill sometimes obese patients every transport or repositioning is very strenuous for the patient as well as for the medical personnel and can be avoided by treating the patient on the ct table . although all the patients in this case series were intubated prior to mt , this setting allows mt in conscious sedation as well . another advantage of this combined ct / c - arm system is the transformation of an interventional suite out of a pre - existing room with x - ray protection . minor reconstruction works , including the installation of the ceiling - mounted monitors and upper body x - ray protection shield , in combination with the cios alpha device turned a regular ct imaging suite into a versatile ct imaging and intervention suite at our institution at relatively low cost . furthermore , in situations where the standard angiographic suite is occupied or out of order for maintenance , this option allows endovascular therapy for procedures other than time - sensitive emergencies . mobile c - arm x - ray devices have been in use for years for procedures such as retrieval of foreign bodies , guidance during spinal surgery , endovascular aortic repair , and coronary bypass surgery.2528 perhaps because of the high image quality and technical prerequisites required , no attempt has been made to use a mobile c - arm x - ray device for mt in ais to date . with the introduction of the cios alpha , technical prerequisites are available to open this new application for mobile c - arm x - ray devices . the cios alpha device is equipped with all necessary angiographic features including fluoroscopic imaging , subtraction imaging , and road - mapping for delineation of the vessels . imaging quality is satisfactory for precise maneuvering of guide wires , catheters , and interventional devices at doses of radiation lower than a biplane angiographic system . moreover , all movements of the c - arm can be done remotely from the ct table so no mechanical adjustments of the c - arm need to be made . the gap between the ct gantry and patient table was increased by 65 cm ( 25.6 inch ) from the standard setting to 95 cm ( 37.4 inch ) to allow the c - arm to be placed between the table and ct gantry . this seemingly small difference allows positioning , rotation , and angulation of the c - arm device for interventional therapy . the ct scanner , patient table , and c - arm device are directly controlled remotely by the neurointerventionalist via two control units . these remote control units are mounted to the side rail of the patient table and fit into the sterile field during endovascular therapy . together with the individually adjustable ceiling - mounted monitors and x - ray protection shield as well as remote controls , this unique setup allows state of the art endovascular therapy in almost the same manner as a primary monoplane angiographic system . most importantly , this setup does not impede diagnostic ct examinations if no intervention is being undertaken . the potential advantages , particularly time saving and ensuing improvement in patient outcome , need to be assessed in a larger study .	backgroundmechanical thrombectomy ( mt ) using stent - retrievers has been proven to be a safe and effective treatment in acute ischemic stroke ( ais ) , particularly in large vessel occlusion . other than patient characteristics , time to recanalization is the most important factor linked to outcome . mt is usually performed in a dedicated angiography suite using a floor- and/or ceiling - mounted biplane angiographic system . here we report our first experience of mt with a new combined ct and mobile c - arm x - ray device setup.methodspatients with ais underwent stroke imaging ( non - contrast enhanced ct , ct perfusion , and ct angiography ) using a commercially available 64-slice ct scanner which was modified for combined use with a c - arm system . in patients with large vessel occlusion , mt was conducted without further patient transfer within the ct imaging suite using a mobile c - arm x - ray device equipped with a 3030 cm ( 1212 inch ) , 1.51.5 k full - view flat detector which was positioned between the gantry and patient table . the safety and feasibility of this new system was assessed in preliminary patients.resultsangiographic imaging quality of the mobile c - arm was feasible and satisfactory for diagnostic angiography and mt . using this setup , time between stroke imaging and groin puncture ( picture - to - puncture time ) was reduced by up to 35 min ( including time for preparation of the patient such as intubation).conclusionsmt using a combined ct / c - arm system is safe and feasible . the potential advantages , particularly time saving and ensuing improvement in patient outcome , need to be assessed in a larger study .
the classical interleukin ( il)-1 family cytokines , il-1 and il-1 , as well as il-18 , are related by mechanism of origin , receptor structure , and signal transduction pathways utilized . il-33 ( also named il-1f11 ) , which belongs to il-1 family , is most closely related in structure to il-18 and il-1 . il-33 is also described as a nuclear factor expressed in high endothelial venules ( nf - hev ) . the human il-33 gene is located on chromosome 9p24.1 , while its mouse counterpart can be found on the syntenic chromosome 19qc1 region . the il-33 cdna sequences encode 270 and 266 amino acid polypeptides for human and mouse , respectively , corresponding to full - length proteins with calculated masses of 30 and 29.9 kda . the proteins do not contain a signal sequence for secretion , similar to the il-1 , il-1 , and il-18 genes [ 4 , 5 ] . the precursor protein ( pre - il-33 ) was cleaved by caspase-1 into a mature 18-kda protein ( il-33 ) in in vitro experiments using a recombinant protein . il-33 signals via the il-1 receptor - related protein st2 [ 2 , 6 ] and il-1 receptor accessory protein activate nf-b and mitogen - activated protein kinases to induce t helper type 2 ( th2)-associated cytokines [ 2 , 8 , 9 ] . administration of il-33 to mice induces production of th2 cytokines and severe pathological changes in mucosal organs . moreover , the ability of il-33 to induce th2 responses has functional relevance in the context of intestinal helminth infection . il-33 can promote survival , adhesion , and cytokine production in human mast cells , but the mechanism behind its involvement in inflammatory conditions remains unclear . it is also a key mediator of immune inflammatory hypernociception normally associated with a th1 type of response , revealing a hitherto unrecognized function of il-33 in a key immune pharmacological pathway that may be amenable to therapeutic intervention . in this study , the cloning and expression of mouse il-33 gene in escherichia coli ( e. coli ) , purification of recombinant protein , and generation of polyclonal antibody against il-33 are described . the prepared antibody can be useful for the study of expression and distribution of il-33 in various tissues at protein level and for the elucidation of its biofunctions and regulation mechanism in inflammatory diseases . e. coli strain bl21 ( de3 ) and ni - nta his - bind resins were obtained from novagen ( madison , wi ) . hotstar taq dna polymerase and qiaquick gel extraction kit were purchased from qiagen ( valencia , ca ) . primescript first strand cdna synthesis kit , restriction endonucleases xhoi , ndei , t4 dna ligase , isopropyl--d - thiogalactoside ( iptg ) , dna marker d2000 , and protein marker were from takara ( dalian , china ) . lysozyme , complete freund 's adjuvant ( cfa ) , and incomplete freund 's adjuvant ( ifa ) were obtained from sigma ( st . louis , mo ) . total cellular rna was extracted from spinal cord of balb / c mouse according to trizol reagent instructions . the coding region of mouse il-33 mature protein was amplified by reverse transcription polymerase chain reaction ( rt - pcr ) . the first strand of cdna was synthesized using the primescript first strand cdna synthesis kit , and the pcr reactions were carried out with a ptc-200 peltier thermal cycler ( mj research , waltham , ma ) . primer 1 ( 5-ggaattccatatg acattgagcatccaaggaac-3 ) and primer 2 ( 5-ccgctcgaggattttcgagagcttaaaca-3 ) were synthesized by invitrogen ( shanghai , china ) . primer 1 was synthesized with an ndei site ( shown in bold ) and an additional atg for initiation of translation in italicized style . the amplification profile included one initial hot - start denaturation step at 94c for 15 minutes , followed by 30 cycles of the following conditions : 94c for 30 seconds , 57c for 30 seconds , and 72c for 1 minute , and a final extension at 72c for 10 minutes . plasmid pet-44 ( novagen , darmstadt , germany ) was digested with ndei and xhoi restriction enzymes . the large fragment was cut and purified from agarose gel using the qiaquick gel extraction kit . a recombinant plasmid was constructed by inserting the amplified fragments ( also digested with ndei and xhoi ) into the ndei and xhoi sites of pet-44 and transformed into e. coli strain bl21 ( de3 ) . the transformants ( pet-44-mil-33 ) were confirmed by colony pcr , restriction enzyme digestion , and dna sequencing . expression in bacteria results in the producing of recombinant il-33 protein containing a hexahistidine tag in the c - terminus . a single colony of successfully transformed e. coli was picked up and cultured overnight at 37c in luria bertani ( lb ) medium , supplemented with 100 g / ml ampicillin . the culture mixture was then inoculated to fresh lb medium ( 1 : 100 dilution ) containing ampicillin and grown at 37c under continuous shaking , until the absorbance at 600 nm reached 0.60.8 . to optimize the expression conditions , il-33 expression was induced by adding 1 mm iptg to the transformed e. coli and the bacteria were incubated at 25c for a period of 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , and 9 hours , respectively . the degree of expression was evaluated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ) . to compare the protein dissolubility , the bacteria with pet-44-mil-33 after 7 hours of induction at 25c , the cells were harvested by centrifugation at 8,000 g for 20 minutes at 4c . the supernatant was discarded and the pellet was resuspended in lysis buffer consisting of 300 mm nacl , 50 mm nah2po4 , 10 mm imidazole , ph 8.0 , and lysed by using lysozyme , and incubated for 30 minutes at room temperature ( rt ) with gentle shaking to assure thorough cell lysis . the cell lysate was further disrupted by sonication on ice with six 10-second pulses at high intensity with a 10 seconds cooling period between each burst . the suspension was centrifuged at 12,000 g for 30 minutes to remove insoluble debris . the supernatant was added to an ni - nta spin column pre - equilibrated with the lysis buffer , and then they were gently shaked on ice for 60 minutes . the column was then washed with native wash buffer ( 300 mm nacl , 50 mm nah2po4 , 20 mm imidazole , ph 8.0 ) . finally , the bound protein was eluted with native elution buffer ( 300 mm nacl , 50 mm nah2po4 , 250 mm imidazole , ph 8.0 ) and analyzed with 12% sds - page . fractions were desalted and concentrated by an ultrafiltration device ( millipore corporation , bedford , ma ) . the new zealand white rabbits were given intradermal injections of il-33 protein ( 500 g / rabbit ) mixed with cfa in 1 : 1 ratio . after 2 week , the rabbits were boosted subsequently 4 times with protein ( 200 g / rabbit ) mixed with ifa in 1 : 1 ratio at 2-weeks interval . before every immunization , blood samples were taken from the marginal vein of the rabbit ear , centrifuged , and the sera were obtained to determine the antibody titer by enzyme - linked immunosorbent assay ( elisa ) . one week after the last injection , antisera of the rabbits were collected and purified by saturated ammonium sulfate ( sas ) and then protein a sepharose affinity chromatography ( amersham biosciences , piscataway , nj ) following manufacturer 's instructions . the titer of antiserum was determined by an indirect elisa . for each well of the 96-well elisa plate , 200 ng of il-33 protein was diluted in 100 l of sodium bicarbonate solution and incubated overnight at 4c . after three washes with phosphate - buffered saline-(pbs- ) tween buffer ( 0.05% tween 20 in pbs ) , the wells were blocked using 100 l of 3% bovine serum albumin for 1 hour at 37c and then incubated with 100 l polyclonal antibodies against il-33 with different dilution ( from 1 : 100 to 1 : 64,000 ) for 1.5 hours . the wells were incubated with 50 l / well horseradish peroxidase-(hrp- ) conjugated goat antirabbit immunoglobulin ( igg , 1 : 250 dilution ) for 1 hour at 37c after thoroughly washed . after final washes , 100 l / well tetramethyl benzidine ( tmb ) solution was added for color development . after 10 minutes , the reaction was stopped by 100 l / well 2 m h2so4 and the absorbance was measured at 450 nm using a plate reader ( molecular devices ) . ratio of antiserum versus control serum absorbency greater than 2.1 was recognized as positive . in order to confirm that the polyclonal antibodies are able to recognize il-33 protein from tissues , we also examined the il-33 protein expression of plasmid pcdna3.1-mil-33 after delivery through hydrodynamics - based gene injection in balb / c mice [ 14 , 15 ] . the plasmid pcdna3.1-mil-33 was constructed as forenamed , but whole mouse il-33 cdna was amplified by pcr and then inserted into mammalian expression plasmid pcdna3.1(+ ) ( invitrogen ) . briefly , slides were dried in an oven ( 60c ) before removing paraffin in several changes of xylene . slides were hydrated through a series of graded alcohols to water , followed by incubation with 0.5% hydrogen peroxide for 5 minutes . sections were incubated with 5% bovine serum albumin ( bsa ) in pbs for 10 minutes at rt to block nonspecific binding . after rinsing with pbs , slides were incubated overnight at 4c with rabbit anti - il-33 polyclonal antibody ( 1 : 1,000 dilution in pbs containing 5% bsa ) . after rinsing with pbst ( pbs containing 0.01% triton x-100 ) , tissue sections were incubated for 40 minutes at rt with hrp - conjugated goat antirabbit igg ( 1 : 100 ) and rinsed with pbst . subsequently , slides were stained with diaminobenzidine ( dab ) and counterstained with hematoxylin , dehydrated , and mounted . in order to express recombinant il-33 in e. coli , the mature protein coding region of il-33 was generated by pcr and was cloned between ndei and xhoi sites of pet-44 vector containing the coding sequence for a hexahistidine - coding sequence in the c - terminal part . amplification of the il-33 gene by pcr produced a single amplified 480 bp dna fragment , coding for a mature il-33 protein of 160 amino acids . the final pet-44-mil-33 construct is able to express the recombinant il-33 protein in fusion with a hexahistidine tag and allow for metal affinity purification . after induction with iptg , e. coli bl21 ( de3 ) transformed with pet-44-mil-33 produced a protein of approximately 18 kda as shown in figure 1 . the size of the protein matched well with its theoretical molecular weight . to determine the optimal induction period , the bacteria were incubated with iptg at 25c for 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , and 9 hours , respectively . the results showed that while the incubation period was below 7 hours , the yield of the ~18 kda protein was increased as the induction time prolonged ; but when the incubation time exceeded 7 hours , the yield of the product was not significantly raised ( figure 1 ) . mmol / l , and the bacteria were cultivated at 25c for 7 hours . induced bl21 ( de3 ) cells at 25c can express the il-33 protein ( figure 2 , lane 5 ) . in contrast , noninduced bl21 ( de3 ) cells did not express this fusion protein ( figure 2 , lane 2 ) . the soluble il-33 was expressed at high levels at 25c ( figure 2 , lane 3 ) , but at low levels at 37c ( data not shown ) . this was most likely due to growth at 37c , which causes some protein to accumulate as inclusion bodies , whereas incubation at 25c decreases the rate of protein synthesis and leads to soluble protein . the target il-33 protein appeared as a single band on sds - page ( figure 2 , lane 1 ) , which is in agreement with the molecular weight reported . polyhistidine tags form high - affinity complexes with immobilized divalent metal ions ( such as ni or co ) , even in the presence of high concentrations of chaotropic agents ( e.g. , urea or guanidine hydrochloride ) , thereby allowing isolation of tagged protein from a crude cellular extract . his - tagged il-33 exists in the bacterial cells in two forms : either soluble protein or inclusion bodies . the soluble protein was incubated with the ni - nta agarose slurry to allow binding of his - tagged il-33 . n - terminal sequencing of the purified il-33 revealed that the first 20 residues were identical to the mouse il-33 sequence deduced from the dna ( tlsiqgtslltqspaslsty ) . after immunizing rabbits with recombinant il-33 according to standard protocol , anti - il-33 serum was purified by sas precipitation and protein a affinity chromatography . the igg fractions showed two bands , a heavy chain ( 50 kda ) and a light chain ( 25 kda ) on sds - page gel after passing through the protein a - sepharose column ( figure 3 ) . the antibody at different dilutions ( 100- to 64,000-fold ) was reacted with an equal amount of the il-33 protein ( 0.2 g ) . the antibody titer is defined as the highest dilution of serum at which the a450 ratio ( a450 of postimmunization sera / a450 of preimmunization sera ) is greater than 2.1 . the antibody titer was found to be approximately 1 : 32,000 ( figure 4 ) . il-33 is a dual - function cytokine that may function as both an intracellular heterochromatin - associated nuclear factor and a potent proinflammatory cytokine . after immunohistochemistry was performed using the anti - il-33 polyclonal antibody in mouse lung and liver tissues , there were brown staining in nuclei or cytoplasm ( figures 5(b ) , 5(d ) , 5(f ) , 5(h ) ) . furthermore , there was a significant increase of il-33 expression in hydrodynamics - injected mice with il-33 cdna plasmid compared with normal mice ( figure 5 ) . bronchial epithelial cells in lung and liver cells revealed the strongest staining with anti - il-33 antibody in hydrodynamics injected mice ( figures 5(h ) , 5(f ) ) . no staining was detected in tissues where no antibody or control igg was used in the primary layer ( igg control , figures 5(a ) , 5(c ) , 5(e ) , 5(g ) ) . our results demonstrated that the endogenous il-33 protein in tissues could be specifically recognized by our prepared anti - il-33 polyclonal antibodies . we have developed a procedure for the efficient expression and purification of il-33 using pet-44 vector and ni - nta affinity chromatography . furthermore , we report the production of specific anti - il-33 antibody and the localization of endogenous il-33 . it has been observed that il-33 mrna is broadly expressed in many tissues but is more restricted at the level of cell type , which includes pulmonary smooth muscle cells , epithelial cells , dermal fibroblasts , keratinocytes , dendritic cells , and activated macrophages . il-33 is also expressed in endothelial cells from chronically inflamed rheumatoid arthritis synovium and crohn 's disease intestine [ 3 , 18 ] and atherosclerotic tissues . however , very little information is available on the expression of il-33 protein in liver and lung tissues . to our knowledge this is the first report so far of production of polyclonal antibody against full - length mouse il-33 and il-33 protein expression in mouse liver and lung determined with anti - il-33 polyclonal antibodies . as il-33 induces gene expression of th2-associated cytokines and pathological changes in the lung , blockade of il-33 may be a new therapeutic strategy for bronchial asthma . we are investigating the effect of exogenously administered anti - il-33 antibody in a murine model of allergen - induced asthma and collagen - induced arthritis . this research may contribute to elucidation of the biological mechanisms of il-33 in human inflammatory diseases in the future .	interleukin ( il)-33 is the latest member of il-1 cytokine family . in this study , the cloning , expression , purification , and polyclonal antibody preparation of mouse il-33 were described . the coding region of il-33 mature protein was cloned into the prokaryotic expression vector pet-44 . the recombinant protein , il-33 containing a hexahistidine tag in the c - terminal , was expressed in escherichia coli . the expressed soluble protein was purified by immobilized metal - ion affinity chromatography using ni2 + -nitrilotriacetic acid agarose . the rabbits were immunized with the purified recombinant protein . the obtained antiserum was precipitated by saturated ammonium sulfate and then purified by protein a affinity chromatography . the sensitivity and specificity of the antibodies were evaluated by enzyme - linked immunosorbent assay and immunohistochemistry . the high titer ( 1 : 32000 ) polyclonal antibodies with high specificity were obtained by immunizing rabbits with the purified recombinant protein . significant expression of il-33 was seen in mouse liver and lung tissues determined with the anti - il-33 . the production of the polyclonal antibody against il-33 provides a good tool for studying the biofunctions of il-33 .
non - invasive automatic methods to detect patient - ventilator asynchrony ( pva ) have been proposed repeatedly in recent years [ 1 - 5 ] . the reason for this increasing interest is the notion that , unlike patients with an asynchrony index ( ai ) of less than 10% , those with high rates of pva ( as defined by an ai of greater than 10% ) are characterized by poorer outcome ( that is , longer durations of mechanical ventilation [ 6 - 8 ] and icu stay , a reduced number of ventilator - free days , a higher rate of tracheotomy , and lower probabilities of survival and home discharge ) . we do not know whether pva itself causes this increased morbidity by prolonging the time spent on mechanical ventilation or instead is a marker of poor respiratory function in sicker patients at increased risk of high morbidity . in the first case , decreasing pva would improve a patient 's outcome , whereas in the second case , pva would just represent a prognostic factor . nevertheless , in both cases , recognizing pva would be a valuable clinical piece of information . in this issue of critical care , gutierrez and colleagues present a new method that is based on airflow spectral analysis for the detection of pva and that is characterized not by synchronous breaths but by a less organized spectral pattern . when this novel approach is correlated to visual inspection of airway pressure and flow waveforms in detecting an ai of greater than 10% , sensitivity and specificity both exceeded 80% , and this led the authors to conclude that this method could be adapted to ventilators for pva monitoring . such a continuous non - invasive monitoring device would facilitate the recognition of pva and help to evaluate the effectiveness of measures adopted to face it , such as reducing sedation , changing ventilator mode , or varying ventilator settings , chiefly by decreasing the amount of assistance or by improving the match between neural and mechanical inspiration or by both means . the studies in which pva is assessed by visual inspection of the ventilator waveforms are commonly performed by trained observers or researchers who have a high level of knowledge in this specific field and who might not reflect the average skill of the icu professionals . new data on the ability of icu physicians to recognize pva at the bedside show that the chance to detect pva by ventilator waveform observation in the ' real world ' is less than anticipated , making automatic methods of pva detection , such as the one proposed by gutierrez and colleagues , even more helpful than expected . however , it is worthwhile to consider the potential limitations of ( a ) the automatic methods of pva detection in general and ( b ) this approach acting in the frequency domain in particular . it is unclear whether inspection of airway pressure and flow waveform represents a valid gold standard . some studies proposing automatic methods of pva detection use indices of inspiratory effort , such as esophageal and trans - diaphragmatic pressure . the latter approach is not susceptible to potential biases arising from neuro - ventilatory uncoupling in those situations in which , although the excitation of the respiratory muscles ( neural effort or drive ) generates pressure ( mechanical effort ) , this pressure is not translated into flow and volume [ 9 - 11 ] . indeed , in extreme cases of poor neuro - mechanical coupling ( that is , impaired transformation of the muscle electrical activation into pressure ) , even the esophageal and trans -diaphragmatic pressures might not be completely effective in ascertaining a patient 's neural drive . our chances of determining the occurrence of neuro - ventilatory and neuro - mechanical uncoupling by visual inspection of the airway pressure and flow waveforms are limited , but it is uncertain to what extent this may actually hamper our ability to detect pva at the bedside . more specifically , to consider a method based on an analysis of the frequency spectrum , further evaluation is necessary to clarify whether such an approach may fit ( a ) modes of ventilation that allow the patient to retain full control of the breathing pattern and that are characterized by high breath - by - breath variability and ( b ) non - invasive ventilation in which asynchronies frequently occur and , given that the patient is usually alert , non - chemical behavioral inputs influence the neural drive and breathing pattern . in the last decade we are now aware that it occurs more frequently than previously perceived and , given its correlation with poorer outcome , is clinically relevant . further research is necessary to address several unresolved issues , and recognition of pva in the clinical setting is surely one of these . the imperfect synchrony between patient and ventilator has become a stand - alone topic within the field of mechanical ventilation . ai : asynchrony index ; icu : intensive care unit ; pva : patient - ventilator asynchrony .	because patient - ventilator asynchrony ( pva ) is recognized as a major clinical problem for patients undergoing ventilatory assistance , automatic methods of pva detection have been proposed in recent years . a novel approach is airflow spectral analysis , which , when related to visual inspection of airway pressure and flow waveforms , has been shown to reach a sensitivity and specificity of greater than 80% in detecting an asynchrony index of greater than 10% . the availability of automatic non - invasive methods of pva detection at the bedside would likely be of benefit in intensive care unit practice , but they may be limited by shortcomings , so clear proof of their effectiveness is needed .
zygomycosis is a group of fungal infections caused by a number of fungi belonging to the class zygomycetes . mucorales are responsible for mucormycosis , more severe form of the disease , seen mainly in immunocompromised host . on the other hand , entomophthoromycosis caused by fungi of the order entomophthorales are of two types : subcutaneous zygomycosis ( b. ranarum ) and rhinofacial zygomycosis ( conidiobolus coronatus ) . b. ranarum are saprophytes found in soil , decaying vegetables , and gastrointestinal tract of frogs . basidiobolomycosis clinically manifests as chronic infection of subcutaneous tissue in immunocompetent host involving trunk and extremities . here we present a case of subcutaneous zygomycosis in a 25-year old immunocompetent lady who responded dramatically to saturated solution of potassium iodide . a 25-year old lady was brought to dermatology opd with a gradually increasing painless swelling over the upper - left arm of 8 month duration . the lesion appeared insidiously as an erythematous nodule which gradually increased in size to attain the present configuration . the patient was nondiabetic and no history suggestive of any major illness in the past especially history regarding immunosuppression could be elicited . on cutaneous examination , we revealed a well - defined , firm to hard , painless , nontender , indurated swelling of approximate 6.5 7.5 cm in size with surface ulceration and crusting at places [ figure 1 ] . well - defined mass with surface crusting and ulceration and positive finger insinuation test routine investigations including complete hemogram , renal , and liver function tests did not reveal any abnormality . x - ray of upper - left arm showed soft tissue shadow without any bony involvement . biopsy sample was taken and sent for histopathology and culture . on direct microscopy with 10% potassium hydroxide mount histopathology from skin lesion showed dense infiltrate of eosinophils , small cluster of epithelioid cells , foci of necrosis with few broad aseptate fungal hyphae surrounded by eosinophilic material ( splendore - hoeppli phenomenon ) [ figures 2 and 3 ] . culture on sabouraud 's dextrose agar demonstrated luxurious , creamy white , membranous , centrally heaped up , radially folded colony with peripheral satellite growth . lactophenol cotton blue ( lpcb ) test could not be performed due to lack of facility . scanner view showing eosinophilic granuloma with cluster of epithelioid cells and foci of necrosis 40 : showing broad aseptate fungal hyphae surrounded by eosinophilic material ( splendore - hoeppli phenomenon ) after confirming the diagnosis as basidiobolomycosis with the help of microscopy and culture , the patient was put on saturated solution of potassium iodide with strict vigilance of thyroid function and signs of iodism . the patient responded remarkably within 2 months . during the first follow up after 1month , lesion reduced to a great extent [ figure 4 ] and on subsequent visit , it almost disappeared leaving some hyperpigmentation [ figure 5 ] . zygomycetes constitute a class of ubiquitous organism which causes clinically relevant fungal infection in humans . members of the order entomophthorales such as b. ranarum are associated with chronic infection of cutaneous and subcutaneous tissue and usually do not disseminate to internal organs . on the contrary , the order mucorales tend to cause invasive disease such as pulmonary , rhinocerebellar and gastrointestinal infection in immunocompromised host . few reports state mucorales as the culprit organism causing cutaneous / subcutaneous infection in immunocompetent individuals . human disease with b. ranarum is prevalent in tropical countries . as far as indian scenario the fungus is a saprophytic one and present in decaying vegetables , intestine of amphibians , reptiles , fish , and insectivorous bat . possible mode of transmission is minor trauma which may be through insect bite , intravenous catheter , or even intramuscular injection . clinically , the disease starts as hardened nodule on extremities or trunk which if untreated may spread and expand locally to form hard , painless , nontender swelling . the mass can be raised up by inserting fingers underneath its smooth and rounded edge . although ulceration of the nodule may be a possible fate , dissemination is extremely rare . previously , clinical isolates of basidiobolus were classified into b. ranarum , b. haptosporus , and b. meristosporus . but recent taxonomic studies using sophisticated techniques such as restriction enzyme analysis prove that b. ranarum is the sole agent causing all the human disease . histopathologically , subcutaneous zygomycosis is characterized by small foci of suppurative granuloma distributed all over the dermis and subcutis . though different types of cells including lymphocytes , histiocytes , plasma cell , and multinucleated giant cells contribute to the composition , eosinophils play the major role . dominance of eosinophils could be attributed to the release of il4 and il10 which help in recruiting eosinophils to the target site . the presence of eosinophilic infiltrate in the granuloma is so characteristic that it is also called eosinophilic granuloma . degranulation of eosinophils leads to formation of eosinophilic sheath ( splendore - hoppelli phenomenon ) surrounding aseptate or infrequently septate thin walled hyphae . though direct examination of the biopsy sample may suggest a diagnosis of this entity , culture is gold standard for confirmation . the possibility of misdiagnosing this entity as neoplasm should be kept in mind as it is a great mimicker of soft tissue tumor , synovial sarcoma and burkitt 's lymphoma . other important differentials are mycobacterial infection , sporotrichosis , and parasitic infection such as onchocerciasis . most of the patients of subcutaneous zygomycosis respond excellently to potassium iodide and azoles . in our patient in a nutshell , early diagnosis and awareness of this entity even in this part of the country is important to prevent disfigurement , misdiagnosis , and avoidance of unnecessary investigations and surgical interventions . patient responded extremely well only with potassium iodide and did not develop any sign of iodism with strict compliance to therapy .	subcutaneous zygomycosis is a rare opportunistic fungal infection caused by basidiobolus ranarum . though this entity is endemic in south india , limited numbers of cases have been reported from this part of the country . we report a case of subcutaneous zygomycosis in a 25 year old lady who presented with a nontender , firm to hard swelling over the upper - left arm . finger was easily inserted below the indurated edge . histopathology revealed suppurative granuloma with aseptate hyphae . patient responded excellently to saturated solution of potassium iodide in subsequent visits .
the introduction of the tumor necrosis factor ( tnf ) antagonists adalimumab , infliximab , and etanercept was a major advance and was highly important and beneficial in treating most rheumatoid arthritis ( ra ) patients who are refractory to a classic treatment with disease - modifying anti - rheumatic drugs ( dmards ) [ 15 ] . the adverse effects of this treatment are infrequent , opportunistic , intracellular infections , especially the reactivation of latent mycobacterium tuberculosis , an exacerbation of demyelinating disorders . it is also possible to induce the production of various types of antibodies , such as antinuclear antibodies ( ana ) or double - stranded dna autoantibodies ( dsdna ) . treatment with biological agents like infliximab can additionally induce synthesis of anti - drug antibodies , such as the human anti - mouse antibodies ( hama ) or human anti - chimera antibodies ( haca ) . the pathogenetic mechanism that changes the humoral response leading to development of autoimmunity during anti - tnf inhibitors therapy is unknown . a possible mechanism leads through the binding of infliximab to the transmembrane and soluble tnf , rapidly lowering tnf level and enhancing apoptotic cell death , which triggers the development of autoantibodies . the other possible mechanisms that may result in autoantibodies production are : a ) tnf - alpha inhibition that causes b - cell activation and production of autoantibodies through the upregulation of interleukin-10 , b ) an increase in th2 activity , and c ) an increase in bacterial infections , which leads to the production of antibodies through molecular mimicry [ 6,1417 ] . only limited data have been published about the induction of antiphospholipid antibodies ( apl ) during treatment using tnf inhibitors [ 1820 ] . antiphospholipid antibodies target phospholipid - binding proteins , and may cause a prolongation of phospholipid - dependent coagulation assays , although patients are at risk for thromboembolic rather than bleeding complications . the most often recognized antibodies from this group are now anti - cardiolipin antibodies ( acl ) and the recently recognized antiphospholipid syndrome ( aps ) criteria anti - b2gp - i antibodies ( b2gp - i ) . the acl that are detected in patients with ra and other autoimmune diseases are directed against negatively charged phospholipids associated with b2-glycoprotein , whereas acl are associated with infection are directed against negatively charged phospholipids alone . in normal populations ( healthy blood donors ) , acl are found in 26% of people , and in an aging population are found in up to 12% and have been associated with the symptoms of aps such as recurrent thromboembolism and fetal loss . in ra patients , their clinical significance in ra is uncertain and their presence has been considered to be a non - specific marker of activation of the immune system . we enrolled 32 infliximab - treated patients with refractory ra ( 28 females and 4 males , medium age 45.4 years , range 1960 years ) . all of them were rf - positive and 25/32 ( 78% ) were accp - positive . patients were treated at the department of rheumatology and connective tissue diseases , medical university of lublin , poland . the patients were allowed to continue dmards , steroids , and non - steroid anti - inflammatory drugs before and during infliximab treatment . no patient had an infectious disease , active or latent tuberculosis , neoplastic disease , heart failure , cytopenia , or a demyelinating disorder . the patients received 3 mg / kg infliximab intravenously at weeks 0 , 2 , and 6 , and every 8 weeks thereafter . in addition to methotrexate , chloroquine ( 250 mg daily ) and steroids ( maximum daily dose 10 mg of oral prednisone or equivalent ) were also permitted . written informed consent was obtained from all patients and the study was approved by the bioethics committee of the medical university of lublin . blood serum samples were collected from all patients at baseline and after 3 and 6 months of anti - tnf treatment . the patients were examined clinically at baseline and after 3 and 6 months of the study by the same physician during each visit for infliximab infusion . the acl and b2gp - i antibodies ( igg and igm classes ) were tested using a commercially available enzyme - linked immunoabsorbent assay ( elisa ) ( euroimmun , germany ) . all the serum samples of ra patients were analyzed in a single session according to the manufacturer s instructions . the antibodies levels were measured in arbitrary units per milliliter and were considered to be positive at a cut off value of 20 u / ml . in further analysis , , we investigate the prevalence of such autoantibodies during 6 months of follow - up in patients with ra successfully treated with infliximab . acl and b2gp - i autoantibodies were evaluated at baseline and at 3 and 6 months after the beginning of infliximab treatment . differences between groups were analyzed using mann - whitney u test . a p value less than 0.05 was considered to be statistically significant . differences between groups were analyzed using mann - whitney u test . a p value less than 0.05 was considered to be statistically significant . we observed 4 acl igm - positive ( 12.5% ) patients before the beginning of infliximab treatment . in these cases there were no acl igg - positive patients at the beginning and after 3 months , but 1 case ( 3.12% ) of seroconversion was observed after 6 months in acl igg class . during the whole study otherwise , we noticed a statistically important increase in the group of b2gp - i igm - positive patients ( p<0.05 ) ( figure 1 ) . there were 2 ( 6.25% ) seropositive patients before the start of the treatment , 1 ( 3.33% ) after 3 months , and the next 3 ( 10.3% ) after 6 months . totally , we observed 4 ( 13.3% ) new b2gp - i igm - positive patients , which was statistically significant . elliott et al . found acl in 1 out of 20 patients with ra treated with anti - tnf ( ca2 ) in the first 8-week open trial on humans . measured the serological effects of repeated doses of the humanized anti - tnf antibody cdp 571 in patients with ra and found that some patients develop positive acl ( igg ) . . showed variations in acl titers over time in etanercept - treated patients with concomitant bacterial infection , where lowering of titers was seen after the treatment with antibiotics . many studies have confirmed induction of ana and anti - dsdna in patients treated with infliximab [ 10,3033 ] . anti - dsdna antibodies are usually igm isotype or igm and iga isotypes together . however , the period of our observation was short and subsequent seroconversions are possible in the near future . we were not able to find an association between new b2gp - i igm and thromboembolism risk . a similar observation was presented by jonsdottir et al . , who noted a statistically significant increase in acl igm positivity after 3 and 6 months of infliximab treatment . . they may be nonspecific markers of the immune system activation and may vanish without a trace . there are some reports that ra is associated with an increased frequency of acl positivity ; the de novo production of apl , especially acl igm , is associated with aps signs , such as thrombosis or vasculitis . according to jonsdottir et al . , a statistically significant increase in frequency of acl igm and igg was induced in patients with ra at 3 months of treatment with infliximab . the aspire trial reported no statistically significant differences in acl igg and igm classes between infliximab + mtx and mtx + placebo groups before or after treatment . fewer than 3% of patients who received infliximab plus mtx and who had negative findings for igg acl at baseline were found to have positive results at week 30 or 54 . for igm acl , a slightly higher proportion of patients who received infliximab plus mtx and had negative findings at baseline were found to have positive results at week 30 or week 54 ( 11.6% ) , as compared with the proportion of patients who received placebo plus mtx ( 7.6% ) . in the ferraro - peyret some authors suggest that the induction of apl appears usually at the beginning of treatment , but morris et al . demonstrated that it is important to check the acl appearance even between the 30 and 54 weeks of observation . emphasize that it is important to obtain serial samples over at least 1 year to ascertain a patient s apl status . further investigations are needed to determine if the new apl appears in patients with 2-gpi gene polymorphisms , such as leucine - to - valine substitution at position 247 , which can lead to conformational changes in 2-gpi protein , leading to apl synthesis . the role of apl in the pathogenesis of aps is still unclear but we should remember the immunogenic aspect of tnf antagonist treatment . therefore , we recommend early detection of apl and observation of the patient , paying special attention to signs and symptoms of thromboembolism . a possible preventive treatment should be discussed in certain patients .	backgroundthe introduction of tumor necrosis factor ( tnf ) antagonists ( adalimumab , infliximab , and etanercept ) was a major advance and was highly important and beneficial in most rheumatoid arthritis ( ra ) patients . the adverse effects of this treatment are infrequent , but include opportunistic intracellular infection ( especially the reactivation of latent mycobacterium tuberculosis ) ; exacerbation of demyelinating disorders ; and the production of various types of antibodies such as antinuclear antibodies ( ana ) or double - stranded dna autoantibodies ( dsdna ) and antiphospholipid antibodies ( apl ) such as anti - cardiolipin antibodies ( acl ) and anti - b2gp - i antibodies ( b2gp - i ) . the aim of the study was to determine the prevalence of acl and b2gp - i in igm and igg classes , using elisa tests , during 6 months of follow - up in patients with refractory ra successfully treated with infliximab.material/methodswe determined the prevalence of acl and b2gp - i in igm and igg classes , using elisa tests , during 6 months of follow - up in patients with refractory ra successfully treated with infliximab.resultswe observed a statistically important increase only in the group of b2gp - i igm ( p<0.05 ) . there are contradictory results concerning the ability of infliximab to induce apl , but most authors confirm this phenomenon.conclusionsfurther investigations are needed to determine if the new apl appears in patients with 2-gpi gene polymorphisms such as leucine - to - valine substitution at position 247 , which can lead to a conformational changes in 2-gpi protein , leading to apl synthesis . the role of apl in pathogenesis of aps is still unclear , but we should remember the immunogenic aspect of tnf antagonist treatment . therefore , we recommend early detection of apl and observation of the patient , paying special attention to signs and symptoms of thromboembolism .
nocturnal enuresis or bed wetting is defined as the persistence of spontaneous and inappropriate voiding of urine during sleep after the age of expected bladder control ( 1 , 2 ) . enuresis is explained in the dsm - iv as the repeated voiding of urine into bed or clothes at least twice a week for at least 3 consecutive months in a child who is at least 5 years of age ( 3 ) . nocturnal enuresis is described as the primary enuresis if the child has not been dry for more than 6 months ( 4 ) . bed wetting is the most common pediatric urologic problem in outpatient clinics ( 1 ) and is 3 times more common in boys than girls ( 2 ) . nocturnal enuresis affects 15 - 20% of 5 year olds , 5% of 10 year olds , 7% of 7 year olds , 2 - 3% of 12 - 14 year olds , and 1 - 2% of individuals aged 15 years and over ( 4 ) . without treatment , enuresis seems to be a multifactorial condition with different contributing factors , including genetics , sleep - arousal dysfunction , overnight polyuria , urodynamic features , developmental delay , and psychosocial problems ( 1 ) . before considering any therapeutic approach , it is important to identify the possible underlying mechanism of bed wetting and rule out other organic causes of enuresis . careful evaluation is required to determine specific urologic , psychosocial , developmental , and sleep - related etiologies . however , most children with primary nocturnal enuresis only need a precise medical history , physical examination , and urinalysis before starting the treatment . management of primary nocturnal enuresis should begin with educating and reassuring the child and parents about the condition . behavioral interventions are the first line of treatment and pharmacotherapy is only indicated for children who are impacted by their enuresis ( 6 ) . medication should not be started in children unless non - pharmacological interventions fail ( 2 ) . three frequently used drugs for the treatment of enuresis are imipramine , desmopressin , and oxybutynin ( 1 , 2 , 7 ) . the study aimed to assess which one of the above pharmacological interventions is more efficacious . we evaluated the outcomes of interventions to determine : 1 ) the rate of initial success as defined by the reduction in the number of wet nights per week , and 2 ) the rate of relapse after discontinuing the medications . the study , also explored the effect of demographic and socioeconomic variables on the response and relapse rate . the study was a randomized clinical trial conducted in 92 otherwise healthy children aged 5 - 14 years , who were referred to the pediatric clinic of amir al momenin hospital in semnan , iran , during 2009 and 2010 . the hospital is a provincial referral general hospital affiliated to semnan university of medical sciences . inclusion criteria included primary enuresis ( two or more wet nights per week and not dry for more than 6 months after the third year of life ) , not receiving any medication for enuresis during the last 6 months , and lack of clinical or laboratory signs suggestive of any underlying disease other than enuresis . both children and their parents were given detailed explanation about the study and treatment rationale , and the side effects of the drugs . then , the parents would be asked to sign the consent form , if they allowed their children to participate in the trial . a simple random sampling method was used to recruit the patients from the children with enuresis referred to the pediatric clinic of amir al momenin hospital . in this regard , 150 children ( 5 - 14 years old ) with primary enuresis were recruited . after detailed history taking and careful physical examination , urinalysis , urine culture , and urinary system , ultrasound were performed for all children . the parents of 19 children refused to participate in the study and other 15 patients withdrew from the study during the first assessment . a further 24 children were excluded from the study because of either history of day wetting , abnormal urinalysis or ultrasound , or urinary tract infection . a simple randomization procedure was used to allocate the remaining 92 patients into 3 treatment groups . the first group was given 1 - 2 puffs ( 10 - 20 g ) of nasal desmopressin ( n = 30 ) , the second group 25 - 75 mg imipramine ( n = 31 ) , and the third group 5 - 15 mg oxybutynin ( n = 31 ) . medications were dispensed at the clinic and participants were asked to fill the follow - up forms every morning and attend a follow up visit every 3 weeks . during the first visit , the detailed explanation of the medication , its possible side effects , dosage , and other necessary information were given , and participants were asked to call the physician if any major side effect was noticed . during the next follow up visits , the children were evaluated for the side effects or any possible difficulties . if participants were affected by the side effects of the medication , the responsible drug would be discontinued . when a child did not show up for the follow up visit , the researchers contacted him or her to find out the reasons and discuss the problems . any child who had not received the medication properly or was affected by major side effects was excluded from the study . the duration of treatment was 6 weeks and all children were evaluated regarding the number of wet nights per week at the end of the 6th week . moreover , all children were followed up for a further 3 months for the number of wet nights after discontinuing the medication ( figure 1 ) . the criterion for successful treatment was lack of wet nights for at least two consecutive weeks . relapse was defined as the reappearance of more than 2 wet nights per week , 3 months after the discontinuation of pharmacological therapy . the study protocol was approved by the ethics committee of semnan university of medical sciences . the trial has been registered in the iranian registry of clinical trials ( irct138807042503n1 ) . statistical analysis were performed by kolmogorov - smirnov , mann - whitney , and chi - square tests to compare demographic and socioeconomic variables , cure rate and relapse rate between the 3 groups using spss 16.0 software . out of 92 participants with enuresis , 30 children received nasal desmopressin , 31 children were given imipramine , and 31 others received oxybutynin . the study revealed a slightly higher rate of response to treatment in the oxybutynin group ( 71.0% ) , compared with the desmopressin group ( 63.3% ) , and the imipramine group ( 61.3% ) . however , the difference between the three groups was not statistically significant ( p = 0.70 ) . after discontinuing the therapy , 30 children presented with bed wetting again , indicating a relapse rate of 50% in general . the oxybutynin group showed a markedly lower relapse rate ( 31.8% ) compared to the other two groups ( desmopressin 57.9% and imipramine 63.2% ) , however , the difference was not statistically significant ( p = 0.095 ) ( table 2 ) . a few children in the oxybutynin group experienced mild headache , dry mouth , or stomach upset , which did not lead to discontinuation of the treatment . a small number of children in the imipramine group had mild drowsiness , and a few of them experienced mild gastrointestinal upset , which was not disturbing . mild headache and minimal nasal congestion were the only side effects in the desmopressin group . as a whole , none of our samples suffered from noticeable side effects due to medications . the study evaluated the treatment outcomes in children enuresis and compared the successful response and relapse rates among children treated with three different medications . we evaluated the outcomes of the interventions by assessing the rate of response , as well as the rate of relapse . the study showed that oxybutynin was successful in 71% of children with enuresis after 6 weeks therapy , followed by desmopressin ( 63.3% success ) and imipramine in ( 61.3% success ) . although the response rate was higher in the oxybutynin group than the desmopressin and imipramine groups , the difference was not statistically significant ( p = 0.701 ) . a huge number of studies have compared the effect of non - pharmacological interventions , desmopressin , antidepressants , or various combination therapies . review of the literature indicates that oxybutynin has been effective in 47 - 71% of children and showed a better response if combined with desmopressin ( 2 ) . nasal desmopressin had been efficacious in 60 - 70% and imipramine in 40 - 60% of children ( 2 ) . a systematic review based on a meta - analysis from the cochrane collaborative stated that desmopressin can result in 1 - 2 fewer wet nights per week compared with the placebo ( 8) . another systematic review showed that imipramine has been associated with a reduction of about one wet night per week during the treatment ( 9 ) . there is not enough evidence - based and reliable information indicating oxybutynin as an efficacious treatment for monosymptomatic enuresis ( 10 ) . some studies recommended oxybutynin for desmopressin resistant children , or those with primary nocturnal enuresis and daytime wetting ( 2 , 11 - 13 ) . several studies stated that oxybutynin is more effective in children who have small bladders , restricted bladder capacity , thickened bladder wall , and hyperactive detrusors ( 11 , 14 ) . in one study , the majority of children enuresis ( 88.2% ) with inadequate bladder storage function were responsive to the 15 mg daily oxybutynin regime , while in patients with normal bladder function this medication was generally unsuccessful for enuresis ( 15 ) . in a study by montaldo et al . ( 11 ) , the responders to combined oxybutynin and desmopressin showed a higher rate of response ( 45% ) compared with the desmopressin plus placebo group ( 17% ) . in a recent study in iran , the cure rate for oxybutynin was only 23% ( 16 ) , while in a randomized double - blind controlled trial the response rate of oxybutynin was 45% ( 11 ) . oxybutynin had a role for treating children with a restricted bladder capacity who did not have lower urinary tract symptoms during the day ( 17 ) . one study concluded that in children with enuresis , bladder capacity during sleep was significantly lower than day time capacity , and these children may not be able to hold urine during sleep ( 18 ) . we did not evaluate children s bladder capacity or function , but we obtained a better response to oxybutynin , which was inconsistent with previous studies . observed a reduction in nocturnal functional capacity of bladder during sleep at night in children who had a normal daytime urodynamic and functional bladder capacity . these children were refractory to treatment with desmopressin ( 19 ) , but they showed a good response to oxybutynin ( 12 , 15 , 17 ) . neveus ( 14 ) in a case - control study concluded that those responding to oxybutynin have small bladders and possibly hyperactive detrusors , whereas children responding to desmopressin or those who need both drugs for staying dry have polyuria . our findings highlight the idea that children with monosymptomatic enuresis without lower urinary tract symptoms during the day may still have problems with their nocturnal or functional bladder capacity . it is well established that treating enuresis can not be successful if the child and their parents do not cooperate , and that treatment with medication will fail if the family s social construct and home circumstances are not supportive . the clear differences between the findings of our study and previous studies may be partly explained by this point . other important factors in treatment success are comorbid psychological , behavioral , and emotional problems as well as age of the child ( 9 ) . a positive family history of enuresis , child s drinking habit , abnormal deep sleep pattern and constipation have a strong impact on the response to treatment ( 20 ) . parents , who experienced bedwetting , may have a more tolerant attitude towards the enuresis of their children ( 20 ) , which can positively affect the child s response to the medication . other risk factors of enuresis , including low socioeconomic level of the family , low school success , inappropriate and forced toilet training , and strict or over protective parents ( 21 ) may also influence the response to treatment . moreover , the various treatment approaches are influenced by the child s environment , and by the background and prejudgments of the family . as suggested by the cochrane database of systematic reviews : not all interventions are suitable for all children ( 9 ) . in our study , the oxybutynin group showed a clearly lower relapse rate ( 31.8% ) compared to the desmopressin ( 57.9% ) and imipramine ( 63.2% ) groups , with the relapse rate in the imipramine group being approximately twice as much as of the oxybutynin group . however , studies evaluating other medications have shown good initial response , albeit a substantial relapse rate ( 80 - 90% ) when children were off medication ( 1 ) . studies on desmopressin have reported the relapse rates as high as 80% ( 22 ) , which is higher than the relapse rate in our study . some rcts have shown no significant difference between desmopressin and placebo in terms of success rates after discontinuing the treatment ; and in comparison with alarms , desmopressin has been less effective when treatment was stopped ( 7 ) . review of the literature has shown that most children would relapse after discontinuing imipramine treatment ( 9 ) . controlled double blind trials have reported a long - term cure rate of 25% for imipramine ( 23 ) . relapse was reported in 60% of patients with enuresis treated with oxybutynin plus imipramine during the follow up period ( 12 ) . only in one study , the relapse rates for imipramine and oxybutynin were 58.6% and 42.3% , respectively ( 16 ) , which was compatible to our findings . in addition , the study conducted by kwak kw and his co - workers ( 24 ) showed that in the full responders to desmopressin and enuresis alarm , 60.7% did not relapse after cessation of treatment . however , when they added oxybutynin , 88.9% did not relapse after discontinuation of treatment . although the relapse rate of the three groups were different , because of the small sample size , the statistical analysis was not significant . the other limitation was that we recruited our samples from a relatively homogenous urban population with rather similar cultural and family circumstances . further clinical trial studies with larger sample size on different populations are needed to produce more reliable results in this field . nocturnal enuresis is a common problem seen by pediatricians and is one of the most common sources of concern for children and their families . despite many years of research , there are still some uncertainties about the benefit of pharmacological treatment and the most efficacious interventions . more studies with larger sample size and longer duration of treatment and follow up are needed to decide which treatment is the most appropriate for enuresis . studies should be performed on children with various cultural and socioeconomic backgrounds and different family circumstances and should take into account the individual characteristic of every child . the study showed that oxybutynin was successful in 71% of children with enuresis after 6 weeks therapy , followed by desmopressin ( 63.3% success ) and imipramine in ( 61.3% success ) . although the response rate was higher in the oxybutynin group than the desmopressin and imipramine groups , the difference was not statistically significant ( p = 0.701 ) . a huge number of studies have compared the effect of non - pharmacological interventions , desmopressin , antidepressants , or various combination therapies . review of the literature indicates that oxybutynin has been effective in 47 - 71% of children and showed a better response if combined with desmopressin ( 2 ) . nasal desmopressin had been efficacious in 60 - 70% and imipramine in 40 - 60% of children ( 2 ) . a systematic review based on a meta - analysis from the cochrane collaborative stated that desmopressin can result in 1 - 2 fewer wet nights per week compared with the placebo ( 8) . another systematic review showed that imipramine has been associated with a reduction of about one wet night per week during the treatment ( 9 ) . there is not enough evidence - based and reliable information indicating oxybutynin as an efficacious treatment for monosymptomatic enuresis ( 10 ) . some studies recommended oxybutynin for desmopressin resistant children , or those with primary nocturnal enuresis and daytime wetting ( 2 , 11 - 13 ) . several studies stated that oxybutynin is more effective in children who have small bladders , restricted bladder capacity , thickened bladder wall , and hyperactive detrusors ( 11 , 14 ) . in one study , the majority of children enuresis ( 88.2% ) with inadequate bladder storage function were responsive to the 15 mg daily oxybutynin regime , while in patients with normal bladder function this medication was generally unsuccessful for enuresis ( 15 ) . in a study by montaldo et al . ( 11 ) , the responders to combined oxybutynin and desmopressin showed a higher rate of response ( 45% ) compared with the desmopressin plus placebo group ( 17% ) . in a recent study in iran , the cure rate for oxybutynin was only 23% ( 16 ) , while in a randomized double - blind controlled trial the response rate of oxybutynin was 45% ( 11 ) . oxybutynin had a role for treating children with a restricted bladder capacity who did not have lower urinary tract symptoms during the day ( 17 ) . one study concluded that in children with enuresis , bladder capacity during sleep was significantly lower than day time capacity , and these children may not be able to hold urine during sleep ( 18 ) . we did not evaluate children s bladder capacity or function , but we obtained a better response to oxybutynin , which was inconsistent with previous studies . observed a reduction in nocturnal functional capacity of bladder during sleep at night in children who had a normal daytime urodynamic and functional bladder capacity . these children were refractory to treatment with desmopressin ( 19 ) , but they showed a good response to oxybutynin ( 12 , 15 , 17 ) . neveus ( 14 ) in a case - control study concluded that those responding to oxybutynin have small bladders and possibly hyperactive detrusors , whereas children responding to desmopressin or those who need both drugs for staying dry have polyuria . our findings highlight the idea that children with monosymptomatic enuresis without lower urinary tract symptoms during the day may still have problems with their nocturnal or functional bladder capacity . it is well established that treating enuresis can not be successful if the child and their parents do not cooperate , and that treatment with medication will fail if the family s social construct and home circumstances are not supportive . the clear differences between the findings of our study and previous studies may be partly explained by this point . other important factors in treatment success are comorbid psychological , behavioral , and emotional problems as well as age of the child ( 9 ) . a positive family history of enuresis , child s drinking habit , abnormal deep sleep pattern and constipation have a strong impact on the response to treatment ( 20 ) . parents , who experienced bedwetting , may have a more tolerant attitude towards the enuresis of their children ( 20 ) , which can positively affect the child s response to the medication . other risk factors of enuresis , including low socioeconomic level of the family , low school success , inappropriate and forced toilet training , and strict or over protective parents ( 21 ) may also influence the response to treatment . moreover , the various treatment approaches are influenced by the child s environment , and by the background and prejudgments of the family . as suggested by the cochrane database of systematic reviews : in our study , the oxybutynin group showed a clearly lower relapse rate ( 31.8% ) compared to the desmopressin ( 57.9% ) and imipramine ( 63.2% ) groups , with the relapse rate in the imipramine group being approximately twice as much as of the oxybutynin group . however , studies evaluating other medications have shown good initial response , albeit a substantial relapse rate ( 80 - 90% ) when children were off medication ( 1 ) . studies on desmopressin have reported the relapse rates as high as 80% ( 22 ) , which is higher than the relapse rate in our study . some rcts have shown no significant difference between desmopressin and placebo in terms of success rates after discontinuing the treatment ; and in comparison with alarms , desmopressin has been less effective when treatment was stopped ( 7 ) . review of the literature has shown that most children would relapse after discontinuing imipramine treatment ( 9 ) . controlled double blind trials have reported a long - term cure rate of 25% for imipramine ( 23 ) . relapse was reported in 60% of patients with enuresis treated with oxybutynin plus imipramine during the follow up period ( 12 ) . only in one study , the relapse rates for imipramine and oxybutynin were 58.6% and 42.3% , respectively ( 16 ) , which was compatible to our findings . in addition , the study conducted by kwak kw and his co - workers ( 24 ) showed that in the full responders to desmopressin and enuresis alarm , 60.7% did not relapse after cessation of treatment . however , when they added oxybutynin , 88.9% did not relapse after discontinuation of treatment . these findings are also in agreement with the results of our study . although the relapse rate of the three groups were different , because of the small sample size , the statistical analysis was not significant . the other limitation was that we recruited our samples from a relatively homogenous urban population with rather similar cultural and family circumstances . further clinical trial studies with larger sample size on different populations are needed to produce more reliable results in this field . nocturnal enuresis is a common problem seen by pediatricians and is one of the most common sources of concern for children and their families . despite many years of research , there are still some uncertainties about the benefit of pharmacological treatment and the most efficacious interventions . more studies with larger sample size and longer duration of treatment and follow up are needed to decide which treatment is the most appropriate for enuresis . studies should be performed on children with various cultural and socioeconomic backgrounds and different family circumstances and should take into account the individual characteristic of every child .	background : nocturnal enuresis is the most common pediatric urologic problem in outpatient clinics.objectives:to assess the effect of various monotherapies , and comparing the effects of desmopressin , imipramine , and oxybutynin in children with enuresis , as well as the influence of socioeconomic and cultural factors of their families on the response and relapse rates.patients and methods : the study was a randomized clinical trial conducted on 92 children aged 5 - 14 years , referred to the pediatric clinic of semnan university hospital in semnan , iran . children with primary nocturnal enuresis were randomly allocated to three different treatment groups : desmopressin ( n = 30 ) , imipramine ( n = 31 ) , and oxybutynin ( n = 31 ) all for 6 weeks . the socioeconomic and demographic characteristics of all participants were recorded . the number of wet nights per week was noted at the end of the 6-week - trial , and children were followed up to three months for relapse.results:children in the oxybutynin group showed a slightly higher response rate ( 71.0% success ) and a lower relapse rate ( 31.8% ) , while in the desmopressin group the response and relapse rates were 63.3% and 57.9% , respectively , and in the imipramine group 61.3% and 63.2% , respectively . however , the difference between the 3 groups in terms of response ( p = 0.701 ) and relapse rates ( p = 0.095 ) was not statistically significant.conclusions:there is no significant difference between monotherapy with desmopressin , imipramine or oxybutynin in children with enuresis . however , oxybutynin showed a higher response rate and a lower relapse rate compared to other medications . more clinical trials with a larger sample size are needed to clarify these uncertainties .
lichen planus ( lp ) is a chronic inflammatory and immune - mediated disease that affects the skin , nails , hair , and mucous membranes . the development of a true koebner phenomenon or the isomorphic response in lp is a well - established fact . rarely , linear papules may be the initial presentation of the disease , presumably reflecting koebnerization into the sites of previous trauma or scratching . this report aims to demonstrate an uncommon clinical presentation of lp , with koebner phenomenon , involving a rare site . a 54-year - old female patient presented with the chief complaints of itchy raised lesions on the hip ( at the site of friction by clothing , inner waist coat ) for a period of two months . she did not have similar lesions elsewhere in the body , including the oral cavity . her past history was insignificant . on examination , well - defined violaceous polygonal flat - topped papules and plaques coalescing with one another were noted , arranged circumferentially from the hip to the lower back on both sides in an interrupted pattern [ figure 1 ] . skin biopsy of a well - defined papule [ figure 2 ] ( h and e section ) showed compact orthokeratosis , focal parakeratosis , wedge - shaped hypergranulosis , irregular acanthosis , vacuolar alteration of the basal layer in the epidermis , and a band - like lymphocytic infiltrate in the papillary dermis , suggestive of lichen planus . the patient was prescribed topical betamethasone diproprionate ointment and advised to wear the drawstring loosely . low - power view showing lichenoid band of infiltrate with focal areas of basal cell vacuolation ( h and e , 10 ) drawstring dermatitis is a type of frictional dermatitis that can result from a traditional tightly worn garments such as the sari orthe salwaar - kameez . the resulting chronic friction may result in dermatoses such as lichenified grooves , postinflammatory depigmentation , or leukoderma . it may also result in koebnerizaton of preexisting dermatoses such as vitiligo and lp . in our patient , this koebnerization due to drawstring is rare and only one case had been previously reported . the other interesting feature is that it was the initial presentation of lp in our patient . this case is unique and demonstrates how sociocultural practices can influence the presentation of common dermatoses such as lp .	drawstring dermatitis is a type of frictional dermatitis that can result from a traditional tightly worn garments such as sari or salwaar - kameez . we report a 54-year - old female patient presented with lichen planus of the drawstring site that was confirmed histopathologically . this case is rare and demonstrates how sociocultural practices can influence the presentation of common dermatoses such as lp .
condylar injuries are often a matter of discussion and controversy among maxillofacial topics ( 1 ) . these traumas constitute 1116% of all facial fractures ( 25 ) and 3040% of mandibular fractures ( 37 ) . this area is superiorly confined to the line passing through the sigmoid notch and anteriorly to the line obliquely connecting the sigmoid notch to the masseter tuberosity ( 8) . this area has a great clinical value due to important components such as the facial nerve and temporomandibular joint ( tmj ) . both of these are prone to functional disability due to either the fracture itself , or the surgical intervention . different stages of dislocation , displacement , comminution and fracture line are dependent on the force magnitude , point of application and transmission , and the patient s mandibular position and occlusion , during the trauma ( 8) . one of the reasons that cause condylar fractures to be of paramount importance in maxillofacial surgery , is that many ensuing complications are connected to a condylar fracture , whether it is treated or not . even though early clinical results are seen to be good , complications such as pain , restriction of movement , muscle spasms , mandibular deviation , malocclusion , pathologic alterations of the tmj , osteonecrosis , facial asymmetry , and ankylosis may be evident later ( 913 ) . other complications related to the condylar fracture are ranged from tympanic bone fracture , fracture of mandibular fossa of temporal bone with or without dislocation of the condylar segment into the middle cranial fossa , injury to the cranial nerves , vascular damage and bleeding , growth inhibition and arteriovenous fistula ( 14 , 15 ) . as there are controversies around management of condylar fractures , different treatment modalities are suggested in literature ( 16 ) . the aim of this study was to review the different treatment strategies of these fractures in adults : closed reduction with maxillomandibular fixation ( mmf ) open reduction with internal fixation ( orif ) endoscopic - assisted reduction with internal fixation ( erif ) closed reduction was historically considered the standard treatment for subcondylar fractures ( 17 ) . the assumption that a closed reduction will result in fewer complications , though the esthetics and functionality will be just the same as an orif treatment , induced this method as the common procedure . the noninvasive nature of this method will consequently result in a very little to no facial nerve damage or scaring ( 18 ) . despite this knowledge , and much debating , authors are still unable to agree on an absolute treatment plan for management of condylar fractures . some publications , state no marked difference between the results of open or closed treatments ( 16 , 1921 ) . closed treatment however , is considered undesirable in some other studies ( 17 , 22 , 23 ) . unfortunately no clinical trial large enough is available on this matter to answer these questions . it is mentioned in different articles that duration of mmf has a range of two to six weeks ( 8) . the shortest time for mmf is chosen by surgeons , to avoid possible ankylosis of the temporomandibular joint ( 24 ) . the precise reason for the ankylosis is yet to be explained but in theory , the bleeding in the joint , hematoma and the following fibrosis and bone generation due to a trauma , could cause the undesired ankylosis ( 25 , 26 ) . it would thus be wise to assume that tmj ankylosis is a result of a direct trauma to the joint capsule or condylar head therefore the risk of ankylosis is minor in subcondylar fractures : hence , longer periods of mmf should be considered for a better union in fractures distant from the joint capsule ( 8) . upon conclusion of the gathered information , the authors suggest 46 weeks of mmf in the subcondylar fractures in which the tmj capsule is not involved . this protocol is also applicable to bilateral fractures in which the fractured segments are not displaced . however in bilateral fractures , segments are more prevalently prone to displacement , thus an orif approach is recommended . since the introduction of internal fixation appliances , open reduction of condylar fractures is becoming more popular . few studies emphasize on the indications of this treatment procedure ( 27 ) , but as a general rule , the need for anatomic reduction in complex fractures is one reason to employ the open reduction and internal fixation approach . nowadays , many of the upcoming articles are discussing the better results of the surgical treatment of the condylar fractures , for better occlusion , bone morphology and tmj function ( 20 , 2831 ) . others however argue that a surgical access to the mandibular condyle , would increase the risk of trauma to the facial nerve and leave unpleasant scaring ( 27 , 3234 ) . hypertrophic scaring in 7.5% of cases are said to be quite tangible and significant ( 27 ) . however the superior anatomic reduction and better functional results , induce orif as the treatment of choice in patients with complex conditions . another advantage would be the immediate capability of the patient to move the injured area , reducing the mmf required time and thus lowering the risk of ankylosis ( 8) . in cases of major displacement and loss of functional occlusion , when a closed reduction is not possible , orif is indicated and the result would overcome the presumptive complications ( 29 ) . to access the fractured area , different approaches are available and surgeons would decide upon a suitable approach , based on experience and personal taste . some of these approaches are periauricular , submandibular , retromandibular antroparotid and retromandibular transparotid . the retromandibular antroparotid with a two centimeter incision under the ear lobule is utilized in this center . post - surgical scaring is less evident in this approach and no entry to the parotid gland occurs , preventing complications such as salivary fistula ( 35 ) . erif is said to be embracing the benefits of both the closed and the open techniques . the resulting scar is very minor , no facial nerve damage is expected and a functional occlusion is ensured with anatomic reduction . an excellent visibility is achieved through the endoscope . this method is also swifter in comparison to other extraoral techniques ( 36 ) . according to literature , it is said that by using the endoscope , a variety of methods are possible . ( 3739 ) , although an intraoral approach is most commonly used by the surgeons utilizing an endoscope ( 40 , 41 ) . this approach is also used in this center , and a small incision on the skin suffices the entry of the trocar . the majority of the subcondylar fractures can be managed by this method , however an orif procedure would be the treatment of choice in cases of significant displacements . despite the preponderance and imperfections of the mentioned methods , the goal is achieving the premorbid and normal function of the patient with minimal damage . pointing out the best procedure is certainly not easy assay and many factors influence the matter , so the aim of this review was to summarize the available methods , and their advantages and disadvantages , in order to help make the decision easier . due to different techniques and materials and more importantly the position and kind of the subcondylar fracture , choosing the right treatment protocol is a challenge every time . despite the many clinical researches available on management of condylar fractures , it still remains a matter of debate ( 36 ) . the modern day surgical techniques such as the endoscope - assisted technology , reducing the morbidity rate , again raises the question of choosing either the open or closed treatment technique ( 42 ) . the closed technique is continues to be the most commonly used for medium to large displacements ( 43 ) . retrospective and prospective studies in literature comparing open and closed treatments , report that the surgical procedure has no superiority to the closed technique as in function , range of motion , occlusion , contour , sensory or motional function retrieval . the most common complaint of patients after six months was chronic pain after the closed reduction and a noticeable scar after the surgical treatment ( 22 , 44 , 45 ) . m. gupta et al . concluded that after both treatment protocols , the patients presented similar occlusion , retention of the reduced part s fixation , trismus index , mandibular movements and chewing sufficiency . the notable differences were the patient s discomfort during mandibular movements , chewing , swelling , neurogenic complications and parotid fistula on occasion after surgery ( 1 ) . however several radiologic studies demonstrate a better anatomical reduction after the orif treatment ( 19 , 4549 ) . the study of conservative treatment of 466 condylar fractures carried out by zachariades et al . , concluded that the closed technique is the treatment of choice for most of the subcondylar fractures , unless when there is nt a recoverable occlusion , severe displacement , loss of height of ramus or an edentate patient where the open reduction is indicated ( 50 ) . ( 48 ) mentioned that the two indicators for surgery are the condylar angle of 1045 and the loss of more than two millimeters of the ramus height . these studies also concluded that despite the surgical technique , anatomic reduction , mouth opening , lateral and forward movements of the mandible and the lower rate of pain and discomfort of the patients were considered beneficial in the orif group . the study by lee jw et al . , and al - moraissi et al . . however there are some studies about complications of open surgeries which indicate that permanent deformity and dysfunction after surgery is very rare ( 27 ) , also the mandibular and condylar mobility is better ( 29 , 30 ) and opening incisor pathway is much more normal ( 31 ) and also due to not decreasing the ramus height , less asymmetry is visible ( 28 ) . kokmueller et al . ( 36 ) in 2012 carried out a study to assimilate the endoscope - assisted transoral treatment and closed reduction of subcondylar fractures . both closed reduction and patients undergoing surgery had more complaints in the short run , but they were presented with fewer symptoms in long term follow ups . patients with endoscopic treatment had better occlusion parameters both in early and late follow up sessions . it is mentioned several times in literature that the surgical complications of patients are far less , when treated transorally by the endoscopic - assisted method ( 19 , 23 , 53 , 54 ) . authors using the endoscopic method to treat subcondylar fractures believe that a single intraoral incision would be sufficient to reduce a laterally displaced condyle . even for lateral dislocations of the subcondylar area , an intraoral approach is suggested , due to the unperceptive scar , no matter how challenging it may be ( 40 , 41 ) . it is wise to emphasize on the short duration of this procedure also ( 41 ) , butfor the surgeons who are newly utilizing this method , there is probably much more time to perform the anatomic reduction and do the surgery than the conventional extraoral methods . but according to the learning curve that is considered for endoscopic treatment , this time will soon be decreased ( 55 ) . for more complicated malocclusions and bigger dislocations closed reduction was historically considered the standard treatment for subcondylar fractures ( 17 ) . the assumption that a closed reduction will result in fewer complications , though the esthetics and functionality will be just the same as an orif treatment , induced this method as the common procedure . the noninvasive nature of this method will consequently result in a very little to no facial nerve damage or scaring ( 18 ) . despite this knowledge , and much debating , authors are still unable to agree on an absolute treatment plan for management of condylar fractures . some publications , state no marked difference between the results of open or closed treatments ( 16 , 1921 ) . closed treatment however , is considered undesirable in some other studies ( 17 , 22 , 23 ) . unfortunately no clinical trial large enough is available on this matter to answer these questions . it is mentioned in different articles that duration of mmf has a range of two to six weeks ( 8) . the shortest time for mmf is chosen by surgeons , to avoid possible ankylosis of the temporomandibular joint ( 24 ) . the precise reason for the ankylosis is yet to be explained but in theory , the bleeding in the joint , hematoma and the following fibrosis and bone generation due to a trauma , could cause the undesired ankylosis ( 25 , 26 ) . it would thus be wise to assume that tmj ankylosis is a result of a direct trauma to the joint capsule or condylar head therefore the risk of ankylosis is minor in subcondylar fractures : hence , longer periods of mmf should be considered for a better union in fractures distant from the joint capsule ( 8) . upon conclusion of the gathered information , the authors suggest 46 weeks of mmf in the subcondylar fractures in which the tmj capsule is not involved . this protocol is also applicable to bilateral fractures in which the fractured segments are not displaced . however in bilateral fractures , segments are more prevalently prone to displacement , thus an orif approach is recommended . since the introduction of internal fixation appliances , open reduction of condylar fractures is becoming more popular . few studies emphasize on the indications of this treatment procedure ( 27 ) , but as a general rule , the need for anatomic reduction in complex fractures is one reason to employ the open reduction and internal fixation approach . nowadays , many of the upcoming articles are discussing the better results of the surgical treatment of the condylar fractures , for better occlusion , bone morphology and tmj function ( 20 , 2831 ) . others however argue that a surgical access to the mandibular condyle , would increase the risk of trauma to the facial nerve and leave unpleasant scaring ( 27 , 3234 ) . hypertrophic scaring in 7.5% of cases are said to be quite tangible and significant ( 27 ) . however the superior anatomic reduction and better functional results , induce orif as the treatment of choice in patients with complex conditions . another advantage would be the immediate capability of the patient to move the injured area , reducing the mmf required time and thus lowering the risk of ankylosis ( 8) . in cases of major displacement and loss of functional occlusion , when a closed reduction is not possible , orif is indicated and the result would overcome the presumptive complications ( 29 ) . to access the fractured area , different approaches are available and surgeons would decide upon a suitable approach , based on experience and personal taste . some of these approaches are periauricular , submandibular , retromandibular antroparotid and retromandibular transparotid . the retromandibular antroparotid with a two centimeter incision under the ear lobule is utilized in this center . post - surgical scaring is less evident in this approach and no entry to the parotid gland occurs , preventing complications such as salivary fistula ( 35 ) . erif is said to be embracing the benefits of both the closed and the open techniques . the resulting scar is very minor , no facial nerve damage is expected and a functional occlusion is ensured with anatomic reduction . this method is also swifter in comparison to other extraoral techniques ( 36 ) . according to literature , it is said that by using the endoscope , a variety of methods are possible . ( 3739 ) , although an intraoral approach is most commonly used by the surgeons utilizing an endoscope ( 40 , 41 ) . this approach is also used in this center , and a small incision on the skin suffices the entry of the trocar . the majority of the subcondylar fractures can be managed by this method , however an orif procedure would be the treatment of choice in cases of significant displacements . despite the preponderance and imperfections of the mentioned methods , the goal is achieving the premorbid and normal function of the patient with minimal damage . pointing out the best procedure is certainly not easy assay and many factors influence the matter , so the aim of this review was to summarize the available methods , and their advantages and disadvantages , in order to help make the decision easier . due to different techniques and materials and more importantly the position and kind of the subcondylar fracture , choosing the right treatment protocol is a challenge every time . despite the many clinical researches available on management of condylar fractures , it still remains a matter of debate ( 36 ) . the modern day surgical techniques such as the endoscope - assisted technology , reducing the morbidity rate , again raises the question of choosing either the open or closed treatment technique ( 42 ) . the closed technique is continues to be the most commonly used for medium to large displacements ( 43 ) . retrospective and prospective studies in literature comparing open and closed treatments , report that the surgical procedure has no superiority to the closed technique as in function , range of motion , occlusion , contour , sensory or motional function retrieval . the most common complaint of patients after six months was chronic pain after the closed reduction and a noticeable scar after the surgical treatment ( 22 , 44 , 45 ) . m. gupta et al . concluded that after both treatment protocols , the patients presented similar occlusion , retention of the reduced part s fixation , trismus index , mandibular movements and chewing sufficiency . the notable differences were the patient s discomfort during mandibular movements , chewing , swelling , neurogenic complications and parotid fistula on occasion after surgery ( 1 ) . however several radiologic studies demonstrate a better anatomical reduction after the orif treatment ( 19 , 4549 ) . the study of conservative treatment of 466 condylar fractures carried out by zachariades et al . , concluded that the closed technique is the treatment of choice for most of the subcondylar fractures , unless when there is nt a recoverable occlusion , severe displacement , loss of height of ramus or an edentate patient where the open reduction is indicated ( 50 ) . ( 48 ) mentioned that the two indicators for surgery are the condylar angle of 1045 and the loss of more than two millimeters of the ramus height . these studies also concluded that despite the surgical technique , anatomic reduction , mouth opening , lateral and forward movements of the mandible and the lower rate of pain and discomfort of the patients were considered beneficial in the orif group . the study by lee jw et al . , and al - moraissi et al . however there are some studies about complications of open surgeries which indicate that permanent deformity and dysfunction after surgery is very rare ( 27 ) , also the mandibular and condylar mobility is better ( 29 , 30 ) and opening incisor pathway is much more normal ( 31 ) and also due to not decreasing the ramus height , less asymmetry is visible ( 28 ) . kokmueller et al . ( 36 ) in 2012 carried out a study to assimilate the endoscope - assisted transoral treatment and closed reduction of subcondylar fractures . both closed reduction and patients undergoing surgery had more complaints in the short run , but they were presented with fewer symptoms in long term follow ups . patients with endoscopic treatment had better occlusion parameters both in early and late follow up sessions . it is mentioned several times in literature that the surgical complications of patients are far less , when treated transorally by the endoscopic - assisted method ( 19 , 23 , 53 , 54 ) . authors using the endoscopic method to treat subcondylar fractures believe that a single intraoral incision would be sufficient to reduce a laterally displaced condyle . even for lateral dislocations of the subcondylar area , an intraoral approach is suggested , due to the unperceptive scar , no matter how challenging it may be ( 40 , 41 ) . it is wise to emphasize on the short duration of this procedure also ( 41 ) , butfor the surgeons who are newly utilizing this method , there is probably much more time to perform the anatomic reduction and do the surgery than the conventional extraoral methods . but according to the learning curve that is considered for endoscopic treatment , this time will soon be decreased ( 55 ) . for more complicated malocclusions and bigger dislocations overall it is safe to declare that endoscopic surgery is certainly a good replacement for approaches through the skin , for subcondylar fractures . avoiding the complications related to the traditional open technique such as the facial nerve damage , scarring and those related to the close technique such as the lengthened mmf , non - anatomical reduction and difficulties associated with mandibular movements , have deemed the endoscopic method a favorite among surgeons . with enhanced experience of the surgeons toward the endoscopic technique the controversies over the right treatment choice would slowly subside .	condylar injuries are often subjected to discussion and controversy in maxillofacial surgery as they constitute many of the facial fractures . the condylar area has a great clinical value due to its important components . vital components in this area are susceptible to functional disability due to either the fracture itself or the subsequent surgical intervention . each of the strategies for the management of these fractures has its advantages and disadvantages . as there are controversies around management of condylar fractures , different treatment modalities are suggested in literature , so this paper reviews different treatment strategies of the following types of fractures in adults : 1-closed reduction with maxillomandibular fixation , 2-open reduction with internal fixation , 3-endoscopic - assisted reduction with internal fixation . in conclusion , we declare that the endoscopic surgery is certainly a good replacement for approaches through the skin , for subcondylar fractures , but still more randomized clinical trials are needed to be carried out on this issue .
primary biliary cirrhosis ( pbc ) is an autoimmune disease , and high - titer antimitochondrial autoantibodies ( amas ) are characteristically present in sera from almost all pbc patients [ 1 , 2 ] . the major autoantigens recognized by ama are the lipoyl - containing e2 subunits of 2-oxoacid dehydrogenase complexes , such as the branched - chain 2-oxoacid dehydrogenase complex ( bcoadc ) and pyruvate dehydrogenase complex ( pdc ) . the multimeric e2 subunits form the structural core of each complex , around which multiple e1 and e3 subunits assemble to form huge macromolecular complexes . in pdc , there is another subunit , termed e3bp , which is involved in the association of e3 with the e2 core . the lipoyl domain of the e2 polypeptide contains the major epitopes recognized by both ama and t cells [ 47 ] , and the significance of these epitopes in eliciting autoimmunity has been firmly established . curiously , however , the non - lipoyl - containing e1 components ( and other subunits ) are also recognized by ama [ 9 , 10 ] . more recently , the frequencies of antibodies against the and subunits of pdc - e1 have been reported to be strikingly high , indicating the significance of anti - e1 autoimmunity in the pathogenesis of ama . furthermore , anti - e1 antibodies are exclusively found in sera from patients with anti - e2 antibodies [ 912 ] , suggesting antibody diversification from e2 to e1 . therefore , the mechanisms underlying anti - e1 autoimmunity may be closely linked to , but clearly distinct from , those of anti - e2 autoimmunity . although such features may reflect the pathogenesis of ama , the potential significance of anti - e1 autoimmunity has been largely overlooked [ 11 , 13 ] . determinant spreading refers to the development of immune responses against endogenous epitopes as a result of tissue damage and plays an active role in ongoing disease pathology . therefore , it is necessary to clarify whether a determinant spreading cascade also underlies the pathogenesis of pbc . in this regard , the reactivity of ama against the non - lipoyl - containing e1 components is suggestive of determinant spreading . however , since very little attention has been paid to the immunogenic roles of e1 proteins [ 11 , 13 ] , the involvement of a spreading cascade in pbc has not been argued extensively . to address this issue , analyses of antibody reactivity against e1 should be carried out in comparison with those against other antigens . we previously characterized the autoreactivity against non - lipoyl - containing subunits by focusing on the e1 molecules , which are the most frequent targets of ama in this category [ 10 , 16 ] . in the present study , we analyzed the relationships between the antibody reactivity against bcoadc - e1 and those against other subunits of bcoadc and pdc . in addition , we carried out antibody epitope mapping of bcoadc - e1 using a multipin elisa , which has been applied to systematically search for epitopes on various antigens [ 1820 ] . the solvent accessibilities of the epitopes were analyzed based on the reported crystal structure of bcoadc - e1 . our findings revealed that anti - bcoadc - e1 antibodies appeared in association with antibodies against other subunits of bcoadc but not of pdc . in addition , the major epitope overlapped with the active center , and multiple b - cell autoepitopes were mapped on the surface of the bcoadc - e1 molecule . thus , a spreading cascade may underlie the pathogenesis of ama , similar to the case for other autoimmune diseases . pbc sera were collected from 30 patients with a well - established diagnosis of pbc . during testing with human liver bcoadc - e1 , 16 of the 30 pbc sera we used these 16 sera for epitope mapping of bcoadc - e1 , all of which were at the time point of first diagnosis . all the subjects were from clinics at fukushima medical university ( fukushima , japan ) , and provided written informed consent . the study protocol was approved by the ethical committee of fukushima medical university and conformed to the ethical guidelines of the 1975 declaration of helsinki . blocks with pins were purchased as part of a mimotope epitope scanning kit ( chiron , clayton , australia ) . fmoc - protected amino acids and dimethylformamide were obtained from wako pure chemical industries ltd . affinity - purified goat anti - human igg conjugated with horseradish peroxidase was purchased from bio - rad laboratories ( richmond , ca ) . a 3,3,5,5-tetramethylbenzidine peroxidase substrate kit for elisa the obtained e1 , e2 , and e3 components of the complex were further subjected to preparative sds - page . proteins were visualized by zinc staining , and the individual areas corresponding to bcoadc - e1 ( 46 kd ) , bcoadc - e1 ( 36 kd ) , bcoadc - e2 ( 52 kd ) , and bcoadc - e3 ( 55 kd ) were excised and recovered by electroelution using an ae-6580 electroelution apparatus ( atto , tokyo , japan ) . the purity of each polypeptide was verified by sds - page followed by silver staining . the bcoadc activity was reconstituted in vitro by mixing the purified e1 , e2 , and e3 components as described previously . before assay , 1.25 g of e1 was incubated for 3 min on ice with sera at a 200-fold dilution . the reactivities of the sera against purified bcoadc - e1 were determined by elisa in ms-3696f 96-well immunoplates ( sumitomo bakelite ) . briefly , 50 ng of a purified antigen was added to each well and blocked with 10% block ace in pbs overnight . sera diluted 1 : 100 to 1 : 1000 with pbst containing 10% block ace were added to the wells and incubated at room temperature for 1 h. after four washes with pbst , horseradish peroxidase - conjugated goat anti - human igg diluted 1 : 1000 in pbst was added to each well and incubated for 1 h. next , the wells were washed four times with pbst , and color development was initiated by the addition of 100 l of 3,3,5,5-tetramethylbenzidine for 5 min and terminated by the addition of 100 l of 2 n h2so4 . for statistical analyses , overlapping peptides were synthesized according to the bcoadc - e1 sequence ( genbank , np_000700 ) . in the following procedure , the ser residue just after the mitochondria - targeting sequence was designated amino acid number 1 . peptides were synthesized on a 96-pin block with the aid of the mimotope software as previously described [ 16 , 17 ] . peptide synthesis was verified by the reactivities of control antibodies supplied by the manufacturer against positive and negative control peptides ( plrq and glaq , resp . ) , both of which were synthesized on every pin block . the reactivities of the sera against the synthesized peptides were estimated by pin elisa as previously described . the bound antibodies were estimated using a model 3550 microplate reader ( bio - rad laboratories ) . the absorbance value for each peptide was transformed into a standard score ( z - score ) that represented the magnitude of deviation from the mean absorbance value . crystallographic data for bcoadc - e1 ( pdb , 2bev ) were utilized for analyses . the solvent - accessible surface areas of the bcoadc - e1 subunit were calculated using the dssp program ( http://swift.cmbi.ru.nl/gv/dssp/ ) . visualization of epitopes on the crystal structure of bcoadc - e1 was conducted with the pymol software ( http://www.pymol.sourceforge.net/ ; delano scientific , south san francisco , ca , usa ) . sequence alignment of amino acids between human bcoadc - e1 and human pdc - e1 was conducted using the blast2 program ( http://www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi/ ) . homology analyses of amino acids among bcoadc - e1 subunits from various species were performed by aligning the protein sequences using the clustalw2 program ( http://www.ebi.ac.uk/tools/clustalw2/ ) . thereafter , the homology index of each residue was calculated by the consurf program ( http://consurf.tau.ac.il/ ) on the basis of x - ray diffraction data ( pdb , 2bev ) . initially , the antibody profile against bcoadc - e1 in sera from patients with pbc was compared with that against bcoadc - e2 . as shown in table 1 , anti - bcoadc - e1 antibodies were always found with anti - bcoadc - e2 antibodies . based on fisher 's exact test , the appearance of anti - bcoadc - e1 antibodies was significantly linked to that of anti - bcoadc - e2 antibodies ( n = 30 , p = 0.037 ) . such a relationship was also found between the appearances of anti - pdc - e1 and anti - pdc - e2 antibodies , and this was also statistically significant ( supplemental table s1 , available online at doi:10.1155/2012/369740 p = 0.004 ) . hence , anti - e1 antibodies for each type of complex were always found in combination with antibodies against the respective e2 subunit , which was consistent with previous observations including ours [ 912 ] . considering that multiple e1 and other components constitute the complex assembly around the e2 core in vivo , there may be additional relationships between the antibody titers against the e1 , e2 , and other antigenic subunits . therefore , we further characterized the relationships among the antibody titers ( od values by elisa ) against each antigenic subunit of both complexes ( table 2 ) . the antibody titer against bcoadc - e1 was strongly correlated with that against bcoadc - e1 ( n = 30 , r = 0.786 , p = 0.018 ) , and weakly but significantly correlated with that against bcoadc - e2 ( r = 0.336 , p = 0.043 ) . however , the antibody titer against bcoadc - e1 did not show any significant correlations with those against pdc - e1 ( r = 0.193 , p = 0.168 ) , pdc - e2 ( r = 0.073 , p = 0.358 ) , or pdc - e3bp ( r = 0.044 , p = 0.413 ) . therefore , the antibody titer against bcoadc - e1 was correlated with those against the other subunits of the bcoadc complex but not with those against the subunits of the pdc complex . on the other hand , the antibody titer against pdc - e1 was weakly but significantly correlated with that against pdc - e2 ( r = 0.339 , p = 0.042 ) , and moderately but very significantly correlated with that against pdc - e3bp ( r = 0.655 , p = 0.000 ) . however , the antibody titer against pdc - e1 did not show any significant correlations with those against the individual subunits of bcoadc . thus , the antibody titer against each e1 subunit was correlated with those against the other antigenic subunits of the complex it belonged to . cluster analysis ( figure 1 ) further revealed that there were two identifiable epitope clusters : one toward the bcoadc complex and the other toward the pdc complex . the distance in the relationship between the two antibody clusters was further than the distances between the antibody reactivities toward the individual subunits in each complex . these observations suggest that anti - e1 antibodies arise in association with the intermolecular diversification of epitopes among the subunits within each individual complex . in the next step , we carried out a multipin elisa to better define the antigenic characteristics of bcoadc - e1. the anti - bcoadc - e1-positive sera were selected for epitope mapping , and representative profiles are shown in figures 2(a ) and 2(b ) . the sera from two symptomatic patients with the strongest titers ( p13 and p18 ) showed similar reaction patterns . the highest reactivity was observed against the region containing amino acids 134168 with z - scores of more than 3.0 ( supplemental figure s1 ) , and all the pbc sera with anti - bcoadc - e1 antibodies reacted with this region ( table 3 ) . therefore , this region was regarded as the major determinant on bcoadc - e1. the significance of this region was also supported by the characteristic antibody - binding profiles as follows . as shown in figure 2(c ) , multiple small epitopes were clustered in the major epitope region . a similar situation was commonly observed among the anti - bcoadc - e1 antibody - positive sera including those from asymptomatic patients . the highly reactive octapeptides with peak od values in the major determinant region were isdlgkgr ( aa 137144 ) , grqmpvhy ( aa 143150 ) , mpvhygck ( aa 146153 ) , ygckerhf ( aa 150157 ) , and vtisspla ( aa 158165 ) ( figure 2(d ) ) . these profiles revealed that the major determinant comprised overlapping epitopes , thus forming nested epitopes . in addition to the major determinant , multiple regions ( designated epitopes 1 to 12 ) were identified ( table 3 ) . among these , the major epitope ( epitope 6 ) was invariably recognized , while the reactivities against the remaining epitopes were variable among the sera . the p13 serum , which showed the highest titer against bcoadc - e1 , reacted with most of the epitopes . the octapeptides exhibiting the peak od values within each region , which represent the core sequences , supplemental table s2 also indicates the presence of additional epitope clusters in regions 7 and 9 . in summary , the above data suggest the occurrence of intramolecular diversification of epitopes on the bcoadc - e1 molecule . subsequently , the epitopes determined by elisa were analyzed using crystallographic data for bcoadc - e1 . using the dssp program , the solvent accessibilities of each residue were calculated . the major determinant ( aa 134168 ) was shown to contain two solvent - accessible surface areas on the bcoadc - e1 22 tetramer ( supplemental figure s2 ) . these two areas ( aa 137142 and 153156 ) were included within two of the defined epitopes ( aa 137144 and 150157 ) that constitute the major determinant region ( figure 2(d ) ) . these observations support the validity of pin elisa for analyzing epitopes , and suggest that these areas are the primary recognition sites . figure 3(a ) depicts a three - dimensional map of the major determinant on bcoadc - e1. the major determinant overlapped with the active center ( figure 3(b ) ) . these observations imply that the major determinant may be structurally important for the enzymatic function . indeed , the sera with anti - bcoadc - e1 antibodies strongly inhibited the enzyme activity in an in vitro reconstitution assay ( supplemental figure s3 ) . next , the dominant and subdominant epitopes were structurally mapped ( supplemental figure s4 ) . although the locations of the epitopes varied among the patients , the epitopes formed weak clusters in each case . using surface representation ( figure 3(c ) ) , the epitopes were found to form clusters on the surface of the molecule , suggesting that the antibodies recognize consecutive surface areas of bcoadc - e1. taken together , these results suggest that the native bcoadc - e1 itself is an immunogen . with respect to the mechanisms underlying the antibody diversification , one possibility is that it results from antibody cross - reactivity against homologous proteins . initially , human pdc - e1 should be considered to be responsible for initiating such cross - reactivity as a candidate homolog of bcoadc - e1. both of these molecules are thiamine pyrophosphate - dependent enzymes involved in the first reaction step for oxidative decarboxylation of 2-oxoacids . they share conserved motifs , namely a tetramer interface , thiamine pyrophosphate - binding site , heterodimer interface and phosphorylation loop region . however , no significant relationship between the antibody titers against bcoadc - e1 and pdc - e1 was found ( table 2 ) . in addition , the appearances of anti - bcoadc - e1 and anti - pdc - e1 antibodies were statistically independent of one another ( table 4 , p = 0.466 by fisher 's exact test ) . figure 4(a ) shows an alignment of the sequences of human bcoadc - e1 ( bcdc - e1 ) and pdc - e1 by the blast2 program , in which the epitope regions are colored in red and blue , respectively . a comparison of the epitopes between the two proteins indicated that they were independent of their counterparts . these data compare well with our previous findings that autoantibodies against bcoadc - e1 and pdc - e1 did not cross - react with each other 's antigen . finally , the amino acid sequence of human bcoadc - e1 was compared with those of its homologs from other species using the consurf program . we found that the epitope regions were independent of the conserved segments ( figure 4(b ) , upper ) . in addition , the two core sequences of the major determinant ( aa 137142 and 153156 ) were specific to the human protein . in this regard , and in contrast to the highly conserved lipoyl - containing e2 subunit , the amino acid sequence of the non - lipoyl - containing e1 subunit was less conserved among species ( figure 4(b ) , lower ) . these observations support the idea that the immune system in patients targets the self bcoadc - e1 antigen . our study has demonstrated that both intermolecular and intramolecular diversifications of autoimmunity are associated with the emergence of anti - bcoadc - e1 antibodies . although it is difficult to chase the autoimmune profiles over time from the beginning of the pathogenesis , combining the intermolecular and intramolecular information would provide clues toward clarifying the underlying sequence of events . from an intermolecular point of view first , they tended to appear in association with antibodies against other subunits constituting bcoadc but not pdc . as a result , a clustering of antibody reactivities toward bcoadc was observed that could be distinguished from that toward pdc . second , the appearance of anti - bcoadc - e1 antibodies always accompanied that of anti - bcoadc - e2 antibodies , suggesting a constant order in their emergence . these observations are understandable by assuming that the native bcoadc complex is the immunogen with the e2 subunit as the primary determinant . conceivably , the immune system may be erroneously induced to attack the self bcoadc complex released from damaged cells , consequently giving rise to intermolecular determinant spreading between the subunits within the complex . according to the literature , intermolecular determinant spreading is generally observed between the components of macromolecular complexes and is characterized by the progressive and ordered appearance of autoreactivities against different components of the complexes [ 32 , 33 ] . the above findings are compatible with this model , and thus anti - bcoadc autoimmunity is a plausible example of this amplification cascade . if this notion is true , breakdown of self - tolerance could be observed against the bcoadc - e1 subunit itself . from an intramolecular point of view first , the major determinant recognized by all the sera with anti - bcoadc - e1 antibodies was composed of nested epitopes , indicating active responses against this region . second , multiple epitopes were observed throughout the polypeptide , suggesting an antigen - driven mechanism . third , the major epitope overlapped with the active center on the molecular surface , which seemed to be easily recognized by the surface immunoglobulins of b cells . finally , antibody reactivities against human bcoadc - e1-specific sequences were observed . taken together , these observations indicate that the native bcoadc - e1 itself is targeted by the immune system . this feature extends the above - mentioned view of an antigen - driven mechanism , which may explain the causal relationship between the intermolecular and intramolecular determinant spreading . there are many examples of autoimmune disorders in which the spreading of autoimmunity plays an active role in the disease pathology [ 14 , 33 ] . therefore , it is worth highlighting the underlying mechanisms whereby spreading takes place in anti - bcoadc - e1 autoimmunity . one of the possible causes of the diversification of the epitopes is molecular mimicry based on structural similarities between the self bcoadc - e1 antigen and an ortholog in either humans or pathogens . however , the present study argues against the possibility of cross - reactivity between human bcoadc - e1 and human pdc - e1 , supporting independent autoimmunity against each e1 molecule . in addition , the anti - bcoadc - e1 antibodies were shown to recognize human - specific sequences . this is probably because the subunit composition and the amino acid sequence of bcoadc - e1 differ considerably among species . in particular , most gram - negative bacteria , including escherichia coli , that have been proposed as causative agents for pbc [ 35 , 36 ] are deficient in bcoadc . nevertheless , it may be possible that conformational epitopes are cross - reactive between human bcoadc - e1 and certain microbial antigens . in contrast to this lack of significant interspecies conservation of bcoadc - e1 at the primary structure level , the conservation is strong between the lipoyl - containing - e2 antigens , where molecular mimicry is considered highly likely . other than the molecular mimicry theory , the prevailing model emphasizes an immune regulatory role for b cells in determinant spreading [ 15 , 38 ] . according to this model , b cell - mediated uptake of the multiprotein complex via surface immunoglobulins followed by processing and presentation to t cells is the underlying mechanism that leads to the breakdown of self - tolerance to each component of the complex . this concept depends on the physical associations among the antigen molecules and is fully compatible with our present observations . thus , it can be hypothesized that this model explains the appearance of autoantibodies against the non - lipoyl - containing subunits , e1 , e1 , and e3 , within the bcoadc and pdc complexes [ 9 , 10 , 39 ] . in contrast , however , the diversification of the autoimmunity between the lipoyl - containing e2 subunits of bcoadc and pdc could be well explained by the standard theory of molecular mimicry . we speculate that the anti - e2 autoreactivity caused by mimicry may , in turn , act as an initiation factor that triggers the determinant spreading to other subunits within each complex . interparticle determinant spreading between the macromolecular complexes in sle [ 15 , 40 ] . the current hypothesis on the etiopathogenesis of pbc implies that this disease is the result of a genetic predisposition that is permissive for still unknown environmental agents , similar to other autoimmune diseases [ 1 , 41 ] . recent genome - wide association studies have demonstrated that the hla region is the most important genetic component for susceptibility to pbc , and that all of the other minor susceptibility variants are in immune - related genes , thereby further strengthening the similarity [ 4244 ] . while determinant spreading is a well - known phenomenon in other autoimmune diseases , further investigations are required to clarify how the diversification of ama is associated with the pathogenesis of pbc .	primary biliary cirrhosis ( pbc ) is characterized by antimitochondrial antibodies ( amas ) that react with the lipoyl - containing e2 subunits of 2-oxoacid dehydrogenase complexes such as bcoadc and pdc . the lipoyl domains of e2 contain the major epitopes essential for immunopathology . however , the non - lipoyl - containing e1 subunits are also frequently targeted . since anti - e1 antibodies always appear in combination with anti - e2 antibodies , the mechanisms underlying the autoimmunity against e1 may be linked to , but distinct from , those against e2 . here , we demonstrate that intermolecular and intramolecular determinant spreading underlies the autoimmunity against e1 . we performed characterizations and epitope mapping for anti - bcoadc - e1 antibodies from both the intermolecular and intramolecular points of view . the antibody reactivities form a cluster against the bcoadc complex that is distinct from that against the pdc complex , and the anti - bcoadc - e1 antibodies arise as part of the cluster against the bcoadc complex . multiple epitopes are present on the surface of the bcoadc - e1 molecule , and the major epitope overlaps with the active center . sera with anti - bcoadc - e1 antibodies strongly inhibited the enzyme activity . these findings suggest that the e1 subunit as part of the native bcoadc complex is an immunogen , and that determinant spreading is involved in the pathogenesis of ama production .
the diseases with high infectivity and mortality are particularly feared , and in the past , people have regarded such diseases as a disaster or a punishment . however , improvements in identifying the etiology of viral infections and knowledge about microbiology , which were followed by the development of various vaccines , have enabled humankind to overcome the irrational fear of death . the invention of vaccination is regarded as one of the biggest triumphs in the history of medicine . although numerous efforts have focused on producing qualified and effective vaccines , there are insufficient barriers to protect populations from diseases that may cause epidemics or pandemics ( e.g. , the ebola virus epidemic in 2014 ) . thus , researchers are trying to increase the numbers of diseases that can be prevented by vaccines and , by doing so , to expand the target populations that will receive the benefits of vaccination in the future . in addition , vaccine development strategies are being tailored to the particular economic and health requirements of specific countries . the products under development and the numbers and types of clinical trials are influenced directly by this trend . this is why physicians and others involved in vaccine development should be alert to the current paradigm . its aim is to increase understanding about vaccine development by providing up - to - date information . the history of the development and application of ' vaccine - like ' substances to humans startedin ancient times . 1 ) . according to this diagrammatic outline , we are living in the modern era of vaccine development , which is more successful and productive than any other period in history . this progress has been dependent on the abundant financial support received ( left column ) . using knowledge based on experience and observation , people in the 12th to 15th centuries practiced ' variolation ' , the first known method of human immunization . powdered scabs or fluid from the pustules of a smallpox patient were inserted into superficial scratches made in the skin of the recipient . many variations of this technique were used in china , the middle east , and africa , and they spread widely throughout europe in the 17th century . the first scientific investigation of this technique was made by edward jenner in 1796 when he used cowpox virus rather than smallpox scabs in a human experiment based on doctrines of the enlightenment . this was the origin of the term ' vaccine ' and the beginning of vaccinology . despite the historic achievement of jenner , because of insufficient fundamental knowledge about microbiology , no new vaccines were developed for more than a century . in the late 19th century , heroic scientists such as louis pasteur , robert koch , emil von behring , and paul ehrlich discovered the basic principles and developed the experimental methodology of immunology and immunotherapy that led to the next stage of vaccinology . following their seminal investigations , many other studies were performed and led to improved regulations ( e.g. , the biologics control act of 1902 ) , which resulted in the development of valid live and/or attenuated vaccines . new vaccines against diseases including rabies , typhoid fever , diphtheria , shigellosis , tuberculosis , tetanus , and pertussis were developed by 1930 . however , during this period , vaccine research was limited to the areas of public and/or military need ( world war i ) because of restricted funding resources . in 1931 , an important transition to vaccine mass production began with good pasture 's findings of viral growth in embryonated hens ' eggs . in addition , the number of large - scale human tests with improved scientific validity increased during this period . the methodologies of randomization , blinding , and use of control groups helped increase the accuracy of the evaluation of the safety and effectiveness of vaccines . between 1930 and 1950 , and especially during world war ii , military purpose remained a powerful motivation for vaccine development . support from other bodies including public agencies and foundations ( e.g. , the world health organization [ who ] and the rockefeller institute ) has arisen since then . vaccines against adenovirus , poliovirus , japanese b encephalitis virus , and influenza virus were developed in this stage . in the second half of the 20th century , defined by hilleman as the modern era , scientific improvements related to the screening and manufacturing of vaccine products enabled the development of new types of vaccines . plotkin and plotkin regarded the same period as the ' golden age ' of vaccine development . this age began with the development of three classical attenuated - virus vaccines against measles , mumps , and rubella ( mmr ) in the 1960s followed by the varicella zoster virus vaccine and inactivated japanese encephalitis virus vaccine in the 1970s . all of these vaccines involved cell culture techniques under controlled conditions for a certain purpose ( i.e. , attenuation ) in their manufacturing processes . inactivated whole hepatitis a virus and cell culture - derived rabies viruses were also developed as vaccine products using similar methodology . it was not until the 1980s that the conjugation of bacterial capsular polysaccharides to proteins was applied in a real - world setting , although it had been proposed in the 1930s by avery and goebel . thanks to this technology , bacterial vaccines against haemophilus influenzae type b , meningococcus , and pneumococcus were introduced . the development of recombinant viral vaccines using genetic engineering is another important step in the evolution of vaccinology . the first example of this type of vaccine was the vaccine against hepatitis b virus .the human papillomavirus vaccine is another important example . these kinds of vaccines brought dramatic improvement in vaccine safety , mitigating the risk of using purified inactivated antigens obtained from infected patients . to meet society 's need for safe and efficacious vaccines , the clinical vaccine development process has been refined for more than a century . similar to that of chemical drugs , the clinical evaluation of a vaccine typically comprises three phases ( fig . the entire process takes 10 - 15 years and requires a budget of about 1 billion us dollars . a vaccine - specific developmental plan should be clearly established to ensure the efficient and successful development before clinical evaluation . this includes the following contents : 1 ) identification of the target population ( mostly healthy people with particular demographic characteristics ) and their sociocultural factors ; 2 ) risk assessment of the target disease and the vaccine itself ; 3 ) understanding of the incidence of the target disease and environmental factors ; 4 ) identification of the dose and route of administration ; 5 ) plans to induce herd immunity ; and 6 ) regulatory strategies . the general characteristics of clinical vaccine development compared with those of conventional drug development are summarized in table 1 . human studies of the acceptable safety and reactogenicity of a vaccine candidate are achieved in ' phase i ' clinical trials . in this phase , safety and tolerability are evaluated at both the local and systemic levels as the primary endpoint . these trials are often designed as randomized , double - blind , placebo - controlled , single - center studies . according to the characteristics of the product , either a crossover or parallel design may be chosen . the statistical analysis is generally descriptive and exploratory in nature because the trials involve only small numbers of participants ( 20 - 80 ) , and thus sufficient information needed for confirmatory tests can not be obtained . in the ' first - in - human ' setting , more attention should be given to live attenuated vaccines because the risks tend to be higher than those of killed vaccines . in ' phase ii ' , the ' proof - of - concept ' ( poc ) of the vaccine product should be ensured . clinical trials of this phase are conducted to demonstrate the immunogenicity of the relevant active component(s ) and the safety profile of a candidate vaccine within the target population and to define the optimal dose , initial schedule , and safety profile of a candidate vaccine . theses purposes are often achieved by separating clinical trials into ' phase iia ' and ' phase iib . ' in designing these clinical trials , multiple variables associated with the host immune response are considered . determinants of clinically applicable vaccine regimens are also included , such as the dose and number of doses , sequence / interval between doses , and route of administration . most of these clinical trials include parallel group comparisons with placebo / active control groups . prospective and confirmatory statistical analyses are performed , and the percentage of responders should be defined and described based on predefined criteria of an immune response ( e.g. , antibodies and/or cell - mediated immunity ) . the final step in the clinical evaluation before product license is the ' phase iii ' trial . this stage is intended to provide a pivotal conclusion needed for marketing approval , and the efficacy and safety of formulation(s ) of the immunologically active component(s ) must be assessed in the large - scale target population . the clinical outcome is strongly recommended as a parameter for comparing efficacy ( e.g. , with placebo / active control groups ) . therefore , serological data are usually collected from at least a subset of the immunized population at predefined intervals . the designs of phase ii and phase iii clinical trials are similar , but the size of a phase iii trial is much larger . in consideration of the modern vaccination strategy - administration of multiple vaccines at the same time - interaction and/or interference with other vaccines are evaluated routinely . it is sometimes not possible to conduct a confirmatory study to determine the protective efficacy of products containing the same antigens that are already used commonly and/or whose target disease has a very low incidence . the information obtained during the developmental processes mentioned above are summarized and filed for submission to regulatory authorities in support of an application for marketing approval . the who and each regulatory authority have their own guidelines to ensure the quality of the information provided . as an example , the united states food and drug administration ( fda ) calls the process ' biologics license application ' ( bla ) . the multidisciplinary fda review team reviews the efficacy and safety information needed to make a risk - benefit assessment and is advise by vaccines and related biological products advisory committee ( vrbpac ) . the appropriateness of label contents and the reliability of the manufacturing process are also reviewed . even though a vaccine may be licensed , the safety information provided for licensure is regarded as insufficient , because at that point , only a few thousand people have likely been exposed to the vaccine . thus , many vaccines undergo postlicensure ( ' phase iv ' ) studies . in the united states , the vaccine adverse event reporting system ( vaers ) one of the most important aspects of vaccinology in the 21st century is the extension of the target population by the development of new vaccines against emerging infections , tumors , and chronic diseases . ultimately , the goal of modern vaccination may be expressed as to prevent or to cure as many diseases with vaccination as possible . 3 ) . meeting this challenge requires increasing both the number of vaccine clinical trials in nontraditional populations worldwide and the scientific expertise necessary for the successful development of new vaccines . many initiatives have been launched recently including the decade of vaccines , the millennium development goals , and the us institute of medicine consensus study identifying and prioritizing new preventive vaccines for development . another focus is to improve the efficacy and safety of vaccines even further beyond the overwhelming successes of vaccines in the past several centuries . the most important keyword from the efficacy viewpoint is ' adjuvant ' . a number of vaccine products are licensed or under development in the form of a mixture of a vaccine and a certain adjuvant ( table 2 ) . the emphasis on the importance of adjuvants is gradually increasing with the aging of the population . because they facilitate the immune response to vaccination in older people , many experts expect that adjuvants will be an essential component for widespread vaccine use in entire populations . in the traditional paradigm , rappuoli has stressed the methodological approaches used to overcome the risks of vaccination in the 21st century ( table 3 ) . in addition , thanks to improvements in genomic techniques , new vaccine - design methods , such as reverse vaccinology , have enabled the high - throughput screening of vaccine candidates with greater confidence in their safety profiles . the characteristics of vaccine recipients are also considered , and there is much focus on developing ways to personalize vaccination , which is termed ' vaccinomics ' . without doubt , the quantity and quality of clinical vaccine development will improve greatly in the future . simultaneously integration of knowledge about microbiology and immunology , establishment of efficient vaccine development strategies , and streamlining of regulatory approval processes may facilitate this trend . doing so will increase the chances that human society will experience the continued benefits of vaccination .	vaccination is regarded as one of the biggest triumphs in the history of medicine . we are living in the most successful period of vaccine development . the accumulation of multidisciplinary knowledge and the investment of massive funding have enabled the development of vaccines against many infectious diseases as well as other diseases including malignant tumors . the paradigm of clinical vaccine evaluation and licensure has also been modernized based on scientific improvements and historical experience . however , there remain a number of hurdles to overcome . continuous efforts are focused on increasing the efficacy and reducing the risks related to vaccine use . cutting - edge knowledge about immunology and microbiology is being rapidly translated to vaccine development . thus , physicians and others involved in the clinical development of vaccines should have sufficient understanding of the recent developmental trends in vaccination and the diseases of interest .
certain strains of lactic - acid producing bacteria are classed as probiotics because their consumption is associated with health benefits , which are mediated via the gut . the current probiotic definition is live microorganisms which when administered in adequate amounts confer a healthy benefit on the host . probiotic bacteria are most frequently of the lactobacillus or bifidobacterium species and usually species that can be found in the normal commensal microbiota . probiotics can be effective in treating some patients with inflammatory bowel disease ( ibd ) [ 37 ] , but the details of which strains confer benefit and their mechanisms of action are only slowly being defined . ulcerative colitis ( uc ) and crohn 's disease ( cd ) , collectively termed inflammatory bowel disease ( ibd ) , result from a dysregulated response of the mucosal immune system to components of the luminal microbiota and breakdown of immune tolerance in individuals who are genetically predisposed to the disease . these processes lead to inappropriate activation of mucosal t cells and production of inflammatory mediators [ 811 ] . dendritic cells ( dc ) recognize and respond to bacteria and bacterial products and generate primary t cell responses . dc also determine whether t cell responses generated are immunogenic or tolerogenic [ 1214 ] . in particular , intestinal dc maintain the delicate balance in the gut between immunogenicity against invading pathogens and tolerance of the commensal microbiota ; alterations in intestinal dc have been found in ibd [ 15 , 16 ] . the effects of probiotic bacteria on dc , which are so pivotal in early bacterial recognition , tolerance induction , and shaping t cell responses , are likely to be central in immunomodulation by these bacteria and are likely to partially account for the reported efficacy of probiotics in ibd [ 37 ] . ibd is associated with a variety of extraintestinal manifestations ( eim ) , with up to a third of ibd patients developing cutaneous manifestations . the causes of eim are poorly understood , but it has been suggested that compartmentalisation of inflammatory processes to different organs ( e.g. , the intestines , skin , or liver ) may be linked to homing and trafficking of immune cells . indeed , dysregulated lymphocyte trafficking has been reported in both uc and cd [ 1922 ] . homing properties are imprinted on t cells upon stimulation by dc , to localise immune responses to specific tissues [ 2326 ] . effector t cells migrating to intestinal sites express high levels of gut - homing molecule 47 , with its ligand madcam-1 being constitutively expressed by postcapillary endothelial cells in the small intestine and colonic lamina propria . skin t cells express e- and p - selectin ligands including cutaneous lymphocyte - associated antigen ( cla ) and ccr4 . the occurrence of eim associated with ibd indicates a systemic disease , rather than immune dysregulation confined to intestinal sites ; however it is currently unclear whether alterations in circulating dc occur in ibd patients , including dc ability to imprint specific homing properties on stimulated t cells . trafficking of immune cells is an area yet to be investigated regarding specific mechanisms of action of immunomodulation by probiotics or dysregulated dc function in ibd . the strain - specific nature of the immunomodulatory effects of probiotics is well established ; some lactobacillus strains induce production of regulatory cytokines , suppress th1 responses , and are thought to be involved in oral tolerance . however , human intervention studies have shown a variety of beneficial immunomodulatory effects associated with consumption of the probiotic bacterial strain lactobacillus casei shirota ( lcs ) specifically , including significant improvement in uc disease activity index ( ucdai ) scores in patients with mild - moderate uc administered lcs orally for 8 weeks , compared to pretreatment and also patients on conventional therapy . the same study demonstrated that lcs reduces production of il-6 from peripheral blood mononuclear cells ( pbmc ) in vitro . other studies demonstrate reduction of gingival inflammation and downregulation of allergic responses following consumption of lcs . to this end , we aimed to determine whether systemic changes exist between healthy controls and patients with active uc , regarding the ability of circulating ( blood - enriched ) dc to generate effector t cell responses and imprint specific homing properties on t cells stimulated . we also aimed to study the immunomodulatory effects of probiotic strain lcs on such dc . human peripheral blood was collected from healthy volunteers with no known autoimmune or inflammatory diseases , allergies or malignancies ( n = 8) or from patients with active uc following informed consent ( n = 6 ) . disease activity for uc was assessed using the uc disease activity index ( ucdai ) ; patients scoring ucdai 412 , alongside diagnosis from clinical parameters , radiographic studies , and endoscopic and histological criteria , were defined as active uc . patients were treatment nave or on minimal treatment : 5-aminosalicylic acid ( 5asa ) and/or azathioprine ( aza ) . peripheral blood mononuclear cells ( pbmc ) were obtained by centrifugation over ficoll - paque plus ( amersham biosciences , chalfont st . human blood - enriched dc ( low density cells or ldc ) were obtained following nycoprep centrifugation of overnight cultured pbmc . these cells were 98%100% hla - dr , with morphological characteristics of dc ( both at optical microscopy and electron microscopy ) , and are potent stimulators of nave t cells . blood ldc have been characterised in detail in previous studies from our laboratory [ 35 , 36 ] and will be referred to as blood dc in this study . tokyo , japan ) was cultured at 37c for 24 hours in mrs broth and grown on mrs agar ( oxoid , hampshire , uk ) for 48 hours at 37c in an anaerobic cabinet ( macs mg 1000 ; don whitley scientific , west yorkshire , uk ) with a gas mixture of 10% h2 , 10% co2 , and 80% n2 by volume . for liquid culture , one pure colony was taken from an mrs nutrient agar plate and grown overnight in 10 ml of prereduced mrs broth ( oxoid ) with 0.05% l - cysteine hydrochloride ( sigma , dorset , uk ) in a shaking incubator at 37c ; 0.5 ml of the overnight culture was inoculated into another 10 ml mrs broth . the bacteria were harvested in the exponential phase , resuspended in phosphate - buffered saline ( pbs ; oxoid ) , centrifuged twice at 1960 g ( sanyo / mse micro centaur , haverhill , usa ) for 5 minutes , and resuspended at the required concentration in rpmi 1640 containing 0.75 mm l - glutamine . bacteria were then heat - killed with viability checks done to make sure that no bacteria survived , and varying concentrations ( 1 10 , 1 10 , or 1 10 ) of heat - killed lcs were used to condition 2.5 10 blood dc in 1 ml total volume of complete medium ( dutch modification rpmi 1640 containing 2 mm glutamine , 10% fetal calf serum , and 100 u / ml penicillin / streptomycin ) for 24 hours . control conditions involved conditioning dc with complete medium only for 24 hours . following conditioning , dc were washed and used in a mixed - leucocyte reaction ( mlr ) with allogeneic t cells . pbmc were suspended in minimacs buffer ( pbs containing 0.5% bsa and 2 mm edta ) and t cells were enriched by depletion of cd14 , cd19 , and hla - dr cells with immunomagnetic beads ( miltenyi biotech , bisley , uk ) following manufacturer 's instructions . carboxyfluorescein diacetate succinimidyl ester ( cfse , invitrogen ltd , uk ) labelled t cells ( 4 10/well ) were incubated for 5 days in u - bottomed 96-well microtitre plates with enriched , previously conditioned , allogeneic dc at 0% , 1% , 2% , or 3% in a mixed leukocyte reaction ( mlr ) . monoclonal antibodies with the following specificities and conjugations were used : cla - fitc ( heca-452 ) , 7 integrin - pe ( fib504 ) , il-12 ( p40/p70)-pe ( c11.5 ) , il-17a - pe ( scpl1362 ) , cd3-percpcy5.5 ( sk7 ) , cd3-pecy5 ( ucht1 ) , il-10-apc ( jes3 - 19f1 ) , ifn ( 25723.11 ) , cla - biotin ( heca-452 ) , and streptavidin - apc were purchased from bd biosciences ( oxford , uk ) ; ccr9 ( either fitc or apc ) ( 112509 ) , ccr7-pe ( 150503 ) , ccr10-apc ( 314315 ) , ccr4-apc ( 205410 ) , and tgf ( ic388p ) were purchased from r&d systems ( abingdon , uk ) . after the staining , cells were fixed with 1% paraformaldehyde in 0.85% saline and stored at 4c prior to acquisition on the flow cytometer , within 48 hours . data were acquired on a facscanto ii cytometer ( bd biosciences ) and analysed using winlist 5.0 software ( verity , me , us ) . proportions of positive cells were measured by subtracting the appropriate isotype - matched control staining from test histogram using superenhanced dmax ( sed ) normalised subtraction . the intracellular cytokine production by stimulated t cells after mlr was measured using superenhanced dmax ( sed ) normalised subtraction upon data analysis following incubation + / monensin , t cell permeabilisation , antibody labeling , and flow cytometry . two - way repeated measures anova and two - tailed paired t - tests were applied as stated in the figure legends . in the case of multiple comparisons , subsequent ad hoc bonferroni correction was applied . we analysed dc stimulation of t cells in a 5-day mixed leucocyte reaction ( mlr ) . t cells from the same donor ( a separate , healthy control ) were stimulated by dc from healthy controls and uc patients , within the same experiments . dc stimulated a strong , dose - dependent proliferative response in both healthy controls and uc patients ; dividing t cells were identified as cfse cd3 lymphocytes , by flow cytometry ( figure 1(a ) ) . however , dc from uc patients ( uc - dc ) stimulated a significantly weaker proliferation of the same cfse - labelled t cells compared with dc from healthy controls ( control dc ; figure 1(b ) ) . we have previously demonstrated that t cells within fresh pbmc expressed either gut - homing molecule 7 or skin - homing molecule cla ; the majority expressed 7 only . freshly purified t cells exhibited the same homing profile , prior to coculture with allogeneic dc . after culture , the expression of 7 on dividing t cells ( cfse ) was the default pathway ; t cells stimulated by both control and uc - dc maintained 7 expression , as did unstimulated t cells . in contrast , cla expression was induced on dividing t cells by both control and uc - dc so that substantial numbers of t cells were identified as double positive for cla and 7 following stimulation ( due to inherent high expression of 7 in all conditions ; figure 2(a ) ) . however , uc - dc exhibited an enhanced ability to prime skin - homing t cells , significantly increasing the proportion of total cla t cells ( figure 2(b ) ) and the proportion of t cells expressing skin - homing molecule ccr4 ( figure 2(c ) ) within the stimulated population . optimisation experiments on healthy control dc determined no significant differences between live or heat - killed ( hk ) lcs regarding ability to enhance dc activation / maturation markers cd80 and cd83 ; both live and hk lcs significantly enhanced cd80 and cd83 expression ( figure 3(a ) ) . we analysed dc stimulation of t cells in a 5-day mixed leucocyte reaction ( mlr ) following dc conditioning with complete medium only or varying concentrations of hk lcs ( 1 10 , 1 10 , or 1 10 cfu / ml ) . a significant , dose - dependent increase in dc stimulatory capacity was observed upon lcs conditioning of both control- and uc - dc ( figure 3(b ) ) . following lcs conditioning , uc - dc levels of stimulation were restored to normal lcs conditioning of dc had differential effects in healthy controls compared with uc , on dc ability to imprint homing properties on stimulated t cells . in healthy controls , lcs conditioning enhanced dc ability to induce a skin - homing profile on t cells , significantly increasing the proportion of stimulated t cells expressing cla , in a dose - dependent fashion ( figures 4(a ) and 4(b ) ) . however , in uc , cla expression on t cells was already enhanced ( figures 2(a ) and 2(b ) ) , and lcs conditioning had no further effects on dc ability to enhance cla expression on t cells ( figure 4(b ) ) . lcs conditioning had no effect on dc ability to induce ccr4 expression in either healthy controls or uc patients ( data not shown ) . cla expression on t cells was enhanced upon stimulation by both untreated uc - dc and lcs - conditioned ( control ) dc . induction of cla on t cells stimulated by lcs - conditioned dc from controls was in conjunction with gut - homing marker 7 . however , cla induction by untreated uc - dc was on the 7 negative fraction of t cells ( figure 4(c ) ) . thus , the proportion of cla7 t cells within the total cla dividing t cell pool was significantly greater upon stimulation with uc - dc , compared to lcs ( 1 10 cfu / ml ) conditioned ( control ) dc ( figure 4(d ) ) . lcs conditioning of dc also had differential effects on dc ability to induce cytokine production by stimulated t cells , in controls compared with uc patients . although there were differences within individual experiments between the ability of control dc and uc - dc ( both untreated ) to induce cytokine production by t cells ( il-10 , tgf , ifn , and il-17a were measured ) , overall there were no significant differences ( figure 5(a ) ) . however , tgf production by t cells was significantly increased , in a dose - dependent manner , when dc were conditioned with lcs in healthy controls but not in ulcerative colitis ( figure 5(b ) ) . we demonstrate for the first time that human circulating dc from uc patients exhibit a restricted stimulatory capacity for allogeneic t cells , and these dc induce a specific skin - homing profile on stimulated t cells that dc from healthy controls do not . our data support studies demonstrating dysregulated dc function in ibd [ 11 , 15 , 16 ] and , furthermore , demonstrate systemic immune dysregulation in ibd patients rather than at mucosal sites only . the occurrence of extraintestinal manifestations ( eim ) associated with ibd indicates that ibd is indeed a systemic disease , and our data provide an explanation for the occurrence of eim affecting the skin . conditioning uc - dc with probiotic strain lcs restored their stimulatory capacity , reflecting that of control dc . lcs had differential effects on dc in healthy controls and uc on dc ability to imprint specific homing profiles on stimulated t cells , and to induce cytokine production by t cells . this is the first study , to our knowledge , to investigate the effects of probiotic bacteria on migratory properties of immune cells . our data supports studies demonstrating multifunctional immunoregulatory activities of lcs , depending on the responding cell types and the local microenvironment . lcs conditioned control dc , but not uc - dc , to imprint skin - homing molecule cla on stimulated t cells . however , unlike the skin - homing profile induced on t cells by uc - dc , cla expression induced via lcs conditioning was in conjunction with gut - homing molecule 7 , suggesting induction of a multihoming profile . the differential effects of lcs on control and uc - dc were further demonstrated by the induction of tgf production by t cells stimulated with lcs - conditioned dc in controls but not uc patients . these data suggest that effects of lcs exerted on human dc are flexible , depending on the responding cell types and the local cytokine environment . the restoration of uc - dc stimulatory capacity by lcs suggests that lcs may partly contribute to restoration / maintenance of homeostasis . lcs may also confer homeostatic properties at intestinal sites ( e.g. , via oral administration ) which could be beneficial in ibd ; gut dc play a central role in immune homeostasis in the gut and exhibit tolerogenic properties . alterations occur in gut dc in ibd [ 15 , 16 ] , leading to loss of tolerance in the gut and dysregulated immune responses to the colonic microbiota , a major contributing factor in the onset of ibd . restoration of homeostatic properties of gut dc by lcs at intestinal sites may account for the reported efficacy of lcs in uc . however , the local microenvironment and responding cell types differ dramatically in the circulation and the gut , for example , gut dc are conditioned by intestinal epithelial cells and epithelial cell - derived products to adopt their tolerogenic function [ 4042 ] . future studies will determine in vitro effects of lcs on gut dc and also on epithelial cell conditioning of gut dc . although the definition of probiotics involves live microorganisms ( which when administered in adequate amounts confer health benefit on the host ) , our data demonstrates immunomodulation by heat - killed bacteria ; furthermore , we demonstrated no significant differences between live and hk lcs regarding their ability to enhance activation marker expression on blood - enriched dc from healthy controls . these data support studies demonstrating immunomodulation by probiotic bacterial products , including the ability of probiotic bacterial dna to induce regulatory il-10 production by human peripheral blood mononuclear cells and dendritic cells and the ability of sonicated probiotic bacteria to induce marked anti - inflammatory effects on blood and intestinal dc . furthermore , our recent studies have demonstrated that an immunomodulatory peptide secreted by lactobacillus plantarum mediates some of the molecular dialogue between intestinal bacteria and dc , inducing immunoregulatory effects in both blood and intestinal dc in vitro . ibd is associated with a variety of eim , with up to a third of patients developing cutaneous manifestations including erythema nodosum ( en ) and pyoderma gangrenosum ( pg ) . the causes of eim of ibd are poorly understood , but it has been suggested that compartmentalisation of inflammatory processes to different organs ( e.g. , intestine , skin , and liver ) may be linked to homing and trafficking of immune cells . for example , ccl25 , the ligand for gut - homing receptor ccr9 , is expressed on epithelium in both the liver and the small intestine . dysregulation of lymphocyte trafficking plays a key role in ibd pathogenesis [ 1922 , 46 ] and ibd therapeutics have previously demonstrated efficacy by abrogating trafficking of effector cells to intestinal sites [ 4751 ] . however , we demonstrate in this study that skin - homing markers cla and ccr4 are aberrantly expressed on 7 t cells stimulated by uc - dc , providing an explanation for the occurrence of eim affecting the skin and supporting previous studies demonstrating that conditioning dc with supernatants from culture of colonic biopsies from uc patients enables them to imprint a skin - homing phenotype on stimulated t cells . blocking trafficking of effector cells to cutaneous sites in patients with eim of ibd may also be of therapeutic benefit . although there were no significant effects of lcs on dc ability to induce t cell cytokine production in uc overall , effects of lcs were variable between individual experiments , depending on whether production of particular cytokines was increased or decreased compared to t cells stimulated by control dc ( data not shown ) normal phenotype and support a multifunctional immunoregulatory role for lcs , returning dysregulated immune functions to the original normal state when the host becomes either immunocompromised or excessively activated . indeed , lcs can have either pro- or anti - inflammatory effects in human intervention studies [ 32 , 53 , 54 ] and in vitro studies [ 5557 ] depending on the context . in summary , our data demonstrate systemic alterations in immune cells in uc , specifically a dysregulated dc function . our data provides an explanation for the occurrence of eim of the skin in uc patients and suggests that the probiotic strain lcs has multifunctional immunoregulatory activities on dc , depending on the disease state and the inflammatory environment . our data supports studies demonstrating probiotic bacterial products , rather than live bacteria , are capable of inducing immunoregulatory effects . the reported therapeutic effects of lcs and other probiotic lactobacilli strains in uc [ 32 , 58 ] may be partly due to promotion of homeostasis , restoring the dysregulated functions of immune cells .	background . dendritic cells regulate immune responses to microbial products and play a key role in ulcerative colitis ( uc ) pathology . we determined the immunomodulatory effects of probiotic strain lactobacillus casei shirota ( lcs ) on human dc from healthy controls and active uc patients . methods . human blood dc from healthy controls ( control - dc ) and uc patients ( uc - dc ) were conditioned with heat - killed lcs and used to stimulate allogeneic t cells in a 5-day mixed leucocyte reaction . results . uc - dc displayed a reduced stimulatory capacity for t cells ( p < 0.05 ) and enhanced expression of skin - homing markers cla and ccr4 on stimulated t cells ( p < 0.05 ) that were negative for gut - homing marker 7 . lcs treatment restored the stimulatory capacity of uc - dc , reflecting that of control - dc . lcs treatment conditioned control - dc to induce cla on t cells in conjunction with 7 , generating a multihoming profile , but had no effects on uc - dc . finally , lcs treatment enhanced dc ability to induce tgf production by t cells in controls but not uc patients . conclusions . we demonstrate a systemic , dysregulated dc function in uc that may account for the propensity of uc patients to develop cutaneous manifestations . lcs has multifunctional immunoregulatory activities depending on the inflammatory state ; therapeutic effects reported in uc may be due to promotion of homeostasis .
coccidioidomycosis is an endemic fungal infection caused by the coccidioides species c. immitis and the recently described , non - california species c. posadasii . about 60% of persons with these infections are asymptomatic , and most of the rest have self - limited pulmonary infections , although a minority of cases in otherwise healthy persons may be severe or disseminated . serologic tests have been used for decades to assist in the diagnosis and management of coccidioidal infection . among serologic tests available for the diagnosis of mycotic illnesses , those used for coccidioidomycosis tests commonly used in our endemic area include complement fixation ( cf ) and immunodiffusion ( i d ) tests of either tube precipitin antibodies ( igm ) or complement - fixing antibodies ( igg ) . in addition , an enzyme immunoassay ( eia ) to detect igg and igm can be performed in local clinical laboratories and can often be confirmed by cf or i d when positive . the eia for igg appears comparable in sensitivity to cf or i d , but the eia for igm may give false - positive results and should be confirmed with other serologic methods . some authors have demonstrated lower rates of seropositivity in immunosuppressed persons [ 79 ] , whereas others have reported evidence that the serologic response is maintained in immunocompromised hosts ; most of these observations were seen in small case series , had no comparison group , or contained no temporal information . in contrast , our report reviews our experience with each of the 3 serologic methods in both immunocompetent and immunosuppressed persons as a function of time since onset of illness . a retrospective chart review was conducted for all patients who had coccidioidomycosis diagnosed between january 1 , 1999 , and october 31 , 2003 , at our tertiary - care academic medical institution . patients were identified by an institutional computer search using icd-9 ( international classification of diseases , 9th revision ) diagnosis codes and by a manual search of the reports made to the arizona department of health services . patients were included only if their records contained adequate documentation of symptoms , laboratory and microbiologic reports , and pathologic findings of coccidioidomycosis . abstracted information included age , sex , race , comorbid conditions , dates and results of serologic tests for coccidioidomycosis , microbiologic tests , radiographs , and histopathologic tests . abstracted information about the clinical coccidioidal syndrome included symptoms , location of infection , treatment , and follow - up . a patient was defined as having confirmed coccidioidomycosis if c. immitis was isolated from the culture of any specimen or if any histopathologic test revealed spherules of coccidioides spp . probable coccidioidomycosis was diagnosed when a patient had compatible symptoms ( e.g. , fever , cough , headache , rash , myalgia , or arthralgia ) in association with compatible radiographic changes and positive serologic findings . intrathoracic coccidioidomycosis infections involved any of the following tissues : lung , pleura , chest wall , or pericardium . disseminated coccidioidomycosis required a positive culture or positive histopathologic finding from a specimen outside the thoracic cavity . a patient was considered to be immunocompromised if any of the following comorbid conditions was present : infection with the human immunodeficiency virus , solid organ or hematologic transplant , hematologic malignancy , or immunosuppression resulting from treatment with corticosteroids , chemotherapy , or other immunosuppressant medications . our local laboratory performed the eia to detect igm and igg antibodies using a kit from meridian bioscience , inc ( cincinnati , ohio ) . positive , indeterminate , and negative results were defined according to the manufacturer s instructions ( negative = absorbance value<0.150 ; indeterminate = absorbance value 0.150 but 0.199 ; or positive = absorbance value 0.200 ) . serum was also sent to the mayo clinic infectious diseases serology laboratory , rochester , minnesota , to perform the cf and i d tests . the laboratory branch cf test of the centers for disease control and prevention was used to detect igg antibodies ; it has been described previously . as part of this protocol the antigen for the cf test was obtained from dr d. pappagianis at the university of california at davis . between january 1 , 1999 , and may 30 , 2002 , the i d test was performed using a kit from gibson laboratories , inc ( lexington , kentucky ) that used antigen f to detect igg antibodies . beginning in june 2002 , the i d test was performed using a test kit ( meridian bioscience , inc ) to detect both tp ( early igm ) antibodies and f ( late igg ) antibodies . to consistently pool the pre- and post-2002 i d results , we excluded the i d igm results obtained after june 2002 from this analysis . occasionally , serum samples were sent to the external laboratory of d. pappagianis for serologic evaluation and confirmation of results . if a patient manifested an anticomplementary cf result , the cf test was excluded , although concurrent i d and eia results were still included in the analysis . on occasion , an indeterminate eia result was identified and counted as negative . to minimize potential false - positive results , we counted a positive eia igm as seropositive only in the presence of another positive result ( either eia igg , i d , cf , or positive microbiologic or histopathologic findings ) . each serologic result was entered as positive or negative in 1 of many time intervals from the onset of symptoms ( weeks 1 through 8 , and months 2 through 12 ) . individual patients were counted only once in any particular interval and were considered positive for that interval if any serum sample was positive during that period . the percentage of positive assays for each period was calculated from the number of positive samples divided by the number of samples submitted at each interval . demographic characteristics were compared between immunocompromised and nonimmunocompromised patients using a test for categorical variables and a 2-sample t test for continuous variables . categorical variables are summarized as number ( percentage ; 95% normal binomial confidence interval ) ; continuous variables are presented as meansd and as median ( range ) . in all cases , most of the curves for the qualitative eia and i d were generated by numerous patients tested in 1 or 2 time periods , and a smaller number of patients had multiple studies performed during the course of a year . the cf curve is a composite of patients receiving serial determinations of this assay to assess for serologic response to infection and treatment . a retrospective chart review was conducted for all patients who had coccidioidomycosis diagnosed between january 1 , 1999 , and october 31 , 2003 , at our tertiary - care academic medical institution . patients were identified by an institutional computer search using icd-9 ( international classification of diseases , 9th revision ) diagnosis codes and by a manual search of the reports made to the arizona department of health services . patients were included only if their records contained adequate documentation of symptoms , laboratory and microbiologic reports , and pathologic findings of coccidioidomycosis . abstracted information included age , sex , race , comorbid conditions , dates and results of serologic tests for coccidioidomycosis , microbiologic tests , radiographs , and histopathologic tests . abstracted information about the clinical coccidioidal syndrome included symptoms , location of infection , treatment , and follow - up . a patient was defined as having confirmed coccidioidomycosis if c. immitis was isolated from the culture of any specimen or if any histopathologic test revealed spherules of coccidioides spp . probable coccidioidomycosis was diagnosed when a patient had compatible symptoms ( e.g. , fever , cough , headache , rash , myalgia , or arthralgia ) in association with compatible radiographic changes and positive serologic findings . intrathoracic coccidioidomycosis infections involved any of the following tissues : lung , pleura , chest wall , or pericardium . disseminated coccidioidomycosis required a positive culture or positive histopathologic finding from a specimen outside the thoracic cavity . a patient was considered to be immunocompromised if any of the following comorbid conditions was present : infection with the human immunodeficiency virus , solid organ or hematologic transplant , hematologic malignancy , or immunosuppression resulting from treatment with corticosteroids , chemotherapy , or other immunosuppressant medications . our local laboratory performed the eia to detect igm and igg antibodies using a kit from meridian bioscience , inc ( cincinnati , ohio ) . positive , indeterminate , and negative results were defined according to the manufacturer s instructions ( negative = absorbance value<0.150 ; indeterminate = absorbance value 0.150 but 0.199 ; or positive = absorbance value 0.200 ) . serum was also sent to the mayo clinic infectious diseases serology laboratory , rochester , minnesota , to perform the cf and i d tests . the laboratory branch cf test of the centers for disease control and prevention was used to detect igg antibodies ; it has been described previously . as part of this protocol the antigen for the cf test was obtained from dr d. pappagianis at the university of california at davis . between january 1 , 1999 , and may 30 , 2002 , the i d test was performed using a kit from gibson laboratories , inc ( lexington , kentucky ) that used antigen f to detect igg antibodies . beginning in june 2002 , the i d test was performed using a test kit ( meridian bioscience , inc ) to detect both tp ( early igm ) antibodies and f ( late igg ) antibodies . to consistently pool the pre- and post-2002 i d results , we excluded the i d igm results obtained after june 2002 from this analysis . occasionally , serum samples were sent to the external laboratory of d. pappagianis for serologic evaluation and confirmation of results . if a patient manifested an anticomplementary cf result , the cf test was excluded , although concurrent i d and eia results were still included in the analysis . on occasion , an indeterminate eia result was identified and counted as negative . to minimize potential false - positive results , we counted a positive eia igm as seropositive only in the presence of another positive result ( either eia igg , i d , cf , or positive microbiologic or histopathologic findings ) . each serologic result was entered as positive or negative in 1 of many time intervals from the onset of symptoms ( weeks 1 through 8 , and months 2 through 12 ) . individual patients were counted only once in any particular interval and were considered positive for that interval if any serum sample was positive during that period . the percentage of positive assays for each period was calculated from the number of positive samples divided by the number of samples submitted at each interval . demographic characteristics were compared between immunocompromised and nonimmunocompromised patients using a test for categorical variables and a 2-sample t test for continuous variables . categorical variables are summarized as number ( percentage ; 95% normal binomial confidence interval ) ; continuous variables are presented as meansd and as median ( range ) . in all cases , most of the curves for the qualitative eia and i d were generated by numerous patients tested in 1 or 2 time periods , and a smaller number of patients had multiple studies performed during the course of a year . the cf curve is a composite of patients receiving serial determinations of this assay to assess for serologic response to infection and treatment . between january 1 , 1999 , and october 31 , 2003 , 360 patients had symptomatic , confirmed , or probable coccidioidomycosis with an identifiable onset of symptoms . these 360 patients had a total of 1,797 serologic tests performed in our laboratory , and 314 of the 360 patients had serologic studies within the first year of symptom onset . table 1characteristics of 360 immunosuppressed and immunocompetent patients with symptomatic coccidioidomycosisvariableich ( n = 62)non - ich ( n = 298)p valueno . ( % ) 95% cipatient characteristicsage , y ( mean sd)64.813.956.816.2<.001male sex39 ( 63)5175157 ( 53)4758.14white race57 ( 92)8599238 ( 80)7584.03patient medical historycancer33 ( 53)416660 ( 20)1625<.001cardiovascular disease36 ( 58)4670120 ( 40)3546.01tobacco use34 ( 55)4267156 ( 52)4758.72diabetes mellitus15 ( 24)143540 ( 13)1017.03hiv infection4 ( 6)0130 ( 0)0 ( 0)<.001rheumatologic illness13 ( 21)113120 ( 7)410<.001organ transplantation7 ( 11)3190 ( 0)0 ( 0)<.001hematologic malignancy14 ( 23)12330 ( 0)0 ( 0)<.001nonhematologic malignancy19 ( 31)194268 ( 23)1828.3coccidioidal illnesslungs only39 ( 63)5175231 ( 78)7382.02limited to thoracic cavity50 ( 81)7190267 ( 90)8693.048extrathoracic dissemination12 ( 19)102931 ( 10)714.048confirmed by positive microbiologic or histologic tests32 ( 52)396480 ( 27)2232<.001ci , confidence interval ; ich , immunocompromised ; non - ich , nonimmunocompromised ; hiv , human immunodeficiency virus . characteristics of 360 immunosuppressed and immunocompetent patients with symptomatic coccidioidomycosis ci , confidence interval ; ich , immunocompromised ; non - ich , nonimmunocompromised ; hiv , human immunodeficiency virus . the figure depicts the temporal sequence curves for eia , cf , i d , and all tests combined for these patients . table 2 summarizes the serologic results for eia , cf , and i d among the different groups of immunocompromised hosts . these results are also compared with those for 252 immunocompetent patients who were tested within the first year of symptom onset . in the immunosuppressed group , no single test was 100% sensitive for all groups , although the cf test was 100% sensitive for the other immunocompromised group ( 10 positive of 10 tested ) . this same test was not sensitive for organ transplantation ( 2 positive of 6 tested ) , and it had an overall sensitivity of only 67% ( 35 of 52 ) ( fig . 1 ) . table 2seropositivity among 62 immunocompromised hosts with serologic confirmation of coccidioidomycosis detected by various serologic testscategory of immunosuppressiontype of serologic testing , no . ( % ) eia ( igm and igg)cfid ( igm or igg or both)any testtestedpositivetestedpositivetestedpositivetestedpositivehematologic malignancy ( n = 14)124 ( 33)106 ( 60)61 ( 17)128 ( 67)cancer and chemotherapy , nonhematologic ( n = 19)1813 ( 72)1812 ( 67)159 ( 60)1918 ( 95)hiv infection ( n = 4)41 ( 25)32 ( 67)32 ( 67)43 ( 75)organ transplantation ( n = 7)75 ( 71)62 ( 33)30 ( 0)75 ( 71)rheumatologic illness ( n = 13)119 ( 82)106 ( 60)84 ( 50)1110 ( 91)other ich illness * ( n = 11)109 ( 90)1010 ( 100)86 ( 75)1010 ( 100)all patients5738 ( 67)5235 ( 67)4021 ( 53)5849 ( 84)healthy patients tested 1 y after symptom onset ( n = 261)244212 ( 87)252188 ( 75)248180 ( 73)261247 ( 95)cf , complement fixation ; eia , enzyme immunoassay ; ich , immunocompromised ; i d , immunodiffusion ; hiv , human immunodeficiency virus . * patients with other causes of immunocompromise include 3 inflammatory bowel disease ( 1 taking infliximab ) , 2 autoimmune blood dyscrasias ( hemolytic anemia and idiopathic thrombocytopenic purpura ) taking prednisone , 1 autoimmune polyneuropathy , and 5 taking corticosteroids long - term for sarcoid , cough , other pulmonary diseases ( chronic obstructive pulmonary disease , interstitial pulmonary fibrosis , or normal interstitial pneumonia). six patients have 2 immunosuppressive illnesses and are represented in each category.figure 1temporal sequence of serologic responses after onset of symptoms in immunocompromised ( ich ) versus nonimmunocompromised ( non - ich ) patients with symptomatic coccidioidomycosis . percentage of patients positive for coccidioidomycosis , a , by enzyme immunoassay for igm ; b , by enzyme immunoassay for igg ; c , by complement fixation ; d , by immunodiffusion ; and , e , by any test . seropositivity among 62 immunocompromised hosts with serologic confirmation of coccidioidomycosis detected by various serologic tests cf , complement fixation ; eia , enzyme immunoassay ; ich , immunocompromised ; i d , immunodiffusion ; hiv , human immunodeficiency virus . * patients with other causes of immunocompromise include 3 inflammatory bowel disease ( 1 taking infliximab ) , 2 autoimmune blood dyscrasias ( hemolytic anemia and idiopathic thrombocytopenic purpura ) taking prednisone , 1 autoimmune polyneuropathy , and 5 taking corticosteroids long - term for sarcoid , cough , other pulmonary diseases ( chronic obstructive pulmonary disease , interstitial pulmonary fibrosis , or normal interstitial pneumonia ) . temporal sequence of serologic responses after onset of symptoms in immunocompromised ( ich ) versus nonimmunocompromised ( non - ich ) patients with symptomatic coccidioidomycosis . percentage of patients positive for coccidioidomycosis , a , by enzyme immunoassay for igm ; b , by enzyme immunoassay for igg ; c , by complement fixation ; d , by immunodiffusion ; and , e , by any test . immune competence plays a clear role in the clinical course and control of coccidioidomycosis , initially with innate immunity and subsequently with adaptive immunity afforded by the th1-mediated immune response . numerous studies have demonstrated an increased likelihood of severe or disseminated infection among persons with compromised immunity . typical symptoms and radiographic findings are often nonspecific and resemble pulmonary infection from other organisms . many patients with coccidioidomycosis have a nonproductive cough , and sputum samples can be difficult to obtain for culture without an invasive procedure . newer diagnostic methods under development include polymerase chain reactions and assays to assess the cellular immune response to coccidioidomycosis ; the ultimate utility of these tests in the everyday diagnosis of this infection is not clear at this time . serologic tests therefore play an important role in the diagnosis and follow - up of patients with coccidioidomycosis . for decades although many assay methods have been used in the past , the most commonly used igm serologic methods now include i d and eia , whereas commonly used igg methods include i d , quantitative i d , cf , and eia . some authors have reported a good correlation of the eia with more traditional assays ; others have noted potential problems with specificity and have emphasized the need for confirmation by i d . the eia and i d assays used in our laboratories are qualitative tests , whereas the cf is a quantitative test that is useful in following the course of illness over time ; quantitative results are best interpreted when specimens from different time points are analyzed simultaneously . some reference laboratories offer a quantitative i d ( idcf ) for late igg , which also allows a quantitative result . titer results may vary from laboratory to laboratory , which can hinder interpretation ; therefore , quantitative studies performed serially are best interpreted if performed by the same laboratory . it is important to note that the sensitivity of any assay in either compromised or healthy persons is modest at best , regardless of when the patient is tested relative to the onset of symptoms . when serologic studies are so heavily relied upon to make a diagnosis , negative studies can delay diagnosis , result in the need for invasive testing , and delay initiation of therapy . since all our patients had confirmed or probable coccidioidomycosis , specificity of the test is not addressed in this study . in a few situations , the sensitivity of the eia appears to be greater than that of the cf or i d tests , raising some concern that these cases represent false - positive findings . however , when we analyzed the subgroup of patients whose diagnosis of coccidioidomycosis was confirmed with positive histopathologic or microbiologic results , the relative positivity among the various serologic methods did not change ( data not shown ) . although the magnitude of the finding is unclear , previous authors have observed a decreased serologic response in immunocompromised hosts who contract coccidioidomycosis [ 3,79,17 ] . other authors reported the ability of immunocompromised hosts to mount an adequate serologic response by cf , but the height of the titer may be blunted when compared with that of immunocompetent patients . our study evaluated 3 different methods , a locally performed eia and a cf and i d sent to an external reference laboratory . for the present study , serum samples for cf were sent to 1 of 2 different reference laboratories even for individual patients during their course of illness , which made the quantitative differences between immunocompetent and immunocompromised persons difficult to interpret ; we subsequently opted not to pursue this line of evaluation . our study summarized the seroreactivity of patients as a function of time since symptom onset and compared the results of various serologic tests between immunocompetent patients and immunosuppressed hosts . the curves for the qualitative eia and i d are composite curves generated by numerous patients tested in 1 or more time periods over the course of a year ; on the basis of our observations in this study , we agree with other authors that there is often little to be gained by repeating a positive qualitative test . the cf curve is a composite of patients receiving serial determinations of this assay to assess for a serologic response to infection or treatment . in nearly every comparison time point , immunocompetent patients had a higher overall rate of seropositivity for each of the different serologic methods but only a few data points achieved statistical significance . this finding likely reflects either no ( or small ) difference between the groups or small or heterogeneous cohorts or both . even when all the test results were combined , we did not find a significantly decreased rate of seropositivity in the immunocompromised group . in fact , there appeared to be a higher rate of seropositivity when all methods were considered than when each test was considered alone . in light of the increased sensitivity of a panel of tests compared with any single test , it may be prudent to test immunocompromised patients by multiple methods before concluding that a patient is seronegative . a long - established method for improving the yield of serologic testing has been to concentrate serum before the test , which is recommended and practiced in the 2 reference laboratories we use . subgroups of the immunocompromised patients had differences in seropositivity , ranging from those with hematologic malignancy ( overall seropositivity , 69% ) to a 100% seropositive rate in the miscellaneous group of immunosuppressed patients . this observation most likely reflects inherent differences in the immune capabilities of each group , but the groups were too small to enable us to draw definitive conclusions . this study did not attempt to correlate the laboratory results to the clinical course of patients . many patients were evaluated by numerous physicians , and the number and choices of serologic studies were those deemed appropriate on a case - by - case basis . the time of the patients initial evaluation at our medical center in relation to the onset of their symptoms varied from a few days to several months . serum samples were taken at various times during the patient s illness , and there was no uniformity in the types of serologic tests ordered by different physicians . therefore , each point in the time line may represent a different cohort of patients and simply reflect the positivity of the test at that particular time for the patients tested . a large prospective study of serologic evaluations over time would address some of these issues . the referral center bias is evident in the number of extrapulmonary infections observed in the healthy population as well as by the large percentage of biopsy- or culture - proven infections . inherent problems also exist when patients with so many different types of immunosuppressant illnesses or treatments are grouped into a single cohort . in this study , the many different illnesses and their immunosuppressant - inducing treatments included hematologic malignancies , cancer chemotherapy , organ or stem cell transplantation , human immunodeficiency virus infection , and numerous systemic illnesses ( such as connective tissue diseases ) requiring therapy with immune modifiers . these patients may have had various immune defects , some of which may or may not be important in the detection or control of coccidioidomycosis . in addition , some patients ( e.g. , organ transplant recipients ) may have varying levels of immunosuppression throughout the course of the illness , with marked immune impairment early on and less impairment over time . none of these differences was taken into account in the analysis of the immunocompromised group in this study , and these differences may be an important barrier to detection of significant differences in seroreactivity . in addition , the immunocompromised group was older and had more comorbid illness than the noncompromised cohort , factors that again impede comparison of the 2 groups . in summary , our evaluation of coccidioidal serologic tests among 360 immunocompetent and immunosuppressed patients using 1,797 serologic samples showed that , for any single serologic test , immunocompetent hosts appeared to be more likely than immunosuppressed patients to be seropositive with active coccidioidomycosis . when the test results from 3 different methods were considered as a whole , the sensitivity of the serologic evaluation increased in the immunocompromised patients , but this overall result was not statistically different for immunosuppressed versus immunocompetent persons . immunosuppressed persons have the ability to mount a serologic response to coccidioidomycosis , but multiple methods may be required to detect that seropositivity .	serologic studies are an important diagnostic tool in the clinical evaluation and follow - up of persons with coccidioidomycosis . numerous types of serologic tests are available , including immunodiffusion , enzyme immunoassay , and complement fixation . we conducted a retrospective review of the results of 1,797 serologic tests spanning 12 months from the onset of coccidioidomycosis in 298 immunocompetent and 62 immunosuppressed persons with symptomatic infection . using the onset of symptoms as a reference point , we plotted the positive or negative serologic results over time for both groups . compared with the immunocompetent group , immunosuppressed persons had lower rates of seropositivity for every type of test during the first year after onset of symptoms for coccidioidomycosis , although many results did not achieve statistical significance . combining the results of these tests increased the sensitivity of the serologic evaluation in immunocompromised patients . immunosuppressed persons have the ability to mount a serologic response to coccidioidomycosis , but in some circumstances , multiple methods may be required to improve detection .
the lumbar spine plays a key role in accommodating downward loads resulting from body weight distribution , muscle action , and external forces . the presence of imbalances in this region leads to instability , pain , and increased energy consumption while performing functional activities1,2,3 . the lumbar fascial system supports and connects all tissues and harmonizes and organizes muscular and gravitational forces in the region4 . in the case of fascial retraction , fascial tensions occur along with abnormal changes in tissue mobility and flexibility , which consequently leads to deterioration of movement . the fibrous connective tissue in this region has numerous mechanoreceptors that are occasionally activated to transmit sensitive and short - lived impulses . if these activations are prolonged , they quickly become oversensitive and even painful . although individuals can withstand the initial painful symptoms , the condition is persistent and has a significant recurrence rate5 . low back pain has a prevalence of 80% in the general population , in which 50% of those affected recover spontaneously within two weeks and 90% recover within six weeks the kinesio taping ( kt ) technique uses a tape with unique qualities , the physiological applications of which include : correcting muscle activity , improving active range of motion , improving blood and lymphatic circulation , decreasing pain by neurological suppression , and repositioning joints7,8,9 . the therapy done with kt using the fascial correction technique described by kenzo kase aims create and/or direct fascia movement in order to guide it along a desired direction or alignment7 . therefore , it is intended to free the fascia of any movement limitations through movement of the skin relative to target muscles by means of the mechanical tension generated by and elastic kt10 . the tension imposed from this material , which deforms the fascia , stretches the bonds between molecules , promoting a gentle flow of electrons and generating a piezoelectric charge . the charge is interpreted by cells , which causes them to respond by increasing , reducing , or changing their local intercellular elements . the fascia is a tissue that will deform plastically if stretching is applied slowly enough4 . currently , there are few studies available kt and lumbar flexibility , especially for patients with low back pain . other research with similar goals that have also used kt observed an improvement in range of motion and in pain reduction11,12,13,14,15,16 . the current work found in the literature differs from the present study with regard to the method of application and location taped , with tape application varying along the lumbar area and in some cases extending down to the posterior region of the lower extremities . the purpose of this study was to evaluate the influence of kt with a lumbar fascia application on the range of motion in forward bending of the lumbar spine . this was a longitudinal study composed of a randomized clinical trial and was conducted in the universidade estadual de gois from august to november of 2011 . it was developed upon approval by the ethics committee for research of the hospital for tropical diseases under protocol number 19/2011 . the sample consisted of 18- to 27-year - old female volunteers and physiotherapy students , who did not present any movement dysfunction and did not perform any regular physical exercises . subjects were excluded from the study if they presented a body mass index ( bmi ) greater than 29 , had musculoskeletal disorders or recently presented low back and spinal pain , were participants in any stretching program , and/or presented allergy to the taping material . all the participants provided informed consent for participation in the research before the study began . initially , the study consisted of 45 participants randomly divided into three groups ( control , kinesio without tension - kwt , and kinesio fascia correction - kfc ) and pared according to age and bmi . however , during the study , one subject was excluded because of an allergy to the kinesio taping , and five others were excluded because they did not return for the last assessments . therefore , the final sample was composed of 39 subjects allocated into three groups containing 13 individuals each . lumbar flexibility was evaluated by the schober test , marking the midpoint of the two posterior - superior iliac spines ( s2 level ) and then other two points 5 cm below and 10 cm above the initial level . the distance between the three points was measured in the standing position , and then the subject was instructed to bend forward for reassessment . the difference between the two measurements is an indication of the magnitude of flexion that occurs at the low back and should be at least five centimeters for lumbar mobility to be considered normal17 . we also analyzed the fingertip - to - floor distance , which consisted of measuring the distance from the distal end of the third finger to the ground when the patient bent the trunk forward . the subject was considered within normal flexibility parameters for this test when the third finger was able to touch the ground18 . the original bandage brand indicated by kinesio taping association international ( ktai ) , kinesio tex gold 5 cm wide , was used to perform the kt technique . i strips while the subjects were in position of maximum forward bending of the spine . this application was performed simply by applying the tape to the skin with 0% tension . the kfc group received the same cut of tape and used the same application position as the kwt group , but the researcher applied the kinesio taping fascia correction technique10 by utilizing short and long oscillations in order to apply a varied amount of tension of 15% to 50% . the same procedure was used for application of the second tape , as shown in fig . kinesio taping application for fascia correction the kwt and kfc subjects left the kinesio tape in place for 48 hours and were reassessed at 24 hours with the tape on , 48 hours with the tape on , and 30 days after tape removal . data were analyzed with the statistical package for the social sciences ( spss ) software , version 15.0 . to verify the normality of the distribution of scalar variables , the shapiro - wilk test was utilized , and for all the variables that had a normal distribution , a parametric test was applied . only the value of bmi showed an abnormal distribution ( p = 0.01 ) , and to check the difference between the average positions of bmi groups , the kruskal - wallis test was used the mann - whitney test with bonferroni correction significance was applied for the differences between pairs . analysis of variance ( anova ) was used to assess the independent sample and found the mean age between the groups . inferential statistics were utilized to compare the means of the groups by means of anova for repeated measures , which compared lumbar flexibility before and after application of kt , as well as the means of the control , kfc , and kst groups . all the tests utilized in this research used a value of 0.05 for statistical significance and a confidence interval of 95% . the mean age of the sample analyzed was 21.23 2.07 years , with the minimum being 18 years and the maximum being 27 years . from comparison of the mean age between the groups , it was concluded that there were no significant differences regarding the mean age of the subjects in each group ( p = 0.169 ) , thus showing age homogeneity in the whole sample . when analyzing bmi , the average was found to be 20.71 1.83 kg / m , with the minimum and maximum values being 17.21 kg / m and 25.81 kg / m . the mann - whitney test with bonferroni correction showed no homogeneity of bmi in the sample and subsequent significant differences between the kwt and kfc groups as result of the randomization process . because the sample was only composed of females , it differs from other studies that also included males . although subjects were compared with in these other studies , it is not ideal to have a mixed sample , since there are differences in flexibility between men and women19 . the average schober test result for all participants , before application of kt , was 6.07 1.2 cm . , there was a reduction in the average schober test result in both the kwt group and the kfc group , as illustrated in table 1 . after 30 days , there was an increase in the averages in all three groups . no statistically significant differences were found between the averages obtained from the schober test before applying the kt , 48 hours with the tape on , and 30 days after tape removal , in any of the groups . the average reduction after application of kt may have occurred as a result of the initial restriction to the lumbar joints due to the tape s elastic resistance . it can be inferred that the augmented flexibility witnessed 30 days after kt removal was due to possible fascia mobilization as well as improvement of local blood flow and lymphatic circulation . this was a sample of young , healthy subjects with normal bmi values and no lumbar issues such characteristics may explain the lack of statistically significant results . therefore , it is necessary to conduct a study with subjects presenting actual dysfunctions and abnormal situations . the results of the present study corroborate those obtained by salvat and salvat ( 2010 ) , who concluded that there was no significant increase in lumbar mobility , since a gain in range of motion as a result of reduction of the coxofemoral angle indicates improved flexibility of the posterior muscles of the lower extremities13 . according to the averages obtained in the control group , as shown in table 1table 1.average of obtained values in centimeters for the schober s test in each grouptime / groupcontrolkwtkfcbefore5.88 3.26.51 5.15.90 5.324 hours5.89 5.16.15 2.55.71 3.748 hours5.833.35.99 2.45.70 3.330 days6.374.26.63 3.16.22 4 , there were no significant gains in flexibility , since these individuals did not receive any taping . table 2table 2.average values for fingertip - to - floor distance ( ffd)groupffd beforeffd at 48 hwith tape onffd at 30 daysafter tape removalcontrol group16.73 2.6316.54 2.7815.85 2.79kinesio without tension ( kwt)22.37 2.6313.88 1.66 * 15.53 1.99kinesio fascia correction ( kfc)17.05 1.8911.74 2.14 * 13.51 1.99*p<0.05shows the results for the kwt group , and illustrates that a significantly different mean , 16.5 cm was obtained 24 hours with the tape on . furthermore , reassessment at 48 hours with the tape on showed improvement of flexibility , with the average distance being 13.88 cm . thus , it can be inferred that the participants showed an average increase of 8.49 cm 48 hours when utilizing kt without tension , which make the results even more relevant . thirty days after tape removal , the participants lost some of the acquired flexibility ( 1.65 cm ) . however , the final average ( 15.53 cm ) remained below the baseline ( 22.37 cm ) value . it should be noted that no significant differences were observed between flexibility values before application of kt and 30 days after its removal . applying kt without tension is a new modality that makes use of the kinesio taping method called no tension taping . when applying the elastic fabric over stretched skin , there is a retraction response from the kinesio tex tape itself , which is accompanied by the return of elasticity generated by the skin . this retraction is generally used with tensions of between 10% and 25% ; however , as shown at the international symposium for research in kinesio taping in japan in 2012 , retraction is present even at 0% applied tension . this kt retraction triggers a series of convolutions that provide improved blood and lymphatic fluid circulation as well as decompressions of the various local mechanoreceptors . the results in table 2 show that the average fingertip - to - floor distance before applying kfc group was 17.05 cm . when reassessed 24 hours with the tape on , there was increased flexibility ; however , its significance was 0.059 . after 48 hours with the tape on , there was an improvement in flexibility , as the average was 11.74 cm . at 30 days after tape removal , the average was 13.51 cm . like the kwt group , the kfc group demonstrated significant differences between the values obtained before applying kt and 48 hours with the tape on . the difference between before kt and after 48 hours was 5.31 cm . during the period of one month without the kt , the individuals lost about 1.77 cm of the flexibility that they had obtained . there were no significant differences between the values obtained before applying kt and those at 30 days after tape removal . the results also show that there were no statistical differences between the kfc and kwt groups . in other words , both groups showed improvement in lower limb flexibility during the same time interval . in a similar study , an improvement in flexibility of 2.15 2.30 was observed and found to be significant12 . however , this result was inferior to that obtained in the present study in the kwt and kfc groups . to compare the averages of the two studies , two factors should be considered : the time the tape stayed on the skin and width of the kt that was used . the authors of the aforementioned study used a thinner tape width ( 2.5 cm ) than in the present study ( 5 cm ) , which consequently would trigger less local mechanical and sensorial effects , and the tape application remained on the skin for only 30 minutes . these two associated factors provide a less than optimal effect than the potential of kt , as perceived in the present study , where the peak of improvement in flexibility occurred at 48 hours with the tape on . considering the aspects mentioned above related to the previous study and also considering the assessments made before and after the tape applications and the lack of regard for chronic influences on flexibility , the gains mentioned in the previous research may have been influenced by the fact that subjects remained in an elongated posture while application of kt was conducted , which could have contributed to a false - positive effect on the results . furthermore , the improvements in flexibility may have been attributed to the repeated motions performed during the sit and reach tests , which promote a reduction in connective tissue resistance that , when combined with stretching , may serve as an elasticity intensifier in the muscle - tendon unit20 . ebber and pijnappel ( 2006 ) measured the range of motion with the sit and reach test . in their study , this test was done before applying the taping technique , immediately after its application , and after spending three days with the tape on . cut form of application , and the tape was initially laid out on the lumbar region with subsequent bilateral positioning of its tails along the path of the sciatic nerve in 99 subjects healthy ; however , they did not mention how much tension was applied . the effect of kt in the lower extremities , in both the experiment mentioned above and in the present study , can be justified by the formation of convolutions in the skin of the lumbar region after tape application . these in turn allow for the reestablishment of fluid flow and stimulation of the sensorimotor system4 , 7 , just as mobilization of the tissue produces distal structural effects , since dermatomes of the lumbar and sacral region extend over to the lower extremities21 . by verifying the influence of kt in the lower extremities , it was deemed unnecessary to apply it to the entire lower limb , since application along the trajectory of the sciatic nerve results in values close to those obtained in this study11 . we verified that the values for flexibility of the lower extremities were more significant in both the kwt and kfc groups at 48 hours with the tape on . this implies that the connective tissue does not respond immediately to external stimuli , and as they become more sustainable and repetitive , structural changes occur . on the other hand , excitable tissues differ in that they respond quickly to an external mechanical stimulus and allow for reduction of pain and increased stretching tolerance21 , 22 . kt , like massage therapy , has an inhibitory effect on the positive reflex amplitude that lasts only during application of the technique21 . when either the stimulus to the mechanoreceptors or the tension to the tissue is discontinued , all aspects of the region return to their baseline values , as observed in the present study during the reassessment at 30 days after tape removal . in general , it can be observed that the control group showed no significant improvement in flexibility and that the kwt group presented better results than the kfc group . such results can be attributed to the method of application and the tension used during the procedure . in the kwt group , the tape was applied without any tension , which allowed the formation of more convolutions in the skin and consequently increased blood and lymphatic flow while at the same time reducing pressure on mechanoreceptors . during the fascial correction technique in the kfc group , the tension applied in kt varied from 15% ( mild ) to 50% ( moderate ) , which somewhat limited the formation of convolutions and hence resulted in a lesser effect than that observed in the kwt group . from 0 to 100% tension can be applied in the kinesio taping method ; for muscle purposes , light tensions is used to generate only sensitive reactions , and moderate to high tension is used to generate more mechanical functions24 . so the above results may include some mechanical responses that could be responsible for the limited flexibility found in the kfc group . furthermore , we emphasize the importance of proper application by an experienced professional in order to allow for frequent and rhythmic motion . having experience and mastery of the technique during application reduces the chances of applying tensions higher than 50% , which cause undesired effects . another important factor to be considered is related to the amount of time that the tape stays on the skin . since the goal in the present study was to mobilize the superficial fascia , the technique required a longer period of time to promote mobilization of this connective tissue , which happens to be time dependent . as this study was performed with female subjects , the menstrual cycle must be considered because it coincides with significant elevations in estradiol levels23 . the kinesio taping method is a promising tool , and its use is expanding because of various therapeutic effects that have been proven through research , particularly those related to the musculoskeletal system . it is a particle technique that can be reproduced from other persons for the clinical and research porpoise . the thoracolumbar fascia is a stable structure because of the large flow of piezoelectric charges , which promote the deposition of collagen fibers and confers resistance to this tissues , and thus requires high tensions to be mobilized and stretched5 . therefore , the present study found that a fascial correction technique and application without tension promoted changes in fascia mobility , allowed for discrete gains in lumbar flexibility , and had significant influences on fingertip - to - floor distance . however , to achieve better results in lumbar fascia mobilization , the use of the kinesio tape for longer periods of time is recommended , which would allow for greater tensions on the tissues and consequently promote better plastic adaptations . it is important to consider that the lumbar fascia is not the only reason for improvement of the lumbar flexibility . since kinesio tape is an elastic bandage with moderate tension , it does not acutely trigger sufficient mechanical stress to have an impact in mobilizing the fascia . further studies are recommended with longer periods of tape application and with subjects that present actual myofascial dysfunctions in the lumbar region .	[ purpose ] the aim of this study was to evaluate the influence of a lumbar fascia kinesio taping technique forward bending range of motion . [ subjects and methods ] this was a longitudinal study with a randomized clinical trial composed of 39 subjects divided into three groups ( control , kinesio without tension - kwt , and kinesio fascia correction - kfc ) . the subjects were assessed by schober and fingertip - to - floor tests and left the tape in place for 48 hours before being reassessed 24 hours , 48 hours and 30 days after its removal . [ results ] in all three experimental groups no significant differences were observed with the schober test , but it was possible to observe an increase in lumbar flexion after 30 days . with the fingertip - to - floor distance assessment , the kfc and kwt groups showed significantly improved flexibility 24 hours and 48 hours after tape removal . [ conclusion ] the kinesio taping influenced fascia mobility , allowing for slight improvement of lumbar flexibility .
end - stage renal disease ( esrd ) is a life - changing condition that can significantly reduce both survival and quality of life ( qol ) despite renal replacement therapy with dialysis . although kidney transplantation provides the best health - related outcomes in esrd , this is not feasible for many patients due to lack of organ availability . in the absence of transplantation , the most widely prescribed dialysis modality is hemodialysis ( hd ) . for 7 decades dialysis has been the lifeline treatment for chronic kidney disease ( ckd ) for > 2 million people worldwide . nevertheless , despite technological advances in the field , hd performed in a conventional thrice - weekly regime continues to carry a sevenfold increased mortality when compared to the general population and a suboptimal qol with a high economic burden to the society.13 in contrast to native kidneys , hd is performed on an intermittent basis , leading to unphysiologic fluctuations of the internal milieu of the patient . the increased mortality observed in the dialysis population is predominantly due to an increased risk of cardiovascular events , although infectious complications also play a significant role.4,5 the increased cardiovascular risk for dialysis patients in the short term is due to hemodynamic instability with episodes of intradialytic hypotension , acute cardiac events , including myocardial infarction , arrhythmias , and myocardial stunning.612 in particular , intradialytic hypotension may even result in further complications of myocardial or cerebral ischemia , especially in patients with preexisting cardiac and/or cerebrovascular disease . in the longer term , dialysis patients suffer from left ventricular hypertrophy ( lvh ) , cardiovascular calcifications , global systolic dysfunction due to repetitive myocardial stunning , and arterial hypertension.8,13,14 impaired fluid homeostasis is also responsible for many of the cardiovascular complications associated with hd as patients have to confront with large swings in fluid overload balance . the 72-hour interdialytic interval for the thrice - weekly hd regimens also poses a threat and is associated with an increased risk of cardiovascular mortality.15 current practice entails that most hd patients receive their treatment in hospital or at a community - based satellite unit , three times a week for a minimum duration of 4 hours per session ( conventional hd ) . home - based hd , with its origins in the 1960s , rapidly waned in practice in the 1980s and 1990s but is undergoing a significant revival over the last 10 years.16 the combination of patient empowerment provided by the flexible nature of home - based hd regimes and the emergence of growing data supporting the association of such regimes with improved outcomes , such as survival , patient - reported qol , and cost effectiveness , are the primary drivers for this resurgence.1719 some of the improved outcomes associated with home hd can be linked with more intensive dialysis regimes and its prescription ( high - dose hd ) enabled by the flexibility of schedule in the home environment . charra et al20 reported a dramatic improvement in survival of dialysis patients in tassin by extending the length of hd session duration . high - dose hd refers to hd regimes that provide > 12 hours per week of hd . this could be in the form of either increased dialysis frequency ( number of treatment days per week ) and/or prolonged session duration beyond the conventional 4 hours per treatment session . there are three predominant high - dose hd schedules that are commonly identified : 1 ) short - daily hd ( sdhd ) is performed 56 days per week utilizing shorter hd treatment session duration ( 23 hours ) ; 2 ) long hd refers to prolonging the dialysis session duration ( > 4 hours , could be up to 8 hours ) ; and 3 ) extended ( long - frequent ) hd involves an increase in both frequency and length of the hd session . both long and long - frequent hd can be provided overnight while the patient sleeps ( nocturnal hd [ nhd ] ) . in some health care systems , sdhd and nhd can be delivered in the hospital , but it is ideal for high - dose hd regimes to be delivered in the home setting ( high - dose home hd ) in order to deliver maximal clinical effectiveness . the amount of dialysis delivered during conventional regime is often limited by the overall weekly treatment time and frequency . to deliver the minimum adequate dialysis , conventional regimes utilize high - efficiency dialysis over a short period of time . the resulting peaks and troughs of both hydration status and uremic solute concentrations between body compartments can lead to sharp changes in the internal milieu . more frequent and/or prolonged hd sessions can reduce the fluctuations of the internal environment by maintaining a more steady state , optimized by subtle adjustments to fluid and uremic toxin removal , and by avoidance of long interdialytic intervals . a summary of the benefits of high - dose hd is presented in table 2.22 the survival benefit for patients undergoing sdhd and nhd has been evaluated in a number of studies . however , no well - powered randomized controlled trial ( rct ) has been performed to date , and the bulk of the data remain observational . the evidence points toward a better survival for patients receiving sdhd and nhd compared to those treated with conventional regimes.2330 some studies would even suggest that the survival benefit of sdhd and nhd is comparable to that of deceased donor transplant recipients,31,32 although in the most recent retrospective cohort study of high - dose home hd patients and kidney transplant recipients , kidney transplantation was associated with superior treatment and patient survival.33 blood pressure ( bp ) control is one of the most consistent benefits of high - dose hd in randomized and nonrandomized studies.3440 one of the first reports of improved bp control by means of high - dose hd came from tassin in france where mean ambulatory bp measurements were shown to be inversely correlated with hd session length.41 sub sequently , the frequent hemodialysis network ( fhn ) study showed a significant reduction in systolic bp of 9.7 mmhg ( range from 16.9 mmhg to 2.5 mmhg ) during 12 months of follow - up among the nhd cohort , with a significant decrease in the number of antihypertensive agents used . the lack of significant difference in bp readings in patients on a conventional hd prescription ( 4 hours , three times per week ) , but performed within the home setting , illustrated the importance of high - dose hd as opposed to location of hd delivery as the main factor driving bp control.37 it is widely believed that nhd reduces bp through lowering of total peripheral resistance and plasma norepinephrine levels.39,40 sdhd can also lead to improved bp control , but the proposed mechanism of action is thought to be through improved fluid balance.36 a number of observational studies of sdhd36,42 and nhd34,36 and an rct of nhd43 have also shown that intensive hd regimes can be linked to improvements in left ventricular mass index and lvh , both factors associated with poorer outcomes in ckd and dialysis patients . these findings are further supported by a recent meta - analysis of observational studies and data from rcts that reported improvement in left ventricular mass index and geometry in both frequent and extended hd groups.44 furthermore , jefferies et al , in a cross - sectional study , demonstrated that more frequent hd regimens were associated with lower ultrafiltration volumes and rates compared with conventional in - center hd . there was an associated significant reduction in episodes of intradialytic systolic hypotension in the sdhd groups , and this was more pronounced in the nhd group . as a consequence , frequent hd regimes were associated with less dialysis - induced myocardial stunning compared with conventional hd.45 however , it is worth mentioning that the different groups in the study were not entirely homogeneous in their characteristics . although the groups were well matched for age , sex , dialysis vintage , and the prevalence of ischemic heart disease , diabetes was more prevalent in the conventional arm . in a randomized crossover study , we recently confirmed improvement of hemodynamic and cardiac stability during extended as opposed to conventional hd sessions.46 the observed hemodynamic stability during longer hd treatments involved not only measurements of peripheral systolic bp but also peripheral diastolic and central bp . cardiac output also exhibited more stability during extended hd sessions , associated with better preservation of relative blood volume during longer dialysis sessions . the cumulative evidence points toward improved management of lvh , vascular calcifications and cardiovascular outcomes , and bone mineral abnormalities by the use of extended hd and , to a lesser extent , sdhd.37,44,47 it is common for patients on long - frequent hd prescriptions to require fewer phosphate - binding medications and on some occasions also need phosphate supplementation in the dialysate.48 in the long term , improved control of hyperphosphatemia and secondary hyperparathyroidism by high - dose hd may translate into risk reduction of lvh and vascular calcification . the impact of high - dose hd on qol has been the subject of multiple studies , and overall results show an increase in kidney - specific domains of qol parameters.35,43,4952 this increase in qol with high - dose hd regimes may be due to increased autonomy and functionality , reduced tablet burden , relaxation of dietary restrictions and fluid intake , considerable reduction to the time spent in hospital and in transit to and from the hospital ( as in home hd ) , the ability to remain in employment ( productivity ) , and a reduction to uremic symptoms . high - dose hd in the home setting has been shown to be associated with mood improvement , an important domain of qol associated with improved outcomes.43 this might be due to improved sleep quality . the freedom study showed a reduction in the prevalence of restless legs syndrome from 35% to 26% after 12 months of sdhd ( p=0.05),53 while nhd has been associated with a reduction in the frequency of sleep apnea episodes.54 another important benefit of high - dose hd is its effects on fertility , particularly relevant to young ckd sufferers . there are emerging observational data from patients on high - dose hd with lower urea levels that show significantly better outcomes.55,56 these findings are also in keeping with the evidence of high - dose hd being associated with the best pregnancy outcomes in patients requiring dialysis.57,58 it is thought that the increased rates of successful conception observed in female patients of childbearing age with esrd receiving high - dose hd may partially be due to restoration of the pituitary hypothalamic axis augmented by improved solute clearance . at the same time , improved fluid balance , bp control , and hemodynamic stability in high - dose hd could positively impact on pregnancy outcomes . in the male population , high - dose hd could also improve fertility , possibly by increasing testosterone levels and decreasing hyperprolactinemia.59 the need for more frequent cannulation for frequent hd may potentially lead to increased vascular access - related complications and failure . in the fhn trial , patients on sdhd had a shorter time to first vascular access - related event compared to the conventional hd group.60,61 most of these events were vascular access interventions as opposed to losses . a similar trend was also observed in the nocturnal arm of the fhn study.41,61 in our recently performed in - depth review of all available studies looking at vascular access complications in frequent hd compared to conventional hd , we found a small but significantly increased risk of vascular access complications in frequent hd ( difference of 6.7 events per 100 patient - years , p=0.009 ) . these events included access dysfunction , access - related hospital admission , access failure , and access - related infection.62 the predominant cannulation method of arteriovenous fistulas and grafts is the rope - ladder technique . buttonhole ( constant - site ) cannulation is an alternative technique whereby needles are inserted each time at exactly the same spot using the same insertion angle and the same depth of penetration for each dialysis session . the initial cannulation is performed using sharp needles , and once a subcutaneous tract has been formed , subsequent cannulations are performed using blunt needles . buttonhole cannulation is commonly used in frequent hd , and it is often preferred by patients because it results in less pain , faster cannulation , and lower risk of hematoma.49,63,64 however , concerns have been raised recently regarding the safety of this method , especially with regard to track - related infections . several studies have indeed highlighted the increased risk of local and systemic buttonhole infections in patients on high - dose hd.49,65,66 preservation of residual kidney function ( rkf ) in dialysis patients is associated with clinical benefits.67 a significant decline in rkf is observed in the first year after dialysis initiation , especially in conventional hd patients.68 recently , the nocturnal fhn study showed a faster reduction in rkf in patients on nhd as compared to conventional hd.69 although this study had significant limitations , these findings were unexpected . the reasons for this observed phenomenon are unclear . based on the concept of organ hypoperfusion , it would have been expected that the rate of loss of residual function would have been higher in the conventional hd group . a number of mechanisms have been proposed for this loss of rkf and include reduction in osmotic load , an increased inflammatory response , and platelet activation.70 more studies are required to understand the reasons behind an accelerated loss of rkf with nhd . in general , dialysis is associated with cognitive decline , affecting domains such as executive function and attention . it could be a result of uremia or cerebral perfusion changes caused during hd . in both of these situations , the fhn study attempted to explore the impacts of intensive dialytics prescriptions but showed no benefit . interestingly , it raised concerns over both global cognition and attention in the nhd group . however , these data are exploratory , and the study was not powered to answer this question . to investigate the effects of intensive hd on cognition , appropriately powered studies are required.71 patient motivation is essential for the feasibility of both high - dose and home hd . the suitability of some vascular accesses can be an obstacle to undertaking high - dose hd , while uncontrolled mental health disease , lack of appropriate home environment , and lack of a caregiver ( when a person requires assistance with treatment delivery ) can all make home hd impossible.72 other known barriers to home hd are lack of motivation / interest , unwillingness to change , learned helplessness , fear of isolation , perceived burden on caregivers , and fear of cannulation.7375 however , these barriers can often be overcome by adequate predialysis education , motivational training of patient and caregiver , nurse - assisted cannulation , nurse - led home visits , a well - defined nursing / technical support system for patients , and provision of respite care.7678 all patients going through predialysis education clinic who are capable and willing could be offered the choice of home hd as a form of a home - based therapy ( figure 1 ) . some of these patients and those established on a dialysis modality could be targeted for consideration of high - dose home hd.79 patients with esrd with persistent extracellular fluid overload , severe hypertension and/or lvh , hemodynamic instability , resistant hyperphosphatemia as well as pregnant women or women on dialysis who wish to conceive are established target populations for consideration of high - dose home hd . sleep apnea , reduced qol , uremic symptoms , and persistent inflammation / oxidative stress are other factors that should also trigger the consideration of high - dose home hd in a dialysis patient ( suggested target population ) . in addition to these target populations , there are emerging patient groups for whom high - dose home hd may also be considered . these range from caregiver - dependent patients ( both patients and caregivers generally prefer a home - based dialysis therapy ) , crash starters , employed patients , patients with failing transplants , patients with failing pd , and morbid obese and elderly patients with esrd.80 although the list of target populations for home high - dose hd is extensive , it is by no means exclusive . high - dose hd may be considered for management of rare and challenging conditions such as severe hyperoxaluria where significant benefits of frequent nhd have been reported.81 in developed nations , home hd is generally cost - effective compared to hospital - based hd . this was recently confirmed by walker et al82 who performed a systematic review of full economic evaluations between 2003 and 2014 to analyze the cost - effectiveness of contemporary home hd modalities compared with facility hd . six such studies were identified.8388 two of them compared home nhd , one nocturnal and daily home hd , and three compared conventional home hd to facility hd . the main finding of this review was that contemporary home hd modalities including high - dose regimes such as nocturnal and daily hd are cost - neutral or cost - effective compared with conventional facility hd . a variety of reasons can explain why home hd modalities are mostly less expensive compared to in - center hd . these include reduced need for expensive hospital space , reduced need for dialysis nurses , increased employment of home dialysis patients , and reduced patient transport costs to and from the dialysis centers . we performed a preliminary economic analysis of hd modalities in the netherlands ( unpublished data 2016 ) based on a markov model , which allows the capture of all possible treatment options for people with esrd in so - called transition states , including pd , hd , and kidney transplantation ( figure 2).89 the objective was to compare the cost - effectiveness of high - dose hd ( both in - center and at home ) with conventional in - center hd . . we found that treating patients at home with conventional hd costs significantly less than treating them in - center ( 21,205 ) with an additional health benefit ( + 0.242 quality - adjusted life - years ) . when delivering high - dose dialysis at home , the added health benefit is higher ( + 0.478 quality - adjusted life - years ) for what we feel as an acceptable added cost ( + 7,795 ) . therefore , we could conclude that in the netherlands , high - dose home hd appears to be a cost - effective alternative to center - based hd . the published work in the field of home therapies and the studied subjects remain small with lack of robust trial - based evidence . further research is necessary and should be focused on addressing perceived barriers and risks to high - dose hd modalities . dialysis patients are reliant on health care service delivery and its design for their survival and qol and productivity . redesigning dialysis care to being based on the home setting could translate into healthier outcomes and more personalized care , with better self - management . convincing patients to dialyze at home a change in attitudes of health care professionals toward home - based dialysis is necessary . a lot of the shortfalls in our approaches may be due to lack of exposure and knowledge of home hd . this could be overcome by incorporating high - dose and home hd education as a core component of training curricula for medical and nursing staff . experience will indicate that once patients get started on in - center hd , the development of learned helplessness makes it more difficult for them to transfer to a home dialysis modality . investment in redesign of infrastructure is necessary from health care providers to create a dedicated environment preferably outside the hospital setting to deliver home hd training , support , education , and clinics , as well as facilities for respite care , rehabilitation , and social and psychological support . an area for reevaluating our current practices might be that of transition from predialysis to initiation of dialysis in the home setting . as we previously discussed , although high - dose home hd may be the most suited strategy for maintaining functionality in the first year after initiation of treatment , mainly by reducing dialysis - related complications,90,91 it has also been linked with accelerated loss of rkf , increased risk of vascular access complication , and cognitive decline.4,5,71,92,93 with this in mind , incremental home hd is a growing practice and reflects the flexibility offered by home therapies . these principles behind incremental dialysis involve initiation of hd two or four times a week for 3 hours ( to eliminate the long break ) and subsequently titrating dialysis duration and frequency based on clinical parameters and individual preferences . clinical parameters to assess the required dialysis dose may include the amount of residual renal function , metabolic control ( acidosis , hyperkalemia , hyperphosphatemia , secondary hyperparathyroidism ) , bp regulation , and volume status . subclinical parameters to evaluate whether an increase in dialysis dose may be required may include various tools such as body composition and hydration status ( bio - impedance ) , presence of myocardial stunning ( echocardiography or positron electron tomography ) , assessment of physical activity ( questionnaires , grip strengths , walking speed , sensewear device ) , levels of nontraditional toxins ( fgf-23 , protein - bound uremic toxins , dicarbonyl stress , and advanced glycation end - products ) , as well as polysomnography and qol questionnaires . recently , kalantar - zadeh et al94 studied the effect of incremental hd using an initiation regime of twice - weekly treatments instead of thrice weekly . the objective of their approach was to delay hd - associated loss of residual renal function by reducing the treatment frequency at the time of dialysis initiation . in this study , the clinical factors that would influence transition to more frequent regimes were rkf , volume status , cardiovascular symptoms , body size , potassium and phosphorus levels , comorbid conditions , hospitalizations , and health - related qol . the cutoff for transition from twice - weekly to thrice - weekly hd was a reduction in urine output below 0.5 l per day or if the nutritional status or general health condition of the patient was to show a deteriorating trend over time . however , almost all patients starting dialysis based on clinical grounds , as suggested by the ideal trial , would by these criteria fail to qualify as suitable candidates to start on twice - weekly hd.95 the potential drawbacks of twice - weekly regimen can be significant due to further prolongation of interdialytic intervals , which has been strongly associated with cardiovascular morbidity and mortality.15,96 furthermore , addressing transition from twice- to thrice - weekly regimens as proposed93,94 can be difficult from a patient perspective , potentially leading to suboptimal outcomes.96,97 the recommendation of a minimum of thrice - weekly dialysis was recently supported by hakim and saha98 in their review of the effects of dialysis frequency versus dialysis time . the authors also suggest a minimum initial dialysis duration of 4 hours per session , which later could be increased to 6 hours or even 8 hours if clinical and/or subclinical parameters dictate . the debate over the optimal dialysis dosing at the time of transition from predialysis to dialysis and the long - term dose adjustment according to the patient s individual need is still at large , and further studies are required to inform our future strategies . therefore it is our view that for the majority of patients , the minimum dialysis frequency should be thrice weekly until further evidence of timely initiation of twice - weekly dialysis regimen and its outcomes have been studied . following their findings , kalantar - zadeh et al94 proposed a series of rcts of incremental versus thrice - weekly hd to further describe the relation between dialysis dosing , frequency , rkf , and patient - related outcomes . high - dose and home hd extends this debate further and provides an opportunity to shift the paradigm from adequate to optimum dialysis .	despite technological advances in renal replacement therapy , the preservation of health and quality of life for individuals on dialysis still remains a challenge . the high morbidity and mortality in dialysis warrant further research and insight into the clinical domains of the technique and practice of this therapy . in the last 20 years , the focus of development in the field of hemodialysis ( hd ) has centered around adequate removal of urea and other associated toxins . high - dose hd offers an opportunity to improve mortality , morbidity , and quality of life of patients with end - stage kidney disease . however , the uptake of this modality is low , and the risk associated with the therapy is not fully understood . recent studies have highlighted the evidence base and improved our understanding of this technique of dialysis . this article provides a review of high - dose and home hd , its clinical impact on patient outcome , and the controversies that exist .
essential thrombocythemia ( et ) , an acquired myeloproliferative neoplasm , is characterized by excessive proliferation of megakaryocytes accompanied by persistent peripheral thrombocytosis . . an increased number of circulating platelets is known to increase the risk of progression of these complications , as well as predispose the subject to thrombotic events . the main treatment strategy for et is to rapidly decrease the patient 's platelet count to a normal level in order to prevent the development of et - related complications . in addition , ideal therapeutic agents should have limited side effects and minimal long - term safety concerns . a number of therapeutic agents have been studied for the treatment of et , including alkylating agents , hydroxyurea , and interferon- [ 2 , 3 ] . anagrelide is an oral imidazoquinazoline agent with a selective effect on the megakaryocyte cell lineage through the inhibition of cyclic - amp phosphodiesterase iii activity , which inhibits platelet aggregation [ 4 , 5 ] . however , it can also directly induce vasospasm of the coronary arteries , and can lead to other serious cardiovascular side effects , including congestive heart failure , arrhythmias , and acute coronary syndrome . acute coronary syndromes are reported in 1 - 5% of patients treated with anagrelide , although the overall incidence of acute coronary syndromes is very rare [ 4 , 7 - 9 ] . here , we describe the case of a 30-year - old woman with et who developed an acute , non - st - segment elevation myocardial infarction ( mi ) with severe chest pain following the use of anagrelide . this patient had no cardiovascular risk factors , suggesting that anagrelide may have been the cause of her acute mi . a 30-year - old female patient presented to the emergency department with dyspnea on exertion and severe left anterior chest pain that had begun about 30 minutes prior to arrival , and which increased in intensity over time . she reported a history of exertional chest pain that had begun 10 days prior to her presentation . previous studies , including electrocardiography ( ecg ) , cardiac biomarkers , echocardiography , and an exercise treadmill test had shown no abnormalities . the patient had no smoking history and had no cardiovascular risk factors such as hypertension , dyslipidemia , or diabetes mellitus . the patient had been diagnosed with et 6 months previously at a workplace health screening , and was being treated with 100 mg aspirin , 500 mg hydroxyurea twice a day , and 1 mg anagrelide three times daily . her et was relatively well controlled without any other complications , and her platelet counts had been within the normal range . initial vital signs were stable , with a blood pressure of 110/80 mmhg , a heart rate of 78 beats / min , and a respiratory rate of 18 breaths / min . s1 and s2 were normal and s4 was faintly audible at the apex . an ecg showed t - wave inversions in leads i , avl , and in v2 through v5 , which were consistent with myocardial ischemia ( fig . the mb fraction of creatinine phosphokinase ( ck - mb ) was 8.5 ng / ml and cardiac troponin i was 3.08 ng / ml ( upper normal limit , 0.07 ng / ml ) . b - type natriuretic peptide ( 100 pg / ml ) was also slightly elevated . a complete blood count showed the following : a leukocyte count of 3.7010/l , hemoglobin of 10.5 g / dl , and a platelet count of 38810/l . an echocardiograph revealed hypokinesis in the anteroseptal wall and a left ventricular ejection fraction of 55% . immediately , the patient was treated with anti - platelet therapy ( 300 mg aspirin and 600 mg clopidogrel ) . in addition , according to the early invasive strategy , coronary angiography was performed , which revealed significant segmental stenosis ( 90% ) of the mid left anterior descending artery ( lad ) ( fig . the segmental lesion in the mid lad did not respond to intracoronary nitroglycerin injection , which excluded coronary spasms as the causative factor ( fig . scan showed a focal soft plaque with a ruptured cap and thrombus formation ( fig . 2c ) . after coronary angioplasty , the chest pain and t - wave inversions on ecg disappeared . anagrelide was discontinued , anti - platelet and anti - anginal agents were added to the treatment , and the patient was discharged without further cardiac symptoms . if an effective cytoreductive therapy is not introduced , acute coronary syndromes resulting from coronary artery involvement may develop in patients with et . however , case reports of mis in well - controlled cases of et are rare , and most reports are of elderly subjects with various cardiovascular risk factors [ 7 , 10 , 11 ] . our patient was a young woman with no smoking history and without hypertension , dyslipidemia , diabetes mellitus , or obesity . although the patient had an underlying disease , et , the disease was well controlled and she had a normal platelet count . considering that the risk of thrombotic events decreases in patients with et who have lower platelet counts , we tentatively suggest the possibility that anagrelide was the cause of the acute mi in this patient . the most common side effects of anagrelide are headache , nausea , diarrhea , edema , palpitations , and an increased number of myocardial contractions [ 4 , 9 ] . potential cardiovascular adverse effects include vasospasm of the coronary arteries , congestive heart failure , arrhythmia , or acute coronary syndrome [ 4 , 6 , 8 ] . in addition , myocarditis , silent mi , and myocardial ischemia have been reported in young patients with et and without cardiovascular risk factors following the administration of anagrelide . although a clear correlation between the administration of anagrelide and these events has not been well established , studies demonstrate that the adverse events disappear after anagrelide is discontinued . in this patient , the segmental stenotic lesion in the mid lad did not respond to an intracoronary nitroglyceride injection ; thus , we can exclude the possibility of a vasospasm . according to the patient 's coronary angiography and ivus , a stenotic lesion within the mid lad resulted from a plaque rupture followed by thrombus formation . furthermore , after percutaneous coronary intervention with stent insertion , the patient 's chest pain and t - wave inversions on ecg disappeared . although mi is a rare event among et patients treated with anagrelide , it may be an important and undesirable effect of anagrelide . the mechanism of acute coronary syndrome in patients treated with anagrelide has been reported to result from vasospasms of the coronary arteries , although this could not be fully elucidated in the present case . close monitoring and prompt reporting of adverse events in clinical practice are important , as they may provide clues as to the mechanism of these undesirable side effects . it has been suggested that the majority of the side effects of anagrelide might develop within 1 month of starting therapy , and that the side effects are dose - dependent [ 12 - 14 ] . although the reduction of side effects might be related to improved tolerance to treatment over time , anagrelide should be discontinued in patients who experience life - threatening adverse events , such as acute coronary syndromes [ 4 , 8 , 9 ] . we treated an acute mi in a young female patient with et who had no cardiovascular risk factors and no history of thrombotic events prior to taking anagrelide . anagrelide is a useful drug for the treatment of et ; however , through this case we emphasize the importance of monitoring cardiovascular function both before starting therapy and during the maintenance of treatment . for young patients with et who have a long life expectancy , the long - term benefits from discontinuing anagrelide should be weighed against the potential cardiovascular risks posed by uncontrolled et . finally , it is probable that combining anagrelide with anti - platelet drugs in patients with et might be useful , as this might allow for a reduction in the dose of anagrelide , which would result in fewer side effects . for improved protection against acute coronary syndrome , it is imperative that anti - platelet drugs are added to anagrelide regimens in patients with et who have cardiovascular risk factors .	essential thrombocythemia ( et ) is a chronic myeloproliferative disorder with a prolonged clinical course . since this disorder is considered to be at increased risk of thromboembolism , therapy is mainly focused on the decreased risk of thrombohemorrhagic events by use of cytotoxic agents . anagrelide is a phosphodiesterase iii inhibitor which is utilized in the treatment of et for the reduction of platelets . however , patients treated with anagrelide might experience cardiovascular adverse effects including myocardial infarction ( mi ) , although these events are rare . herein , we report a case of a 30-year - old female with well controlled et by anagrelide , who eventually developed an acute non - st elevation myocardial infarction ( mi ) . there has no found any cardiovascular risk factors in this et patient , strongly suggesting that anagrelide might be the cause of mi . therefore , cardiovascular function should be monitored in those patients prescribed with anagrelide .
the past three decades of studies have unveiled some of the genetic underpinnings of human disease . for complex diseases , those with obscure genetic roots , discoveries have accelerated recently owing to a bloom of genome - wide association studies ( gwass ) . nevertheless , even for the most successful cases ( such as inflammatory and ulcerative bowel disease ) , discoveries account for only a fraction , often small , of the disease 's heritability . these yet to be discovered genetic variants comprise the ' missing heritability ' or the genetic ' dark matter ' for disease . heritability , the proportion of trait variability explained by genetic factors , has two somewhat different meanings . if genetic variation were all to act additively , the best predictor of an offspring 's trait value would be the average of his / her parents ' values . hunting for dark matter for a trait such as human height will be more straightforward than for a disease such as schizophrenia , for which the evidence for substantial gene - gene interaction is compelling . yet when researchers refer to heritability of human height , they implicitly mean narrow - sense heritability ; for schizophrenia , it is heritability in a much broader sense . greater understanding of the genetics equals greater understanding of molecular etiology and , with it , eventually more cogent treatments . however , the origins of some human diseases , especially those of the mind , can be mysterious . for diseases of the mind , few environmental or genetic risk factors are understood ; instead the hope is that identified genetic factors will lead to a subtler understanding of why diseases such as schizophrenia arise and how they can be treated effectively . even for cardiovascular disease , for which environmental risk factors are well characterized , new insights into its genetics could produce more targeted treatment this leads to the other expectation - that greater genetic knowledge will pave the way for ' personalized ' medicine . the rapid technological advances in genomics will soon make it feasible to sequence whole genomes at relatively low cost . the idea that each individual will have meaningful sequence variation in their medical records and will have interventions tailored to their risk profile and likely treatment response is quite appealing . the goal of personalized medicine , however , is hindered because so much molecular etiology remains in the dark . one way to explain more of the dark matter is to develop more efficient ways to use existing data . they apply these ideas to a gwas of obesity , as measured by body mass index ( bmi ) . studies estimate bmi 's heritability at 45 to 85% , but identified genetic variants explain about 1% of the total variance . to discover more variants , the authors examined gene expression of adipose tissue in a sample of monozygotic ( mz ) twins discordant for bmi and in a sample of unrelated individuals . because mz twins are genetically identical , or nearly so , the authors reasoned that genes showing expression differences between twins are ' reactive ' genes with differences that are due to regulatory or epigenetic changes in response to environmental factors . by contrast , genes uncovered in unrelated individuals are a combination of reactive and genetically ' causal ' genes . by contrasting results from the unrelated sample and discordant mz twins , the authors identified 27 causal genes that were differentially regulated . they then tested 197 single nucleotide polymorphisms ( snps ) falling in and around these genes in a sample of 21,000 subjects . they discovered a significant excess of small p - values in this set of snps . neither the set of snps defined by reactive genes nor the individual snps in the reactive set were associated with bmi . notably , this work identifies a new gene , f13a1 , which encodes the coagulation factor xiii a chain , with variation that affects bmi . this gene has also been identified by meta - analysis of 12 studies of venous thromboembolism . obesity is well known to predispose to vein thromboses ; however , the study of naukkarinen et al . reveals a potential biological pathway for the relationship between obesity , thrombosis and cardiovascular risk . the methods advanced by naukkarinen and colleagues require discordant mz twins , which were available for bmi . this experimental design could prove highly informative for similar quantitative traits , for which extremes are easily identified and by which the pathology or phenotype of interest is defined . for some diseases , especially diseases of the brain , quantitative traits that map precisely onto risk are not yet available . in addition , because reactive genes are environment - dependent , successful implementation of this design might require a sample exposed to a homogeneous environment , limiting its generality . regardless , this study shows how innovative research can cast more light on dark matter . moreover , the study design could also inform us about pathways of correlated gene expression and how much these correlations are influenced by genetic and environmental variation . many other methods and designs are available to illuminate dark matter [ 8 - 15 ] . one appealing approach teams gene - expression results with genome - wide association data to produce targeted hypothesis tests . one possibility is to organize tests by expression quantitative trait loci affecting genes in pathways meaningful for the disease . statistical methods for targeted testing are available , whether on the basis of prior information of the likelihood of an association between a snp and the phenotype or on the basis of plausible disease pathways . genetic variants with parental origin effects , or whose effects depend on the parent from whom they were inherited , could be part of the dark matter ; methods are now available to determine the parental origin of alleles and haplotypes even in the absence of genotyped parents . studies of copy number variants and their inheritance in families could also reveal insight into plausible biological pathways for disease . it is also safe to say that rare variants account for some of the dark matter , possibly the majority of it in some cases . next - generation sequencing promises to fill some of our void in knowledge by identifying more penetrant but rarer variants . let 's reconsider human height . we know numerous rare variants and about 50 common variants that have an impact on height . thus far , known genetics account for roughly 5% of the variance . using many snps from gwas analysis that are not significantly associated with height , yang et al . estimated the proportion of variance in height explained by snps as 0.45 and even got close to the heritability estimate of 0.84 after correcting for incomplete linkage disequilibrium between snps genotyped and causal variants . in spirit , this approach is similar to the allele score method , which seeks a predictive model for disease status on the basis of thousands of snps with modest evidence for association . if their results are correct , both studies suggest that the effects of snps are small and will be difficult or impossible to detect from simple analyses of gwass , at least for current sample sizes . these intriguing approaches have some drawbacks : they shed no new light on the molecular etiology of phenotype ; and inherent in the calculations are assumptions that could prove difficult to validate . there are other complex pathways for the transmission of a phenotype across generations without the transmission of a specific common or rare variant , namely through epigenetic factors that can result in the inheritance of gene expression patterns without an alteration of the dna sequence . gene - environment interactions could also affect the estimates of heritability and when they are in play , they can explain as much of the variance in the phenotype as genetic factors . concerted effort will almost surely be required to understand the genetic architecture of most complex diseases . 's novel study design illustrates the impact that concerted effort can have in advancing our knowledge of the genetic etiology of such diseases . there remains ample room for novel analytic methods and study designs to shed light on the genetic dark matter of disease . it is entirely possible , 10 years hence , that we will realize that much of the missing heritability was hiding in plain sight in common variants . gwas : genome - wide association study ; mz : monozygotic ; snp : single nucleotide polymorphism bd is associate professor of psychiatry and human genetics , university of pittsburgh school of medicine , pittsburgh . nm is assistant professor of psychiatry , university of pittsburgh school of medicine , pittsburgh . this work was supported by r01 grants ( mh057881 , bd ) and a k01 grant ( mh077930 , nm ) from the national institute of mental health . the national institute of mental health did not participate in the preparation , review or approval of the manuscript .	discoveries from genome - wide association studies have contributed to our knowledge of the genetic etiology of many complex diseases . however , these account for only a small fraction of each disease 's heritability . here , we comment on approaches currently available to uncover more of the genetic ' dark matter , ' including an approach introduced recently by naukkarinen and colleagues . these authors propose a method for distinguishing between gene expression driven by genetic variation and that driven by non - genetic factors . this dichotomy allows investigators to focus statistical tests and further molecular analyses on a smaller set of genes , thereby discovering new genetic variation affecting risk for disease . we need more methods like this one if we are to shed a powerful light on dark matter . by enhancing our understanding of molecular genetic etiology , such methods will help us to understand disease processes better and will advance the promise of personalized medicine .
included were all consecutive patients , 65 years or older , that underwent peg insertion from january 1997 to december 2000 at the endoscopy unit at karolinska university hospital , huddinge . a retrospective survey of four different patient chart systems was carried out and the last day for the follow - up was in april 2002 . data on diagnosis , indication for peg , operating time , removal time , or time of death were collected . the peg insertion was performed in the endoscopy suite using intravenous conscious sedation and local anesthesia . the pull procedure was used to place a 20 fr silicon tube with a semirigid inner bumper under endoscopic control . thirteen patients that received their peg for drainage of an obstruction or for medical supplementation were excluded . for one patient , the time of insertion of the peg was not noted . in 10 of the remaining 201 patients , data are presented as mean ( sd ) and median with range and 95% confidence intervals . for survival analysis we used the log - rank test and kaplan meier curves ( spss and statistica software programs ) . there were 201 patients , 65 years or older , that received a peg for nutritional support between 1997 and 2000 at the karolinska university hospital , huddinge , 107 males , 94 women with a mean age of 79 years ( range 6595 ) . the patients were categorized into seven diagnosis groups : stroke , dementia , mb parkinson , other neurological diseases ( mainly amyotrophic lateral sclerosis [ als ] ) , malignancies with or without dysphagia , and miscellaneous . moreover , they were categorized into three main groups of indications : dysphagia , inability to eat ( as a consequence of extreme weakness , motor and mobility problem , and fatigue due to the illness ) , and nutritional support ( en was used in combination to oral intake ) . stroke was the most common diagnosis ( 49% ) and malignancies with dysphagia were the second most common ( 16% ) . in 86% of the patients dysphagia was the indication for peg . number of patients , age ( sd ) , and indications for peg in the various diagnosis groups cancer of esophagus ( 20 ) , lung ( 3 ) , pharynx ( 2 ) , larynx ( 2 ) , tonsils ( 2 ) , mouth ( 2 ) , thyroid ( 1 ) , and vocal cords ( 1 ) . the 30-day mortality in the total patient population was 22% and the 90-day mortality was 42% . there were 44 and 33% of the patients that were still alive after 6 months and after 12 months , respectively . patients with mb parkinson and dementia had the longest survival while the patients with other neurological diseases and malignant esophageal obstructions had the shortest , with a 1-year mortality of 77% ( table 2 ) . median survival in days and 1-,3- , 6- , and 12-month mortality for various diagnostic groups , n=191 when patients were dichotomized according to age over and under 80 years there was no difference in 1-year mortality in the whole group , i.e. 70 and 64% , respectively ( ns ) . except for patients with dementia , where the corresponding figure for 1-year mortality was 33 and 73% , respectively ( p=0.025 ) there was no age - related difference in the various diagnostic subgroups ( data not shown ) . twenty patients had their peg removed after a mean of 300 days and a median of 200 days . thirteen of these 20 patients were still alive at the time of the last follow - up in the year 2002 . this retrospective survey over older patients that received peg for nutritional treatment found a just stroke to be the most common diagnosis and dysphagia to be responsible for around 90% of the indications . most studies on this subject include adults of all ages and the mean age is usually around 65 years . there seems to be little agreement on whether to report indication or diagnosis for a decision to place a peg . there were different indications for peg insertion within each diagnoses and knowing the indication seems important for planning of medical and nursing resources needed after the hospital stay . in this study both indications and diagnoses are reported . several of the findings confirm what others have reported regarding underlying disease , survival , and mortality . neurological disease was the dominating cause for peg insertion followed by malignancies in the upper gi tract ( 5 , 11 ) . in the study by callahan on 150 elderly patients , 41% suffered from stroke , 35% had neuro - degenerative disorders , and 13% had cancer . when it comes to survival it appears that results from the present study deviates from previous reports . this may reflect different treatment strategies . not only the decision to start nutritional support by peg , but also the timing of this decision in relation to the patients clinical status is of great importance for the survival outcome . in our study ( 6 ) the median survival was 210 days in 175 patients , all above 65 years of age . the 95 patients with stroke had a median survival of 119 days , which is less than half of the median survival time of 305 days reported by james et al . ( 12 ) in 126 patients with stroke and dysphagia and a similar mean age of 80 years . such differences may reflect a tendency to decide on peg insertion later in the disease process , rather than variations in the care of the patients after the surgical procedure . the effect of timing and method of enteral feeding for dysphagic stroke patients was studied in the food trial ( 13 ) . the authors concluded that early tube feeding might reduce case fatality but at the expense of increasing the proportion surviving with poor outcome . therefore early initiation of peg feeding was not supported ( 13 ) . since only those patients were enrolled in whom the responsible clinician was uncertain of the best feeding practice , the results from this multicentre trial have to be interpreted with caution . the results from the food trial and other studies on en in stroke patients with dysphagia are reviewed and analyzed in the espen guidelines on enteral nutrition for geriatric patients . treatment with en is recommended as soon as possible unless there are compelling reasons against it in patients with neurological dysphagia . for long - term nutritional support peg should be preferred to ngt , since it is associated with less treatment failures and better nutritional status ( 1 ) . studies on the natural course of dysphagia after stroke show that spontaneous remission of the swallowing difficulty occurs 714 days after the acute event in 73%86% of the patients ( 4 , 14 , 15 ) . intensive swallowing therapy is also recommended to accompany the treatment ( 1 ) . requests for peg in order to facilitate care are prevalent ; that is , the staff in geriatric and elderly care institutions tend to prefer peg to a temporary naso - gastric tube or to the time - consuming oral feeding . it has been reported that one - third of patients received a peg due to demands from the community ( 16 ) . the decision to place a peg in a patient with dementia must be taken with ethical considerations ( 11 , 1721 ) . espen guidelines do not recommend enteral nutrition to persons with severe dementia due to more risks than benefits for persons with severe dementia and only occasionally in early and moderate dementia to ensure adequate energy and nutrient supply and to prevent undernutrition ( 22 ) . a cochrane review by sampson et al . confirmed this and concluded that there is insufficient evidence to suggest that enteral nutrition is beneficial in patients with advanced dementia ( 23 ) . the 244 days of median survival does not support previous observations of a worsened prognosis for patients with dementia and this may reflect a tendency for selecting mainly mildly demented otherwise healthy patients for peg insertion . the tripled short - term mortality in patients combining dementia and age above 80 years indicate that high age should be a precaution for the decision of peg even in patients with milder forms of dementia . a limitation in this survey is that we have no information about the patients cognitive status at the time when the peg insertion took place . further , our data do not allow any conclusions about quality of life for the patients that received peg . to our knowledge no studies so far have addressed this question . in accordance with previous reports , a diagnosis of malignancy carried a higher risk of short survival ( 6 , 9 , 24 ) . however , the difference was less accentuated in the present study ( 106 vs. 130 days ) , when for example compared to a study by fisman et al . ( 6 ) that reported a survival of 137 days in patients with malignant disease versus 321 days in patients with non - malignant disease . it should be acknowledged that peg insertion due to malignant esophageal obstruction could be justified by palliative reasons even when survival is expected to be short . patients with als usually present with a long survival after peg insertion ( 9 , 25 ) . in our study the 13 patients with other neurological diseases ( 12 with als ) had the shortest survival time of all . however , the current patients were notably older ( 758 years ) compared to those presented by mitchell and tetroe ( 9 ) and chio et al . not unexpectedly , patients older than 80 had a slightly worse prognosis than those younger than 80 , i.e. in line with other reports ( 4 , 2628 ) . otherwise the age effect appeared so small that it would not be advisable to identify age as a clear unfavorable factor for peg in this unselected consecutive group of elderly patients . the overall 30-day mortality of 22% is well in accordance with published data ( 6 , 29 ) , whereas there are reports on even higher mortality figures of up to 28% ( 11 , 30 ) . the high short - term mortality in this elderly population may not be due to procedure - related complications , but rather to the decisions of peg placement taken late in the disease course . the 90-day mortality of 42% and 180 day figure of 56% also corresponds well to published data of 44 and 52% ( 11 ) . in the review by tetroe and mitchell ( 9 ) the 1-year mortality was 62% , well in line with our figure of 67% and other reports of 61 and 66% ( 9 , 6 , 30 ) . we may conclude that there is a major variability in survival after peg insertion in elderly patients that suffer from various diseases . the high 30-day mortality of 22% in this study is in accordance with published data , but median survival time appeared to be shorter than previously reported . dementia was not found to be a negative prognostic factor for survival in this population , especially not in those below the age of 80 years . however , the limited access to patient data , such as disease severity and nutritional state , does not allow a deeper analysis on which clinical variables , which would be discriminative for selecting patients , would benefit most from the procedure . according to the recent study by blomberg et al . , it should not be recommended to make peg insertion in patients with crp levels and low albumin indicating ongoing inflammation ( 10 ) . there is still an urgent need for developing ways to decide the risk / benefit ratio in the individual patient in order to optimize the timing and route of nutritional support . especially in this field , ethical consideration is a challenge for the scientific approach , but such efforts would need prospective randomized controlled studies , which most likely are not accepted due to ethical considerations . however , future studies that include questions regarding quality of life are possible to perform and are needed to elucidate the ethical aspects of enteral nutrition treatment in geriatric patients . the authors have not received any funding or benefits from industry or elsewhere to conduct this study .	backgroundmany diseases striking old adults result in eating difficulties . indications for selecting individuals for percutaneous endoscopic gastrostomy ( peg ) are unclear and everybody may not benefit from the procedure.objectivethe aim of this study was to evaluate indications for and survival after peg insertion in patients older than 65 years.design and methodsa retrospective analysis including age , gender , diagnosis , indication , and date of death was made in 201 consecutive individuals , 94 male , mean age 797 years , who received a nutritional gastrostomy.resultsdysphagia was present in 86% of the patients and stroke was the most common diagnosis ( 49% ) . overall median survival was 123 days and 30-day mortality was 22% . patients with dementia and mb parkinson had the longest survival ( i.e. 244 and 233 days ) , while those with other neurological diseases , and head and neck malignancy had the shortest ( i.e. 75 and 106 days ) . there was no difference in mortality in patients older or younger than 80 years , except in patients with dementia.conclusionsold age should not be a contraindication for peg . a high 30-day mortality indicates that there is a need of better criteria for selection and timing of peg insertion in the elderly .
splenic tumours are an uncommon entity and include a wide range of both benign and malignant conditions . haemangiomas are the most common benign splenic tumours , while lymphoma is the most frequently encountered malignancy . less common conditions include littoral cell angioma , hamartoma and metastases . while ultrasound , ct and mri are useful modalities for detecting splenic tumours , definitive diagnosis can usually only be made following splenectomy . in this case report we present the clinical and pathological features of an inflammatory pseudotumour of the spleen , associated with epstein barr virus and review the current classification of such splenic spindle cell lesions . a 69-year - old lady with a history of breast and uterine carcinoma was found to have a mass at the splenic hilum on surveillance ct . in 2003 she had a hysterectomy for a uterine carcinoma but required no adjuvant treatment . in 2011 she presented with a right sided malignant breast lump and underwent a wide local excision and sentinel node biopsy . ct scan of the abdomen demonstrated a 7.2 cm 7.4 cm mixed attenuation , solid rim - enhancing mass lesion in the hilum of the spleen ( figs . 1 and 2 ) . at the time of her ct scan she was virtually asymptomatic with the exception of some fullness in the upper abdomen. her case was discussed at the oeophago - gastric multi - disciplinary team meeting and a differential diagnosis of lymphoma or metastatic deposit was given . percutaneous biopsy was not felt appropriate given the risk of bleeding although fna and needle core of the spleen has been described . on sectioning the normal sized spleen there was a rubbery , well - defined tumour measuring 8 cm 6.5 cm 5 cm ( fig . 3 ) . histology showed a tumour composed of spindle cells , intimately admixed with lymphocytes , plasma cells and occasional eosinophils ( fig . there were areas of coagulative tumour necrosis but minimal evidence of nuclear atypia or anaplasia . barr virus encoded rna ( eber ) in all of the spindle tumour cells ( fig . 5 ) . the follicular dendritic cell marker cd21 was negative but cd23 , cd35 and fascin were focally positive . she had an uncomplicated post - operative recovery and at her 6-month follow - up appointment she was well and symptom free . inflammatory pseudotumor ( ipt ) is a term that has been used to describe a variety of tumefactive lesions of varying aetiology and different behaviour in a variety of anatomical sites including inflammatory myofibroblastic tumour ( imt ; a low grade malignancy commoner in younger patients and associated with abnormal alk-1 expression ) infective mycobacterial spindle cell proliferations and the tumefactive complications of igg4 related disease . follicular dendritic cell tumour is a rare , low grade malignancy that most commonly occurs as a primary lymph node tumour . however , it has been reported in a variety of extra nodal areas such as the oral cavity , lung , tonsil , pharynx and thyroid where it is usually ebv negative . a specific association between hepatic and splenic ipts and epstein barr virus has been proposed , with 4067% of cases testing positive for the virus . in a study of 18 cases arber et al . the spindle cells were uniformly vimentin positive and in some cases they were smooth muscle actin positive ( sma ) , suggestive of myofibroblastic differentiation . in 2001 neuhauser notably 3 of the 10 cases were associated with a past medical history of carcinoma and 2 of the 10 cases were positive for the follicular dendritic cell marker cd21 . in 2001 , cheuk reported 11 splenic tumours where the spindle cells showed positive staining for eber and the follicular dendritic cell markers cd21/cd35 and cd23 and proposed the term ebv related inflammatory pseudotumour like follicular dendritic cell tumour of the spleen which were characterised by indolent behaviour ( although there were some recurrences ) and a female predeliction . 1n 2003 , krishnan and frizzera proposed categorising splenic spindle cell tumours into three groups:(1)ipt like fdc tumour histology of dispersed fdcs in a prominent inflammatory background ; and consistent evidence of ebv infection of the spindle cells.(2)imt of spleen similar to imts in other sites . a good proportion ( 66% ) follow up for splenic imts uneventful compared to other locations.(3)ipt of spleen predominant spindle cells are not sma positive mfs . ipt like fdc tumour histology of dispersed fdcs in a prominent inflammatory background ; and consistent evidence of ebv infection of the spindle cells . imt of spleen similar to imts in other sites . a good proportion ( 66% ) splenic stromal tumour has been proposed as an alternative term for this heterogenous group of tumours and indeed other authors have observed that such lesions may show variable follicular dendritic or myofibroblastic differentiation and that this may be associated with the levels of ebv expression . ebv positive inflammatory pseudotumour of the spleen is an uncommon spindle cell tumour of the spleen which can show variable myofibroblastic or follicular dendritic differentiation and variation in expression levels of ebv these lesions may be associated with prior malignancy and in the era of increased surveillance with cross sectional imaging may present in asymptomatic patients with radiological findings suggestive of lymphoma or metastatic disease . written informed consent was obtained from the patient for the publication of this case report and images and a copy is available for review . dr paula loughlin , dr eimear devlin , dr aidan brady , dr damian mcmanus and professor roy spence all contributed to the writing , editing and literature review .	introductionepstein barr virus positive inflammatory pseudo - tumour ( ipt ) of the spleen is an uncommon , frequently asymptomatic entity , which is typically picked up as an incidental finding on imaging.presentation of casewe present a case of ebv positive ipt of the spleen which presented as an incidental finding on ct in a patient with a history of malignancy . splenectomy was performed.discussionipts are benign spindle cell lesions of varying aetiology , which can arise in a variety of tissues , including the spleen . in situ hybridisation showed strong staining for epstein barr virus rna in our case , in common with many similar lesions described in the literature . the differential diagnosis of such spindle cell tumours is discussed.conclusionradiologically , ebv positive spindle cell tumours are indistinguishable from malignant lesions such as lymphoma and diagnosis is made on histology , usually at splenectomy .
severe complications have been observed more frequently in transvenous implantation of implantable cardioverter - defibrillator ( icd ) lead than in that of pacemaker , particularly in the dual - chamber rather than single - chamber icd.1)2 ) also , perforation of the right ventricular ( rv ) wall is a rare complication in icd lead implantation with an estimated incidence of 0.6% . this complication mostly occurs in the perioperative period and low incidence of delayed rv wall perforation in the subacute phase has been reported.3 ) detection of rv wall perforation by standard imaging modalities is often challenging . multidetector computed tomography ( mdct ) is emerging as an optional modality for establishing an accurate diagnosis of lead perforation , identifying the extent of accompanying complications such as pericardial effusion and planning the lead extraction strategy . we present a rare case of delayed rv perforation that manifested 14 days after icd implantation and was clearly displayed by mdct . an 81-year - old man was referred to our hospital for presyncope , dyspnea and palpitation . he had a previous history of inferoposterior myocardial infarction treated by percutaneous coronary intervention with drug - eluting stents ( des ) . on arrival , he was conscious and alert but his heart rate was 190 beats / min and the electrocardiogram ( ecg ) revealed sustained ventricular tachycardia . intravenous administration of adenosine or lidocaine did not stabilize the rhythm , and electrical cardioversion finally restored a normal sinus rhythm . after the recovery of sinus rhythm , the ecg did not show ischemic st - t changes and serum creatine kinase as well as troponin t levels were not elevated . the echocardiography revealed left ventricular ( lv ) dilatation with lv end - diastolic / end - systolic dimensions of 66/57 mm , decreased lv ejection fraction of 35% and severe ischemic mitral regurgitation . the implantation of icd with an atrial passive - fixation lead ( model 5076 , capsurefix , medtronic , minneapolis , mn , usa ) and a standard - caliber ventricular active - fixation lead ( model 6947 , sprint quattro 8.6-french lead , medtronic , minneapolis , mn , usa ) was performed through the left subclavian vein without any complications . the two leads were placed in the right atrial appendage and right ventricular apex , respectively ( fig . the rv lead parameters were satisfactory ( r - wave amplitude 6.9 mv , pacing threshold 0.4 v at 0.5 ms , pacing impedance 380 ohms ) . after the procedure , dual anti - platelet therapy ( aspirin 100 mg and ticlopidine 200 mg per day ) was commenced immediately because of des implantation 9 months ago . echocardiography performed on post - implant day 7 showed no significant change without any evidence of pericardial effusion . fourteen days after icd implantation , the patient presented to the emergency room with intermittent chest pain since the last 2 days . the chest pain appeared to be independent of exercise and lasted for 10 to 20 seconds . the ecg as well as circulatory biomarker measurements such as creatine kinase and troponin t indicated no signs of acute coronary syndrome . the chest x - ray showed cardiomegaly and minor changes in the ventricular lead tension and ventricular lead tip position ( fig . interrogation of the icd showed that the sensed r wave measured 6.0 mv , the ventricular pacing threshold had risen to 8 v at 0.5 ms and pacing impedance had dropped to 282 ohms . thus , rv perforation by the icd lead was suspected and 64-slice mdct was subsequently performed with a msct scanner ( vct , ge healthcare , milwaukee , wi , usa ) according to the following protocol : 120 kv , 300 ma , rotation time of 350 to 400 msec and collimation of 640.625 mm but without contrast - media to avoid further deterioration of renal function ( fig . mdct confirmed complete lead perforation , and because of the high bleeding risk due to dual anti - platelet therapy , cardiac surgery under general anesthesia rather than manual lead extraction was indicated ( fig . 2 ) . lead extraction , closure of rv perforation site and re - implantation of the rv lead onto the rv outflow tract were successfully performed . the patient had an uneventful recovery and has been doing well since the last 6 months after discharge . mdct allows ready identification of the course of the lead , relationship to adjacent anatomy and adverse sequelae such as pericardial effusion.4 ) this case highlights the role of mdct as an extremely useful imaging modality to confirm the diagnosis of lead perforation and planning the lead extraction strategy , even without contrast enhancement . mechanisms responsible for rv lead perforation generally involve lead - related factors such as the stiffness and diameter of the lead as well as myocardium - related factors such as morphology of the right ventricular wall and contraction during each heart beat.5 ) patients with thinning of the ventricular wall are at a higher risk of lead perforation . several reports have demonstrated that delayed lead - related complications such as lead perforation and lead failures were more frequent with the use of small - caliber icd leads , especially the st . jude riata leads,6 ) than with standard - caliber icd leads,7 ) thereby suggesting the important contribution of lead - related factors . in the present case , thinning of rv wall due to an old myocardial infarction caused by occlusion of the proximal right coronary artery might have critically contributed to the occurrence of lead perforation , since the standard - caliber active fixation lead ( model 6947 , sprint quattro 8.6-french lead , medtronic ) was implanted at the rv apex . the incidence of icd lead perforation has been reported to be approximately 0.5% in two independent studies including 593 and 3,340 cases , respectively.8)9 ) as previous reports have shown , lead perforation commonly occurred within 7 to 10 days after implantation , mostly occurring at the time of implantation or within the first 24 hours.6)8 ) in addition , small - caliber icd leads were used in many of the recently reported cases of lead perforation . given the type of implanted icd leads used and the timing of rv perforation , this is a rare case of icd lead perforation clearly displayed by mdct . with respect to the treatment of lead perforation , transvenous lead extraction under transesophageal echocardiographic monitoring might be a standard procedure , in case of active - fixation lead.1 ) however , we considered the present case to be at a high risk for hemodynamic and/or uncontrollable bleeding complications , because of decreased lv function with severe mitral regurgitation and dual anti - platelet therapy . thus , we selected open surgery via median sternotomy as the safest approach . operators of icd implantation should always keep in mind that pericardial effusion due to lead perforation may develop without the typical manifestations of cardiac tamponade in the chronic phase .	we present an unusual case of a delayed right ventricular perforation by a single standard - caliber implantable cardioverter - defibrillator lead , which manifested 14 days after implantation . multidetector computed tomography could clearly display the lead perforation , and allow for identification of the associated sequelae such as pericardial effusion and planning the lead extraction strategy .
ventricular septal rupture ( vsr ) is a well - known and potentially fatal complication following acute myocardial infarction . in the pre - thrombolytic era , the incidence was 1 - 2% but has since declined to 0.17 - 0.31% with the advent of reperfusion therapy . vsrs have been reported to occur 16 - 24 h after acute myocardial infarction in the reperfusion era as opposed to 3 - 5 days previously [ 1 , 2 ] . vsr should be suspected clinically in patients with recent myocardial infarction who present with signs of cardiac failure , a new holosystolic murmur over the precordium . a 54-year - old indonesian male with a 48 pack years smoking history , hypertension , diabetes mellitus , and dyslipidemia saw his local doctor for chest pain and dyspnea . he presented to our center in singapore a week later with persistent dyspnea , orthopnea and lower limb swelling . clinical examination revealed a grade 3/6 holosystolic murmur loudest over the left lower sternal edge , reduced breath sounds bibasally and inspiratory crepitations bilaterally to the mid zone . 1 ) showed q waves in the inferior leads with 2 mm st elevations and t - wave inversions , suggestive of a recent inferior myocardial infarction . q waves in the inferior leads with 2 mm st elevations and t - wave inversions , suggestive of a recent inferior myocardial infarction . transthoracic echocardiography showed a thinned and aneurysmal inferoseptal and inferior left ventricular wall from basal to mid - level . a vsr was also seen within the aneurysm with left to right shunt ( fig . 3 ) . we proceeded to perform a cardiac mri which revealed vsr at mid inferoseptal wall ( fig . 4 ) , with a basal to mid inferior wall aneurysm and significant intracardiac shunt ( qp / qs 3.3 ) . a diagnostic angiogram was performed which showed triple vessel disease with diffuse stenosis in the lad and circumflex vessels and 100% stenosis of the mid rca ( fig . lv short axis view shows vsr is at the mid inferoseptal wall with a basal to mid inferior wall aneurysm and significant intracardiac shunt ( qp / qs 3.3 ) . ( a ) diffuse stenosis was seen in the left anterior descending and circumflex vessels . after consultation with the cardiothoracic team , early coronary artery bypass graft and vsr repair were planned . however , the patient deteriorated rapidly into cardiogenic shock . dopamine infusion was started , an intra - aortic balloon pump was inserted and he was brought for urgent operation . during the operation , the inferior myocardial infarct was evident by hemorrhagic myocardium between the right posterior descending artery ( rpda ) and right posterolateral artery ( rpl ) vessels . the large vsr was next to the tricuspid valve septal attachment rendering the valve incompetent . coronary artery bypass grafting was performed , saphenous vein graft ( svg ) to left anterior descending coronary artery ( lad ) and svg to obtuse marginal branch ( om ) . ventriculotomy was made into the infarcted tissue between the rpda and rpl and patch closure of vsr was performed . the patient also developed complete heart block requiring dual chamber permanent pacemaker insertion with right atrial lead and right ventricular lead placed through the coronary sinus . this patient is the first in our center s series where there was inadequate healthy margins to place the vsr patch without compromising the tricuspid valve leaflets and conduction pathway , thus necessitating a tricuspid valve replacement . from our surgical series of 47 patients since 2000 , mortality rates were higher , 7/13 ( 54% ) for inferior vsrs compared to 11/34 ( 32% ) for anterior vsrs . this could be due to a combination of factors such as difficult surgical access , right ventricular dysfunction , increased risk of conduction block and a potentially higher incidence of residual shunts . it is believed that this is the first vsr repair case that required permanent pacing postoperatively , likely due to the compromised conduction pathway from the infarcted tissue and vsr patch placement . furthermore , in view of the friable nature of the infarcted right ventricle and bioprosthetic tricuspid valve , the right ventricular lead of the pacemaker had to be inserted via the coronary sinus . timing to surgery should be decided on a case to case basis taking into account the hemodynamic stability of the patient [ 1 , 3 ] . as reported in the society of thoracic surgeons national database , improved outcomes were seen with delayed surgery with 54.1% mortality ( surgery within 7 days ) vs. 18.4% ( surgery after 7 days ) as those requiring early surgery are likely sicker with greater hemodynamic instability [ 1 , 4 ] . in our center , only a selected subset ( three of 47 ) of patients in our series were stable enough to undergo surgery more than 2 weeks after diagnosis . with the advent of early reperfusion strategies , the incidence of vsr has decreased . however , it is still a serious complication that should not be overlooked especially in cases of myocardial infarction which presents late . authors have nothing to disclose with regard to conflicts of interest to this case report .	ventricular septal rupture ( vsr ) is a well - described and potentially fatal complication of acute myocardial infarctions . in the era of early reperfusion therapy , the incidence has been on the decline . we describe a case of delayed presentation inferior myocardial infarction complicated by vsr requiring early surgery and review our center s experience .
among the modern radiotherapeutic techniques , brachytherapy is an effective approach for delivering high - dose radiation to a target . as a result , we have been treating prostate cancer patients with high - dose - rate ( hdr ) brachytherapy combined with hypofractionated external beam radiotherapy since 1999 [ 1 , 2 ] . the dosimetric accuracy of hdr brachytherapy relies on catheter positions being accurately reproduced to match the original positions in the planning ct dataset for all fractions of treatment . however , caudal catheter displacement is often observed prior to the first fraction [ 3 , 4 ] and between fractions [ 510 ] . this may lead to not only a significant risk of under - dosage at the target , but also over - dosage to the organs at risk . several studies have demonstrated urethral stricture as the predominant late urinary toxicity of prostate hdr brachytherapy [ 2 , 11 ] . with significant caudal displacement , a high dose is likely to be erroneously delivered to the bulbo - membranous urethra . sullivan et al . reported that 92% of all strictures occurred in the bulbo - membranous urethra , which lies distal to the prostatic apex . bulbo - membranous urethral stricture formation may , therefore , represent a clinical manifestation of caudal catheter displacement during hdr brachytherapy . the purpose of this work was to determine catheter displacement prior to the delivery of hdr brachytherapy based on measurements from pre - treatment ct scan compared to the original planning ct scan with an implanted apex gold marker as representative of the bulbo - membranous urethra . patient characteristics values are number or median ( range ) patients in the operating room were placed in a lithotomy position under epidural anesthesia . multiple 24 cm long , closed - end , 6-f hollow plastic catheters were inserted transperineally using a syed - neblett plastic template ( alpha - omega services , bellflower , ca , usa ) under transrectal ultrasound guidance . routinely , 18 catheters were implanted independently of prostate size . twelve catheters were inserted in the peripheral portion , and six catheters were inserted in the central portion of the prostate . the needle tips were left within the urinary bladder 1.5 cm above the sonographically or cystoscopically defined base of the prostate . a ct scan was obtained for ct - based planning . the volumetric scans with reconstructed slice thickness of 1.25 mm were obtained using multi - detector row ct ( optima ct580 , ge - healthcare , wi , usa ) . the planning target volume ( ptv ) was defined as the prostate gland with or without proximal seminal vesicle , with a manually drawn margin between 3 - 5 mm in all directions . reference points for the ptv were automatically distributed on the surface of the ptv . the dose limitation ( maximum dose ) was set as 8 gy per fraction for the urethra , and 4 gy per fraction for 5 mm behind the edge of the anterior rectal wall . a dose of 7.5 gy per fraction to the ptv was prescribed , unless the dose limitation was violated , using inverse planning and geometric optimization . after ct - based planning performed on the oncentra treatment planning system ( nucletron , elekta ab , stockholm , sweden ) , the first treatment session of hdr brachytherapy was conducted using the nucletron microselectron hdr ir remote afterloading system . the first treatment session was conducted on the day of implantation , with the subsequent four treatment sessions administered twice daily on subsequent days . basically , the time differences between plan ct and 1 , 2 , 3 , 4 , and 5 fractions were 6 h , 24 h , 30 h , 48 h , and 54 h , respectively . six days after completion of hdr brachytherapy , patients received ebrt using a dynamic - arc conformal technique , administered with high - energy photons comprising 10-mv x - rays ( mhcl-15tp , mitsubishi electric , tokyo , japan ) to a total dose of 30 gy . the radiation field was limited to the prostate gland with or without proximal seminal vesicles with a 7 mm leaf margin using multileaf collimators . during catheter insertion , gold markers ( visicoil ; iba - dosimetry , schwarzenbruck , germany ) were also implanted in the apex and base of the prostate . the apex - marker was used as a representative reference point for prostate and bulbo - membranous urethral position ( fig . obturator with 3 mm marker and 10 mm spacer was inserted into every catheter ( fig . a ct scan was acquired at 1.25 mm slice thickness prior to 1 fraction in order to measure the catheters displacement relative to apex marker . the actual displacement was calculated by multiplying the thickness of the ct slice with the difference in number of ct slices between the slice of the apex marker and the marker of obturator of each catheter ( fig . gold markers were implanted at the apex ( a ) and base ( b ) of the prostate gland . catheter displacement was calculated by multiplying the thickness of the ct slice with the difference in number of ct slices between the slice of the apex gold marker ( c ) and the reference slice detected by the pattern of the obturator markers ( d ) the slice of an apex marker and an obturator maker were defined as the most cranial slices that showed the top of marker without artifact . in this study , measurements of each catheter position were done for only the first pre - treatment scan . other catheter displacements were calculated by slice - specific pattern as a representative for all 18 catheter positions . the obturators in 18 catheters make slice - specific patterns on ct - images ( high and low density in the catheter hollows ; fig . 1d ) , because no catheter was inserted in completely equal depth , and that make it possible to recognize a representative slice for 18 catheters positions . therefore , this slice - specific pattern on ct - image was used as a representative for all catheter position instead of measuring each 18 needles , because measuring all catheter positions in all treatments was not practical . flow chart of the catheter adjustment protocol where there was enough space between the first dwell position and distal end of the catheter hollow ; dwell position was adjusted by changing indexer length at the treatment console . where there was no space and displacement > 2 mm , the positions of gold markers implanted at the apex and base of the prostate were also checked by comparing with soft tissue anatomy on the ct images . the length of total catheter displacement was evaluated as follows : where dactual is actual displacement of the catheter calculated from difference between original planning ct and the first pre - treatment ct , dmanual is actual length of manual catheter advancement calculated from difference between cts scanned before and after manual advancement , and dtotal is the sum of the two . multiple 24 cm long , closed - end , 6-f hollow plastic catheters were inserted transperineally using a syed - neblett plastic template ( alpha - omega services , bellflower , ca , usa ) under transrectal ultrasound guidance . routinely , 18 catheters were implanted independently of prostate size . twelve catheters were inserted in the peripheral portion , and six catheters were inserted in the central portion of the prostate . the needle tips were left within the urinary bladder 1.5 cm above the sonographically or cystoscopically defined base of the prostate . a ct scan was obtained for ct - based planning . the volumetric scans with reconstructed slice thickness of 1.25 mm were obtained using multi - detector row ct ( optima ct580 , ge - healthcare , wi , usa ) . the planning target volume ( ptv ) was defined as the prostate gland with or without proximal seminal vesicle , with a manually drawn margin between 3 - 5 mm in all directions . reference points for the ptv were automatically distributed on the surface of the ptv . the dose limitation ( maximum dose ) was set as 8 gy per fraction for the urethra , and 4 gy per fraction for 5 mm behind the edge of the anterior rectal wall . a dose of 7.5 gy per fraction to the ptv was prescribed , unless the dose limitation was violated , using inverse planning and geometric optimization . after ct - based planning performed on the oncentra treatment planning system ( nucletron , elekta ab , stockholm , sweden ) , the first treatment session of hdr brachytherapy was conducted using the nucletron microselectron hdr ir remote afterloading system . the first treatment session was conducted on the day of implantation , with the subsequent four treatment sessions administered twice daily on subsequent days . basically , the time differences between plan ct and 1 , 2 , 3 , 4 , and 5 fractions were 6 h , 24 h , 30 h , 48 h , and 54 h , respectively . six days after completion of hdr brachytherapy , patients received ebrt using a dynamic - arc conformal technique , administered with high - energy photons comprising 10-mv x - rays ( mhcl-15tp , mitsubishi electric , tokyo , japan ) to a total dose of 30 gy . the radiation field was limited to the prostate gland with or without proximal seminal vesicles with a 7 mm leaf margin using multileaf collimators . during catheter insertion , gold markers ( visicoil ; iba - dosimetry , schwarzenbruck , germany ) were also implanted in the apex and base of the prostate . the apex - marker was used as a representative reference point for prostate and bulbo - membranous urethral position ( fig . obturator with 3 mm marker and 10 mm spacer was inserted into every catheter ( fig . a ct scan was acquired at 1.25 mm slice thickness prior to 1 fraction in order to measure the catheters displacement relative to apex marker . the actual displacement was calculated by multiplying the thickness of the ct slice with the difference in number of ct slices between the slice of the apex marker and the marker of obturator of each catheter ( fig . gold markers were implanted at the apex ( a ) and base ( b ) of the prostate gland . catheter displacement was calculated by multiplying the thickness of the ct slice with the difference in number of ct slices between the slice of the apex gold marker ( c ) and the reference slice detected by the pattern of the obturator markers ( d ) the slice of an apex marker and an obturator maker were defined as the most cranial slices that showed the top of marker without artifact . in this study , measurements of each catheter position were done for only the first pre - treatment scan . other catheter displacements were calculated by slice - specific pattern as a representative for all 18 catheter positions . the obturators in 18 catheters make slice - specific patterns on ct - images ( high and low density in the catheter hollows ; fig . 1d ) , because no catheter was inserted in completely equal depth , and that make it possible to recognize a representative slice for 18 catheters positions . therefore , this slice - specific pattern on ct - image was used as a representative for all catheter position instead of measuring each 18 needles , because measuring all catheter positions in all treatments was not practical . flow chart of the catheter adjustment protocol where there was enough space between the first dwell position and distal end of the catheter hollow ; dwell position was adjusted by changing indexer length at the treatment console . where there was no space and displacement > 2 mm , the positions of gold markers implanted at the apex and base of the prostate were also checked by comparing with soft tissue anatomy on the ct images . the length of total catheter displacement was evaluated as follows : where dactual is actual displacement of the catheter calculated from difference between original planning ct and the first pre - treatment ct , dmanual is actual length of manual catheter advancement calculated from difference between cts scanned before and after manual advancement , and dtotal is the sum of the two . table 2 shows actual displacements between original planning scan and 1 pre - treatment ct scan of 18 needles for each patient . standard deviations of 18 catheters for each patient ( variation within one single patient ) were < 1 mm except one . these small variations among 18 catheters permit our slice - specific pattern recognition . regarding total catheter displacement in the adjustment protocol , mean dtotal values were 6 4 mm , 12 6 mm , 12 6 mm , 12 6 mm , and 12 6 mm from plan to 1 , 2 , 3 , 4 , and 5 fractions , respectively . mean displacement was 6 mm between the plan and 1 , and 1 and 2 fractions displacements ( mm ) between original planning ct and 1 pre - treatment ct of 18 needles for each patient sd standard deviation within each single patient manual catheter adjustments were needed in 31 of 150 treatment fractions . 3 ) . on the second pre - treatment ct , however , actual advancement was 4 3 mm . no displacement of apex fiducial markers compared to soft tissue anatomy was seen on ct images . meanwhile , two patients displayed base - marker migrations of 20 mm and 25 mm just after implantation , and we failed to implant a base marker in 1 patient . in the remaining 27 patients , distances between apex and base markers gradually increased over time . mean increases of distance between the two gold markers were 0 3 mm , 2 4 mm , 3 4 mm , 4 2 mm , and 3 4 mm from plan to 1 , 2 , 3 , 4 , and 5 fractions , respectively . table 2 shows actual displacements between original planning scan and 1 pre - treatment ct scan of 18 needles for each patient . standard deviations of 18 catheters for each patient ( variation within one single patient ) were < 1 mm except one . these small variations among 18 catheters permit our slice - specific pattern recognition . regarding total catheter displacement in the adjustment protocol , mean dtotal values were 6 4 mm , 12 6 mm , 12 6 mm , 12 6 mm , and 12 6 mm from plan to 1 , 2 , 3 , 4 , and 5 fractions , respectively . mean displacement was 6 mm between the plan and 1 , and 1 and 2 fractions displacements ( mm ) between original planning ct and 1 pre - treatment ct of 18 needles for each patient sd no displacement of apex fiducial markers compared to soft tissue anatomy was seen on ct images . meanwhile , two patients displayed base - marker migrations of 20 mm and 25 mm just after implantation , and we failed to implant a base marker in 1 patient . in the remaining 27 patients , distances between apex and base markers gradually increased over time . mean increases of distance between the two gold markers were 0 3 mm , 2 4 mm , 3 4 mm , 4 2 mm , and 3 4 mm from plan to 1 , 2 , 3 , 4 , and 5 fractions , respectively . because of the 1.25 mm ct slice thickness used , the lower limit of accuracy of our measurement was 1.25 mm . as a result , our measurement data were written as single figures , such as 1 mm . several investigators have already reported caudal catheter displacement during hdr brachytherapy ( table 3 ) [ 37 , 9 , 10 ] . although they used 3 - 5 mm ct slice thickness , mean displacements resembles our results were reported . reported data for catheter displacement during high - dose - rate brachytherapy in our previous protocol , catheter displacements were checked by x - ray films and adjusted if displacement was > 5 mm relative to implanted markers . however , 10% of our patients suffered from grade 3 genitourinary toxicity including urethral stricture . because caudal catheter displacement could be one of the reasons for urethral stricture [ 8 , 12 ] , we changed our action level from 5 mm down to 2 mm with the aim of achieving a lower urethral dose . with this new protocol , exposure of the bulbo - membranous urethra to high - dose radiation could be avoided , and the urethral toxicity rate could be reduced . however , a longer follow - up time for the current patient cohort is required . our results also confirmed that the distances between apex and base markers were increased , suggesting that the prostate gland was gradually swelling throughout the course of treatment . herrmann et al . also reported displacement of markers after hdr brachytherapy , especially in the superior - inferior direction ( mean : 3 mm ) . they discussed possible reasons for displacement such as localized bleeding into the prostate gland , or dislocation of prostate tissue inside the gland due to needle insertion . although we added a 3- to 5-mm margin around the prostate , some parts of the ptv may not have received sufficient dose in some cases . re - planning may be needed for patients with an excessively enlarged prostate after catheter insertion . the apex is , in comparison to other structures around the prostate , less dependent on internal influences such as swelling of the prostate , therefore it may represent a suitable position for marker implantation compared to the base of the gland . examined the impact of catheter displacement on tumor control probability ( tcp ) in patients with prostate cancer receiving hdr , and advised that action levels to correct for catheter displacements should be 3 mm . according to their calculations , median relative tcp was 0.998 , if catheter displacement was 3 mm . another interesting point we found in this study was the difficulty of manually adjusting the catheter . physically advancing the catheter 1 cm into the patient resulted in an actual advance of only approximately 0.4 cm relative to the apex marker , as the prostate itself was also pushed along with the catheter . this result suggests that a combination with computerized adjustment of dwell position in software is needed when manual catheter advancement is performed . in conclusion , frequent catheter displacements relative to the apex of the prostate and bulbo - membranous urethra were confirmed by measurement prior to each treatment fraction on pre - treatment ct images with 1.25 mm slice thickness . our results indicate that catheter positions must be confirmed and if required , adjusted , prior to every treatment fraction for the precise treatment delivery of hdr brachytherapy , and to potentially reduce over - dosage to the bulbo - membranous urethra .	purposethe purpose of this work was to report measured catheter displacement prior to the delivery of high - dose - rate brachytherapy ( hdr ) in the treatment of prostate cancer.material and methodsdata from 30 prostate cancer patients treated with hdr brachytherapy were analyzed retrospectively . eighteen transperineal hollow catheters were inserted under transrectal ultrasound guidance . gold marker seeds were also placed transperineally into the base and apex of the prostate gland . five treatment fractions of 7.5 gy each were administered over 3 days . the patient underwent ct scanning prior to each treatment fraction . catheter displacement was measured from the pre - treatment ct dataset reconstructed at 1.25 mm slice thickness.resultsmost of catheters were displaced in the caudal direction . variations of 18 catheters for each patient were small ( standard deviations < 1 mm for all but one patient ) . mean displacements relative to the apex marker were 6 4 mm , 12 6 mm , 12 6 mm , 12 6 mm , and 12 6 mm from plan to 1st , 2nd , 3rd , 4th , and 5th fractions , respectively.conclusionsour results indicate that catheter positions must be confirmed and if required , adjusted , prior to every treatment fraction for the precise treatment delivery of hdr brachytherapy , and to potentially reduce over - dosage to the bulbo - membranous urethra .
since a robotic surgical system was developed in the early 1990s and the first robotic - assisted radical prostatectomy ( rarp ) was reported in 2001 , robotic surgery has spread in many surgical specialties , changing surgical management . in the united states , rarp has increased steeply from 8% of radical prostatectomies in 2004 to 67% in 2010 . likewise , the number and indications for robotic gynecological surgery have continued to expand . over the past decade , robotic surgery has undergone substantial changes for pelvic procedures and been applied to colorectal surgery . colorectal surgeons expected that robotic surgery would resolve technical difficulties associated with laparoscopic surgery , which has a steep learning curve . since the first report of robotic - assisted colectomies by weber et al . in 2002 , a variety of colorectal procedures have used robot assistance for a range of diseases [ 5 - 8 ] . currently , compared to other colorectal procedures , robotic surgery appears to offer great benefits for total mesorectal excision ( tme ) for rectal cancer . for example , abdominal cavity procedures such as right hemicolectomy and high anterior resection are relatively uncomplicated and can be performed easily by laparoscopic surgery . according to a recent study that analyzed nationwide data on current surgical management for colorectal cancer surgery in korea , robotic surgery was used for 185 cases of rectal cancer in 2008 , increasing steadily to 490 cases in 2013 . in contrast , robotic colon cancer surgery decreased from 110 cases in 2009 to only 10 cases in 2013 . most relevant recent reports have focused on the clinical benefits of robotic rectal cancer surgery compared with laparoscopic surgery [ 10 - 27 ] . however , no clear advantage supporting the use of this expensive technology has been demonstrated . for robotic rectal cancer surgery to become the preferred minimally invasive option , it must demonstrate that it does not have the technical difficulties and steep learning curve of laparoscopic surgery . therefore , we reviewed current evidence to compare surgical , pathological , functional , and oncological outcomes of robotic and laparoscopic surgery for rectal cancer . we searched the medline database with pubmed for all articles published between 2002 and 2015 using the keywords robot * , da vinci , colo * , and * rectal . we also manually searched articles using references from related articles . when multiple studies used the same patient population , the most recent publication was used . short - term surgical outcomes of robotic surgery for rectal cancer were reviewed and compared with laparoscopic or open rectal cancer surgery ; results are in table 1 . initially , a longer operating time was generally accepted to be a drawback of robotic rectal cancer surgery compared with laparoscopic procedures . however , recent studies report equivalent or even shorter operating times for robotic surgery for rectal cancer . as most studies were published by highly experienced laparoscopic colorectal surgeons , the main reason underlying the initially reported lengthy operating times for robotic surgery was likely a difference in the level of mastery for the two operative techniques . operating time does not appear to be an issue after surgeons accumulate experience and overcome the learning curve . an excellent conversion rate is consistently reported in initial studies of robotic surgery . because converted patients have higher complication rates and worse oncological outcomes , the low conversion rate for robotic rectal cancer surgery suggests expected better postoperative courses and improved oncological outcomes [ 28 - 31 ] . pathological outcomes for several prognostic indicators for robotic and laparoscopic surgery groups are in table 2 [ 10 - 27 ] . it is well established that increasing the number of retrieved lymph nodes has a strong impact on prognosis . circumferential resection margin ( crm ) involvement in surgical specimens is a well - known prognostic factor for local recurrence . the length of the distal resection margin and tme completeness are thought to reflect surgical quality and affect long - term oncological outcomes . most studies did not find significant differences between robotic and laparoscopic rectal cancer surgery in number of retrieved lymph nodes . based on recommendations from the college of american pathologists about the minimum number of nodes to be dissected , the reported mean and median numbers of retrieved lymph nodes are acceptable . the length of distal resection margins was not significantly different for robotic , laparoscopic ( or open ) surgery for patients who underwent rectal cancer surgery . the rate of crm involvement was also not different between the two groups in all except one series . macroassessment and microassessment of surgical specimens quality are made by pathologists to determine tme completeness , another predictor of local recurrence . . indicated that the macroscopic completeness of tme for surgical specimens represents tme quality more precisely than crm involvement , because crm involvement is influenced by surgical quality and location , size and progression of the tumor and metastatic lymph nodes . reported that macroscopic tme grading of specimens was significantly better in a robotic surgery group ( 92.9% for robotic vs. 75.4% for laparoscopic surgery , p=0.033 ) . also reported significantly better tme quality in a robotic than a laparoscopic group ( complete tme : 95% vs. 55% , p=0.0003 ) and a trend for a lower rate of crm involvement in the robotic group ( 10% vs. 25% , p=0.1 ) . however , other studies failed to demonstrate the superiority of robotic over laparoscopic surgery for tme quality or reported quantitatively poorer tme quality for robotic than laparoscopic surgery [ 12 - 14,17 ] . because demonstrating the clinical benefits of robotic surgery for rectal cancer is difficult , some studies have evaluated tme quality as the primary endpoint . however , no evidence indicates that better tme quality translates into better survival from robotic rectal cancer surgery . cho et al . performed case - matched analyses of long - term oncological outcomes for fully robotic and laparoscopic tme patients , excluding hybrid robotic surgery . no significant differences were found between the two groups for 5-year overall , or disease - specific , disease - free , or local recurrence - free survival . another study from the same institution by park et al . compared 133 hybrid - robotic surgeries and 84 laparoscopic low - anterior resections for rectal cancer by a single surgeon . the 5-year overall survival and disease - free survival rates were not different between the two groups . cumulative incidence of local recurrence was excellent in both groups : 2.3% in the robotic group and 1.2% in the laparoscopic group ( p=0.649 ) . five - year overall , disease - specific , and disease - free survival rates were not different between the robot and open groups ; however , the 5-year cumulative local recurrence rate was significantly lower in the robot group ( 3.2% vs. 16.1% , p=0.024 ) . at present , no evidence supports that robotic surgery offers major benefits in surgical or oncological outcomes . currently , several randomized controlled trials are underway to address whether robotic surgery has more favorable surgical and oncological outcomes compared with laparoscopic surgery . however , oncological outcomes should be equal , regardless of surgical approach , with strict adherence to surgical principles . for prostate cancer , multiple rarp series have demonstrated the safety , efficiency , and reproducibility of the procedure , although it does not have superior oncological outcomes compared to laparoscopic or open surgery [ 38 - 40 ] . despite the higher medical cost and lack of advantages , we anticipate an increase in rectal cancers treated with robotic surgery without waiting for clear demonstration of its oncological benefits . voiding and sexual dysfunctions after rectal cancer surgery are common and well - known sequelae . the incidences of postoperative urinary and sexual dysfunction have decreased notably since the introduction of tme and autonomic nerve preservation techniques . nonetheless , these adverse events remain a serious problem , with 4%-10% of tme patients reporting urinary dysfunction and 5%-30% reporting sexual dysfunction [ 41 - 44 ] . regardless of the advantages of a laparoscopic approach such as magnified view and direct visualization of the low pelvis , the technical difficulties associated with laparoscopic surgery increase the risk of inadvertent damage to autonomic nerves . in the mrc clasicc trial , laparoscopic rectal resection did not cause voiding dysfunction but tended to adversely affect male sexual function . particularly , tme and conversion to open surgery were identified as independent risk factors for worse male sexual function . robotic tme is hypothesized to achieve better preservation of urinary and sexual functions than laparoscopic tme . technological advantages in robotic surgical systems , including advanced three - dimensional views , finer dissection using the freely articulating endowrist , and stable traction with a third robotic arm are expected to result in greater preservation of autonomic nerves than with laparoscopic surgery . the low conversion rate of robotic tme compared to laparoscopic tme supports this hypothesis and currently available evidence is promising . kim et al . compared voiding and sexual function before and after robotic or laparoscopic tme for rectal cancer . the study demonstrated that voiding and sexual function recovered earlier in a robotic tme group than a laparoscopic group . park et al . compared functional outcomes of robotic and laparoscopic intersphincteric resection for low rectal cancer , and reported a trend toward earlier recovery of male sexual function in the robotic than the laparoscopic group . a report by dannibale et al . demonstrated that erectile function was preserved in all sexually active patients who underwent robotic tme . however , function was preserved only in 43% of patients who underwent laparoscopic tme and this difference was statistically significant ( p=0.045 ) . these results should be interpreted with caution because of the relatively small number of patients enrolled in the three studies . an example is the robotic stapler ( da vinci endowrist stapler 45 ) , which we have used in our recent rectal surgery practice . this stapler is fully controlled from a surgeon s console , provides a full cone of articulation , and features smartclamp feedback . software detects adequate closure of the stapler jaws on the target tissue based on tissue thickness . we are interested in determining whether use of this instrument results in better clinical outcomes , particularly for anastomotic leakage . firefly fluorescence imaging with current surgical robotic systems provides integrated fluorescence images using nearinfrared technology . the images allow surgeons to distinguish cancerous from normal tissue and evaluate organ and tissue perfusion . although indications for fluorescence imaging are limited to assessment of anastomotic perfusion in colorectal surgery , future technological advances and development of new fluorescent agents will expand its clinical applications [ 48 - 50 ] . undoubtedly , future robotic systems will have little resemblance to the machines that are in use today . the extent to which current robotic surgical systems will undergo evolutionary changes over the next decades is unclear . the development of artificial intelligence , miniaturization of surgical robots , incorporation of nanotechnology , and development of telepresence surgery with improved communication systems are all likely to impact robotic surgery in the near future . although robotic rectal cancer surgery is expensive , its safety and feasibility are well established . its lower conversion rate and better surgical specimen quality compared to laparoscopic surgery may potentially improve survival . although initial reports are promising , randomized clinical trials should be performed to definitely determine the safety , efficacy , and long - term oncological and functional benefits of robotic rectal cancer surgery . as the technology for surgical robots continues to advance rapidly , future robotic systems will become increasingly affordable and more refined . adoption of new technology occurs over time , and technological advantages will ensure that robotic surgical systems are eventually part of the normal armamentarium for routine surgical tasks .	during the last decade , robotic surgery for rectal cancer has rapidly gained acceptance among colorectal surgeons worldwide , with well - established safety and feasibility . the lower conversion rate and better surgical specimen quality of robotic compared with laparoscopic surgery potentially improves survival . earlier recovery of voiding and sexual function after robotic total mesorectal excision is another favorable outcome . long - term survival data are sparse with no evidence that robotic surgery offers major benefits in oncological outcomes . although initial reports are promising , more rigorous scientific evaluation in multicenter , randomized clinical trials should be performed to definitely determine the advantages of robotic rectal cancer surgery .
oral cancer is one of the most familiar forms of cancers in the indian subcontinent . from the beginning of twentieth - century , oral squamous cell carcinoma ( oscc ) is more commonly noted in men usually above 40 years of age and only about 0.4 to 3.9% of the patients are affected by oscc earlier than 40 years . controversies exist in literature regarding aggression and prognosis of oscc among young patients ( below 40 years of age ) . general view is that oscc among young patients is more aggressive and has worse prognosis . few studies suggest that 5-year survival rate of patients below 40 years is better than that of patients above 40 years and some other authors state that young patients have a similar clinical course and their survival rate is no different from other age - groups . various histopathological grading systems of oscc have been discussed in literature and broder 's grading has been popular since a long time . advances in diagnostic technology has led to introduction of multifactorial histopathological grading systems such as anneroth et al 's grading system , which is considered appropriate since it provides valuable diagnostic and predictive information of oscc . this study attempts to evaluate aggression of oscc histopathologically by both broder 's and anneroth et al 's . , ( furthermore , an attempt is made to assess the prognosis of oscc in younger patients by matching 5-year recurrence rate among patients > 40 years with 40 years age - group . files of the patients reported with oscc from 1997 to 2005 were retrieved from the records section of the department of oral pathology of a dental institution in india . only those patients whose archival formalin fixed paraffin embedded specimens were available for further processing were considered for this study . patients who did nt take treatments , who had other systemic diseases and who were diagnosed with histopathological variants of oscc were excluded from the study . final number of patients in the study was 75 of which 50 patients were > 40 years of age were randomly selected to make group - i and 25 patients who were 40 years of age were similarly selected to make group - ii . more patients in the elder group were used to increase the statistical power for comparison . archival formalin fixed paraffin embedded tissue blocks of these patients were subjected to soft tissue microtomy for preparation of 5 m thick tissue specimens . subsequently they were stained by hematoxylin and eosin ( h and e ) and microscopic examination was carried out for the purpose of histopathological grading based on broder 's criteria to classify as well - differentiated squamous - cell carcinoma ( wdscc ) , moderately differentiated squamous - cell carcinoma ( mdscc ) and poorly differentiated squamous - cell carcinoma ( pdscc)anneroth et al . , ( 1987 ) criteria which include six parameters assessing are as follows : tumor cell population : degree of keratinization , nuclear polymorphism and mitotic figures [ figures 13]tumor - host relation : pattern of invasion , depth of invasion and lympho - plasmacytic infiltration [ figures 46 ] . further , individual patients were given an overall grade using the sum total grades of six parameters and classified into one of the following three grades ; grade i : 6 - 12 , grade ii : 13 - 18 and grade iii : 19 - 24 . broder 's criteria to classify as well - differentiated squamous - cell carcinoma ( wdscc ) , moderately differentiated squamous - cell carcinoma ( mdscc ) and poorly differentiated squamous - cell carcinoma ( pdscc ) anneroth et al . , ( 1987 ) criteria which include six parameters assessing are as follows : tumor cell population : degree of keratinization , nuclear polymorphism and mitotic figures [ figures 13]tumor - host relation : pattern of invasion , depth of invasion and lympho - plasmacytic infiltration [ figures 46 ] . each parameter was graded from 1 - 4 based on severity . further , individual patients were given an overall grade using the sum total grades of six parameters and classified into one of the following three grades ; grade i : 6 - 12 , grade ii : 13 - 18 and grade iii : 19 - 24 . tumor cell population : degree of keratinization , nuclear polymorphism and mitotic figures [ figures 13 ] tumor - host relation : pattern of invasion , depth of invasion and lympho - plasmacytic infiltration [ figures 46 ] . further , individual patients were given an overall grade using the sum total grades of six parameters and classified into one of the following three grades ; grade i : 6 - 12 , grade ii : 13 - 18 and grade iii : 19 - 24 . , criteria ( h&e stain , 50 ) photomicrograph showing grade 2 nuclear polymorphism of oscc according to anneroth et al . , criteria ( h&e stain , 400 ) photomicrograph showing grade 2 mitotic figures of oscc according to anneroth et al . , criteria ( h&e stain , 1000 ) photomicrograph showing grade 1 mode of invasion of oscc according to anneroth et al . , criteria ( h&e stain , 100 ) photomicrograph showing grade 3 depths of invasion of oscc according to anneroth et al . , criteria ( h&e stain , 100 ) photomicrograph showing grade 3 lympho - plasmacytic infiltrate of oscc according to anneroth et al . , criteria ( h&e stain , 50 ) recurrence of oscc among study subjects within five years of treatment was also evaluated . recurrent lesions are those , which showed local reappearance of oscc or cervical lymph node metastasis or distant metastasis after treatment of the initial lesion . comparison of all the parameters between group - i ( > 40 years ) and group - ii ( 40 years ) was carried out statistically using chi - square test . distribution of study subjects according to broder 's classification is shown in table 1 ; oscc comparison between the younger and older group of patients did nt demonstrate any relevant difference . according to anneroth et al . 's six histopathological parameters , three parameters showed significant difference between the age - groups . all the parameters which exhibited variations were in higher grades among younger age group ( tables 24 ) . distribution of study subjects according to broder 's criteria distribution of subjects according to anneroth et al . , criteria - tumor cell population distribution of subjects according to annerothc et al . , criteria tumor host relationship distribution of study subjects according to overall grade from six parameters total number of patients reported with recurrence in 5 years was 17 out of 75 irrespective of the age - groups . comparison of 5 years recurrence rate between group - i and group - ii did nt appear statistically significant [ table 5 ] 24% of patients showed recurrence in group - i as against 20% in group - ii . while oral cancer accounts for only 2% of all malignancies in uk and usa , it is one of the most common cancers in south east asia , and in india the incidence is as high as one third of all malignancies . about 95% of oral cancers are squamous - cell carcinomas and are commonly seen in elder adults ( > 40 years ) . there is a long - standing debate over the aggression and prognosis of oscc in young patients as compared to older patients and studies have suggested the need for additional research on this topic . comparison of oscc between younger and older age - groups in this study according to broder 's classification did nt show any significant difference . most of the patients among young age groups were diagnosed with mdscc ( 40% ) followed by wdscc ( 36% ) and pdscc ( 24% ) . reports of similar studies such as sasaki et al . , in uk , farnaz et al . , in india suggest wdscc as the most common type of oscc followed by mdscc and pdscc . the difference between wdscc and mdscc cases in these studies and the present study is only nominal suggesting that the common histopathological type of oscc is either wdscc or mdscc irrespective of age . broder 's grading of oscc is a time - honored pathologic tool , but it is slowly expelled from the standard therapeutic planning strategies due to lack of consensus regarding its prognostic value . hence , grading of oscc based only on broder 's criteria will be inconsistent and therefore needs to be reconfirmed with other acknowledged systems . there are various advanced grading systems of oscc proclaiming to be better than broder 's but most of them are done at molecular level and are expensive therefore difficult to adopt in low socioeconomic strata where the incidence of this disease is high . however histopathologic grading that is sensitive and provides valuable diagnostic information that can effectively be applied in the pastoral areas of indian subcontinent is suggested by anneroth et al . in the present study three of the six parameters according to anneroth et al . , criteria showed statistically significant difference between the younger and older groups . differences in nuclear polymorphism , mitosis index and depth of invasion were more obvious in higher grades among young patients when compared to old patients . a study by sasaki et al . , ( 2005 ) used five of the six parameters of anneroth et al . , excluding nuclear polymorphism and found no significant difference between the age - groups . furthermore , when the overall grades of anneroth et al . , parameters were evaluated maximum number of patients in study irrespective of age - groups fell under grade 2 ( 54.66% ) followed by grade 1 ( 40% ) and grade 3 ( 5.33% ) . studies describe that the histopathological parameters of anneroth et al . , are key indicators of tumor aggressiveness and prognostication since they reflect the relationship of the tumor with the host , based on this our results are reminiscent of greater aggression of oscc in younger patients when compared to the older . five - year recurrence rate irrespective of age in the study was lesser compared to other studies and the number of reported recurrences did nt show much variation between the groups . pitman et al . , ( 2000 ) have reported that 45% of their patients showed recurrence in 3 years period with older as well as younger age - groups showing equivalent distribution . similarly pytinia et al . , ( 2004 ) in their study on disease - free survival rates of patients < 40 years and > 40 years of age have not come across any difference . conversely , lacy et al . , ( 2000 ) have reported higher number of older patients with 5-years recurrence in their study ( 56% of older patients compare to 35% of younger patients ) . these results are indicative of either similar or better survival rate for younger patients when compared to older . outcomes of the study are evocative of more aggressive oscc in younger patients which did nt affect the recurrence rate when compared with older patients . the question that needs to be answered is the actual relationship of aggression and prognosis of oscc among young individuals when compared with old individuals . hence , further studies and research on genetics , diet , demographics and tumor host factors will be of a greater value to unveil the reason associated with oral squamous cell carcinoma affecting younger patients .	background : oral squamous cell carcinoma ( oscc ) is commonly noted in elder men , when occurring in younger individuals , its aggression and prognosis is questioned due to biased data in literature . traditionally , various histopathological grading systems have been used for assessing aggression and prognosis of oscc . however , multifactorial grading of anneroth et al . , is considered effective.materials and methods : in this retro - prospective study , files of 75 oscc patients were retrieved from oral pathology department ; among this 50 patients were > 40 years and 25 patients were 40 years of age . archival formalin fixed paraffin embedded tissue blocks of these patients were used to prepare hematoxylin and eosin ( h and e ) stained sections for grading oscc based on broder 's and anneroth et al . , criteria . further , recurrence of oscc among study subjects within 5 years of treatment was evaluated . chi - square test was used to compare the disease in patients who were > 40 years with 40 years.results:comparison according to broder 's classification did nt show any relevant variation . three of the six parameters and overall grading according to anneroth et al . , criteria showed statistically higher grades of oscc in the younger age - group ; however , there was no significant difference in 5-year recurrence rate.interpretation and conclusion : results of the study are suggestive of aggressive oscc among young patients when compared to older . conversely , this aggression did nt affect the recurrence in younger patients . further studies on genetics , diet and demographics of patients below 40 years of age affected by oscc will be of greater value .
penile cancer although rare , is one of the common genitourinary cancers in india affecting mostly aged uncircumcised males with a history of smoking . the age adjusted incidence in rural india is as high as 3 per 100,000 men accounting for more than 6% of malignancies in this population . penile cancer is mostly seen involving glans ( 48% ) and prepuce ( 21% ) . it may arise from the coronal sulcus in 6% of the cases and rarely from the shaft ( < 2% ) . since this superficial lesion in early stages seldom interferes with voiding or erectile function , patients tend to delay seeking medical attention until it has progressed with invasion of deeper tissue and caused infection and necrosis . patients presenting with an early stage preputial lesion t1 - 2 , n0 may require only circumcision for treatment . for small superficial lesions < 3 cm restricted to glans , surgery , radical external radiation or brachytherapy ( bt ) may be offered , the latter being preferred as it allows organ and function preservation . radical bt may be mould therapy or interstitial bt ( low- or high - dose - rate ) . reported the testicular morbidity namely reproductive and sexual is also a concern but rarely reported . from the available literature on fractionated radiation delivered to male pelvic region , it has been demonstrated that doses up to 2 - 3 gy may result in permanent azoospermia while it takes up to 20 gy for affecting testosterone secretion [ 2 , 3 ] . however , the effect of bt doses on testicular function has not been adequately described . with an aim to minimize and document the doses to testis after adequate shielding during high - dose - rate ( hdr ) radical interstitial bt for penile cancers , we undertook this study in two patients and forms the basis of this report . two patients aged 30 and 51 years with early penile cancer t1/t2 lesion - limited to the glans were treated with radical hdr ir bt using interstitial implant . a total of 7 - 8 tubes were implanted in two planes , parallel to the penile shaft . american brachytherapy society ( abs ) and groupe europen de curiethrapie of the european society for radiotherapy and oncology ( gec - estro ) recommend a template based multiplanar implant with the needles placed orthogonally to the longitudinal direction of the penis . however , we prefer non template based insertion of tubes along the longitudinal direction of the penis with adequate lateral margin , hence , avoiding the soft tissue injury caused due to template and needles being in situ for a long time , generally between 5 to 7 days . freehand insertion has another advantage of maintained target position relative to the tubes as the post procedure edema settles . two planes , deep and superficial , sufficiently cover the tumor thickness with margin while restricting doses to the urethra . brachytherapy treatment planning was done on oncentra tps and treatment delivered with mhdr v3 remote afterloading system ( nucletron , an elekta company , elekta ab , stockholm , sweden ) . a total dose of 44 - 48 gy was delivered in 11 - 12 fractions of 4 gy each delivered twice daily , which is higher than recommended by crook et al . 38.4 gy at 3.2 gy per fraction , twice daily 6 hours apart over 6 days , but well tolerated in our patient population . owing to the proximity of the implant to testes , an attempt was made to minimize the scattered dose to this organ by interposing a lead shield between the penile shaft and scrotum . the shield was made of lead sheets 14 cm in length and 6.5 cm in width . the cumulative thickness was 1.1 cm , approximately equivalent to 4 - 5 half value layer ( hvl ) for ir . the testicular dose was measured using lif thermoluminescent dosimeters ( tld ) , a 1 cm 1 cm polyethylene pouch consisting of approximately 40 mg of tld-100 ( lif : mg , ti ) powder ( the harshaw chemical co. solon , ohio , usa ) in it , so that at least three readings could be procured from it . prior to each irradiation , the tld powder was annealed using thermal cycle 400c ( 5c ) for 1 h , cooling for 5 min and 100c for 2 h in programmable muffle furnace ( model-126 , fisher scientific co , pittsburgh , usa ) , and then cooled to room temperature . testes were palpated and the first set of tlds placed on the anterior scrotal skin on either side immediately anterior to the center of the testes ( entry dose ) . similarly , the second set were placed immediately posterior to the testes ( exit dose ) . the average of the two value sets was a fair estimate of the dose received by the testes . the lead shield was then placed over the scrotum and a tld placed in the center over the shield , facing the posterior aspect of shaft . this would act as a surrogate for the testicular dose received in the absence of shielding . these 5 measurements were carried out for 3 fractions for each patient and mean values computed ( figures 1 and 2 ] . schematic representation of thermoluminescent dosimeters placement patient a without ( a ) and with ( b ) lead shield in place a constant time gap of 24 h was maintained between irradiation and readout . usa ) was used to record tl output at maximum acquisition temperature of 280c using constant heating rate of 14c / sec . for each readout , hence , 4 readings were obtained from each tld pouch and the mean value of net tl output per unit weight of these readings was used for calculation of absorbed dose . dose response curve for the tld-100 powder was generated in co gamma ray beam ( equinox 80 , mds nordion , canada ) . irradiation was performed in a virtual water phantom and the measuring point had at least 10 cm of scatter material on all sides to provide full scattering condition . to measure the efficacy of the shield in reducing testicular dose , a wax phantom of size 3 3 cm with separation the source was positioned on the anterior side and irradiated for 2 minutes with single dwell position . the tlds were placed on the posterior aspect , just below the source and above the first lead sheet . the lead sheets were arranged , one below the other , and tlds placed below each sheet subsequently to measure their individual attenuation . the measured dose was plotted against the lead thickness to study the dose attenuation and determine half value thickness ( hvt ) for mhdr ir source . a total of 7 - 8 tubes were implanted in two planes , parallel to the penile shaft . american brachytherapy society ( abs ) and groupe europen de curiethrapie of the european society for radiotherapy and oncology ( gec - estro ) recommend a template based multiplanar implant with the needles placed orthogonally to the longitudinal direction of the penis . however , we prefer non template based insertion of tubes along the longitudinal direction of the penis with adequate lateral margin , hence , avoiding the soft tissue injury caused due to template and needles being in situ for a long time , generally between 5 to 7 days . freehand insertion has another advantage of maintained target position relative to the tubes as the post procedure edema settles . two planes , deep and superficial , sufficiently cover the tumor thickness with margin while restricting doses to the urethra . brachytherapy treatment planning was done on oncentra tps and treatment delivered with mhdr v3 remote afterloading system ( nucletron , an elekta company , elekta ab , stockholm , sweden ) . a total dose of 44 - 48 gy was delivered in 11 - 12 fractions of 4 gy each delivered twice daily , which is higher than recommended by crook et al . 38.4 gy at 3.2 gy per fraction , twice daily 6 hours apart over 6 days , but well tolerated in our patient population . owing to the proximity of the implant to testes , an attempt was made to minimize the scattered dose to this organ by interposing a lead shield between the penile shaft and scrotum . the shield was made of lead sheets 14 cm in length and 6.5 cm in width . the cumulative thickness was 1.1 cm , approximately equivalent to 4 - 5 half value layer ( hvl ) for ir . the testicular dose was measured using lif thermoluminescent dosimeters ( tld ) , a 1 cm 1 cm polyethylene pouch consisting of approximately 40 mg of tld-100 ( lif : mg , ti ) powder ( the harshaw chemical co. solon , ohio , usa ) in it , so that at least three readings could be procured from it . prior to each irradiation , the tld powder was annealed using thermal cycle 400c ( 5c ) for 1 h , cooling for 5 min and 100c for 2 h in programmable muffle furnace ( model-126 , fisher scientific co , pittsburgh , usa ) , and then cooled to room temperature . testes were palpated and the first set of tlds placed on the anterior scrotal skin on either side immediately anterior to the center of the testes ( entry dose ) . similarly , the second set were placed immediately posterior to the testes ( exit dose ) . the average of the two value sets was a fair estimate of the dose received by the testes . the lead shield was then placed over the scrotum and a tld placed in the center over the shield , facing the posterior aspect of shaft . this would act as a surrogate for the testicular dose received in the absence of shielding . these 5 measurements were carried out for 3 fractions for each patient and mean values computed ( figures 1 and 2 ] . schematic representation of thermoluminescent dosimeters placement patient a without ( a ) and with ( b ) lead shield in place rexon ul-320 tld reader ( tld systems inc . , usa ) was used to record tl output at maximum acquisition temperature of 280c using constant heating rate of 14c / sec . for each readout , hence , 4 readings were obtained from each tld pouch and the mean value of net tl output per unit weight of these readings was used for calculation of absorbed dose . dose response curve for the tld-100 powder was generated in co gamma ray beam ( equinox 80 , mds nordion , canada ) . irradiation was performed in a virtual water phantom and the measuring point had at least 10 cm of scatter material on all sides to provide full scattering condition . to measure the efficacy of the shield in reducing testicular dose , a wax phantom of size 3 3 cm with separation 4 cm was used . the source was positioned on the anterior side and irradiated for 2 minutes with single dwell position . the tlds were placed on the posterior aspect , just below the source and above the first lead sheet . the lead sheets were arranged , one below the other , and tlds placed below each sheet subsequently to measure their individual attenuation . the measured dose was plotted against the lead thickness to study the dose attenuation and determine half value thickness ( hvt ) for mhdr ir source . as detailed in material and methods , two patients underwent tld measurements for 3 fractions of hdr and extrapolated to the remaining fractions . patient a : a total of 44 gy in 11 fractions was delivered at 4 gy per fraction , twice daily 6 h apart . the dose to left and right testes was 2.88 0.10 cgy and 3.89 0.74 cgy , respectively , resulting in a cumulative dose of 31.68 1.10 cgy and 42.79 8.14 cgy . actual thermoluminescent dosimeters readings and calculated parameters obtained for the two patients readings r1 , r2 , and r3 obtained on tld 1 ( penis post ; ventral surface of penile shaft ) , tld 2 ( lt ant ; anterior surface of left testis ) , tld 3 ( lt post ; posterior surface of left testis ) , tld 4 ( rt ant ; anterior surface of right testes ) , and tld 5 ( rt post ; posterior surface of right testes ) . lt mid calc and rt mid calc denote calculated mean of the doses to the anterior and posterior surfaces of the left and right testes , respectively , as measured in 3 fractions . patient b : a total of 48 gy in 12 fractions was delivered at 4 gy per fraction , twice daily 6 h apart . the dose to left and right testes was 1.83 0.40 cgy and 1.94 0.07 cgy , respectively , resulting in a cumulative dose of 21.96 4.80 cgy and 23.28 0.84 cgy . the mean dose measured at the posterior aspect of penile shaft was 65.65 6.1 cgy and 67.3 5.6 cgy for the two patients , amounting to 16.4% and 16.8% of the total delivered dose . this dose being a surrogate for the unshielded testicular dose , one can safely assume that unshielded , the cumulative testicular dose would be 722.15 67.1 cgy and 807.72 67.44 cgy for two patients , respectively . hence , the application of lead shield 1.1 cm thick reduced testicular dose from 722 - 808 cgy to 21.96 - 42.57 cgy , an absolute reduction of 95.99 1.5% . the measured hvt for mhdr ir source using phantom dosimetry was found to be 2.8 mm of lead , which is in accordance with the published data of 2.1 to 2.8 mm . patient a : a total of 44 gy in 11 fractions was delivered at 4 gy per fraction , twice daily 6 h apart . the dose to left and right testes was 2.88 0.10 cgy and 3.89 0.74 cgy , respectively , resulting in a cumulative dose of 31.68 1.10 cgy and 42.79 8.14 cgy . actual thermoluminescent dosimeters readings and calculated parameters obtained for the two patients readings r1 , r2 , and r3 obtained on tld 1 ( penis post ; ventral surface of penile shaft ) , tld 2 ( lt ant ; anterior surface of left testis ) , tld 3 ( lt post ; posterior surface of left testis ) , tld 4 ( rt ant ; anterior surface of right testes ) , and tld 5 ( rt post ; posterior surface of right testes ) . lt mid calc and rt mid calc denote calculated mean of the doses to the anterior and posterior surfaces of the left and right testes , respectively , as measured in 3 fractions . patient b : a total of 48 gy in 12 fractions was delivered at 4 gy per fraction , twice daily 6 h apart . the dose to left and right testes was 1.83 0.40 cgy and 1.94 0.07 cgy , respectively , resulting in a cumulative dose of 21.96 4.80 cgy and 23.28 0.84 cgy . the mean dose measured at the posterior aspect of penile shaft was 65.65 6.1 cgy and 67.3 5.6 cgy for the two patients , amounting to 16.4% and 16.8% of the total delivered dose . this dose being a surrogate for the unshielded testicular dose , one can safely assume that unshielded , the cumulative testicular dose would be 722.15 67.1 cgy and 807.72 67.44 cgy for two patients , respectively . hence , the application of lead shield 1.1 cm thick reduced testicular dose from 722 - 808 cgy to 21.96 - 42.57 cgy , an absolute reduction of 95.99 1.5% . the measured hvt for mhdr ir source using phantom dosimetry was found to be 2.8 mm of lead , which is in accordance with the published data of 2.1 to 2.8 mm . the reported tvl value for ir ranges from 9.2 to 11.9 mm of lead . radical bt is a viable option for penile preservation in selected patients with t1-t2 lesions < 4 cm in diameter . results of bt for penile carcinoma are most often reported in the form of institutional case series [ 7 , 8 , 9 , 10 , 11 ] . a meta - analysis by hasan et al . of 17 such studies reports 5 year overall survival of 73% , 5 year local control of 79% with an organ preservation rate of 74% in a cohort of 673 patients . although penectomy provides better control , there is no survival benefit , implying that in most cases salvage surgery is a feasible option in case of recurrence . nearly 20 - 30% of patients develop side effects , such as telangiectasia , depigmentation , fibrosis , sclerosis , and less frequently urethral stenosis and necrosis . besides the urethra , it is also important to minimize the doses to testes . testes are very sensitive to radiation and side effects are dose dependent . literature reports many adverse effects of scattered dose to testis , including oligospermia , azoospermia , temporary or permanent , testicular atrophy , leydig cell dysfunction with impairment of testosterone production , and genetic risk of hereditary disease or developmental impairment of the offspring of irradiated patients [ 3 , 13 , 14 , 15 , 16 ] . elaborating on radiation sensitivity of testes , they reported that with fractionated delivery , doses of 70 - 90 cgy result in oligospermia with recovery at 1 - 1.5 years . single fraction spermicidal doses are 0.1 - 0.2 gy for spermatogonia , 2 - 3 gy for spermatocytes and 4 - 6 gy for spermatids . with doses of 6 - 8 equally important would be to measure and document the doses during treatment and systematically correlate these with toxicities . in the present study , we attempted to define two issues . firstly , we utilized the lead shielding to minimize the doses to testes . secondly , radiation dose measurements by using tld at anterior and posterior surface of the scrotum were done to compute testicular doses . with the use of a simple lead shield as described , we were able to effectively reduce testicular dose from spermicidal ( 6 - 8 gy ) range to oligospermic ( < 0.7 gy ) range with possible reversibility . the backscatter from lead shield is estimated to increase the measured dose at posterior shaft by about 1 - 2% , hence the actual dose at that point in the absence of lead shield would be about 707 - 791 cgy . the advantage of tld is its linearity of response to dose , relative energy independence , and sensitivity to low doses . have published reports emphasizing the need to address the dosimetric uncertainties in brachytherapy , hence , this discussion would be incomplete without addressing this issue ( table 2 ) . taking the dosimetric uncertainties into account , the upper limit of dose received by the testes would be about 46 cgy , in the presence of shielding . direct measurements of doses to testes using tld during high - dose - rate bt for penile cancers allows for objective documentation , which could be helpful for better correlation with late toxicities .	purposepenile cancer , although rare , is one of the common genitourinary cancers in india affecting mostly aged uncircumcised males . for patients presenting with small superficial lesions < 3 cm restricted to glans , surgery , radical external radiation or brachytherapy may be offered , the latter being preferred as it allows organ and function preservation . in patients receiving brachytherapy , testicular morbidity is not commonly addressed . with an aim to minimize and document the doses to testis after adequate shielding during radical interstitial brachytherapy for penile cancers , we undertook this study in 2 patients undergoing brachytherapy and forms the basis of this report.material and methodstwo patients with early stage penile cancer limited to the glans were treated with radical high - dose - rate ( hdr ) brachytherapy using interstitial implant . a total of 7 - 8 tubes were implanted in two planes , parallel to the penile shaft . a total dose of 44 - 48 gy ( 55 - 60 gy eqd2 doses with / = 10 ) was delivered in 11 - 12 fractions of 4 gy each delivered twice daily . lead sheets adding to 11 mm ( 4 - 5 half value layer ) were interposed between the penile shaft and scrotum . the testicular dose was measured using thermoluminescent dosimeters . for each patient , dosimetry was done for 3 fractions and mean calculated.resultsthe cumulative testicular dose to left and right testis was 31.68 cgy and 42.79 cgy for patient a , and 21.96 cgy and 23.28 cgy for patient b. for the same patients , the mean cumulative dose measured at the posterior aspect of penile shaft was 722.15 cgy and 807.72 cgy , amounting to 16.4% and 16.8% of the prescribed dose . hence , the application of lead shield 11 mm thick reduced testicular dose from 722 - 808 cgy to 21.96 - 42.57 cgy , an absolute reduction of 95.99 1.5%.conclusionswith the use of a simple lead shield as described , we were able to effectively reduce testicular dose from spermicidal range to oligospermic range with possible reversibility .
diabetes is one of the major risk factors for kidney damage , and people with diabetes have significantly increased risk for ckd . the prevalence of diabetes nephropathy in asian type 2 diabetes mellitus patients was as high as 58.6% , and 63.9% of the chinese patients diagnosed with type 2 diabetes ( t2d ) had ckd with varying stages . it remains controversial whether common anthropometric measures of obesity , including body mass index ( bmi ) , waist circumference ( wc ) , and waist - to - hip ratio ( whr ) , are related to the risk of ckd . a recent research found u - shaped associations of these three obesity measures with urinary albumin - to - creatinine ratio ( uacr ) or microalbuminuria in the general population . another study showed that , among healthy men , higher baseline bmi was associated with increased risk for ckd . in contrast , another research reported that whr , but not bmi , was associated with incident ckd . the relationship of these obesity measures to ckd is even less clear in patients with t2d , and findings in the literature are largely inconsistent and vary by populations . in the look ahead ( action for health in diabetes ) study , increased bmi and wc were associated with albuminuria in overweight and obese adults with t2d . however , an epidemiology study in uk showed that bmi was not related to the progression of ckd in patients with diabetes . a study of 125 overweight t2d patients reported that waist - to - height ratio , rather than waist circumference , was associated with ckd . compared to caucasians , asians have significantly higher percentage of body fat at lower mean bmi and are more likely to suffer obesity related diseases such as diabetes . prior studies on asian patients showed that central obesity measures such as wc , but not bmi , were associated with 24-hour urinary albumin excretion rate and decline in renal function [ 11 , 12 ] . to the best of our knowledge , there is only one study that investigated the association between anthropometric measures of obesity and ckd in patients with t2d in the chinese population in hong kong . in this study , we aimed to investigate the association of bmi , wc , and whr with the risk of ckd in t2d patients in mainland china . this cross - sectional study included t2d patients who were treated in the outpatient department between october 2012 and may 2013 . patients were diagnosed with t2d if they ( 1 ) had no episodes of ketoacidosis ; ( 2 ) were diagnosed with diabetes over 30 years of age ; and ( 3 ) were not administrated with insulin during the first three years since the diagnosis of diabetes . for each patient , a personal interview was conducted to gather basic demographic data , including age , sex , education , smoking ( never versus ever ) , and drinking status ( never versus ever ) . information about past medical history and time since the diagnosis of diabetes ( months ) was also collected . the initial sample size consisted of 886 t2d mellitus patients who were 30 years of age and older . four patients were excluded due to missing values for serum creatinine and/or urinary albumin , and an additional 12 patients had missing data on demographics or anthropometric measures . as a result , a total of 870 patients were included in this study . the study was conducted in accordance with the declaration of helsinki , and informed consent forms were obtained from all the patients . height was measured to the nearest 0.1 cm using a digital ultrasound instrument , and weight was measured to the nearest 0.1 kg in light clothing and without shoes using standard digital scales . wc was measured to the nearest 0.1 cm between the lower rib margin and the iliac crest in the horizontal plane , with the subject standing comfortably with weight distributed evenly on both feet . hip circumference was measured to the nearest 0.1 cm and was recorded as the greatest circumference around the gluteal region . bmi was calculated as the weight in kilograms divided by the square of height in meters . whr was calculated as the ratio of wc divided by hip circumference . in this paper , we focus on the analysis of the three most common anthropometric measures , including bmi , wc , and whr . patients fasted for 812 hours overnight before their urine specimens and blood samples were collected in the morning . blood samples were taken from the cubital vein of the patients in the supine position and prepared for immediate analysis or stored at 80c for further analysis . plasma total cholesterol ( tc ) and triglyceride were measured by enzymatic methods . hemoglobin a1c ( hba1c ) was measured by high - performance liquid chromatography ( hplc ) using the bio - rad d10 hemoglobin testing system . urinary albumin was measured from fresh morning spot urine by the immunoturbidimetric assays and urinary creatinine was measured by the colorimetric method . urinary albumin - to - creatinine ratio ( uacr ) was calculated as uacr = urinary albumin concentration ( mg / l)/urinary creatinine concentration ( mmol / l ) . when the two uacr measurements differed by more than 10% , another collection was made to assess the adequacy of the collections . ckd was defined as estimated glomerular filtration rate ( egfr ) < 60 mg / min/1.73 m or the presence of albuminuria . here , egfr was calculated using the cockcroft - gault equation : [ ( 140-age ( years ) ) body weight ( kg)/72 serum creatinine ( mg / dl ) ] 0.85 ( if female ) , and albuminuria was defined as uacr 2.7 mg / mmol . blood pressure ( bp ) was measured from the left arm with a mercury sphygmomanometer with the patient in a sitting position and after a 5-minute rest . hypertension was defined as systolic blood pressure 140 mmhg or diastolic blood pressure 90 mmhg or the existence of a previous diagnosis of hypertension . chi - square test was used to compare the categorical variables , and continuous variables were compared using student 's t - test or wilcoxon rank - sum test as appropriate . we imputed missing data based on the other variables in the data and repeated the process 10 times , resulting in 10 imputed datasets . the relation of anthropometric obesity measures to ckd was examined using multivariate logistic regression models with the presence of ckd as the binary outcome , adjusting for age , sex , education , smoking status ( ever versus never ) , duration of diabetes , hba1c , hypertension , and triglyceride . this was done using the 10 imputed datasets with the zelig package [ 15 , 16 ] . all data analyses were performed using r ( http://www.r-project.org/ ) or sas version 9.3 ( sas institute inc . , cary , nc ) and we used a nominal threshold of p < 0.05 for statistical significance . the mean age was 60.2 9.6 years ( range : 30 to 88 years ) , and 40% were males . on average , they had 8.0 3.1 years of education ( range : 0 to 20 years ) . the mean time since the diagnosis of t2d was 9.9 7.1 months ( range : 091 months ) . briefly , patients with ckd were , on average , older in age ( p < 0.0001 ) and had fewer years of education ( p = 0.001 ) . patients with ckd had higher wc and whr ( both p 's < 0.0001 ) . interestingly , we did not find a significant difference in bmi between patients with and without ckd ( p = 0.493 ) . furthermore , compared to patients without ckd , patients with ckd were more likely to have hypertension ( p < 0.0001 ) ; and they also had higher systolic and diastolic bp ( both p 's < 0.0001 ) . we found that patients with ckd had higher levels of blood glucose , both fasting and postprandial ( both p 's < 0.0001 ) , and higher hba1c ( p < 0.0001 ) . levels of serum triglyceride ( p = 0.0003 ) and total cholesterol ( p = 0.049 ) were also significantly higher among patients with ckd . we conducted logistic regression analysis to examine the association of bmi , wc , and whr with ckd ( table 2 ) , adjusting for age , sex , education , smoking status ( ever versus never ) , time since diagnosis of diabetes , hba1c , hypertension , and triglyceride . we did not find a significant association of bmi with ckd ( or 1.010 , 95% ci : 0.9781.043 , p = 0.550 ) . similarly , whr was not associated with ckd ( or 1.191 , 95% ci : 0.9261.532 , p = 0.174 ) . on the other hand , higher wc was associated with greater odds for ckd such that , for every 1 cm increase in the circumference , the odds of having ckd increased by approximately 2% ( or = 1.019 , 95% ci = 1.0021.036 , p = 0.030 ) . surprisingly , patients with small wc ( < 90 cm in males and < 85 cm in females ) had a relatively high proportion of ckd . in males , the proportion of ckd reached a plateau for those with wc 100 cm , while in females , among those with wc 85 cm , the proportion of cdk increases as wc increases . we performed sensitivity analysis by excluding male subjects with wc < 90 cm and female subjects with wc < 85 cm and found similar association between wc and ckd ( or 1.028 , 95% ci 1.0061.051 , p = 0.012 ) . a further examination by age indicates that the effect of wc on ckd risk is most significant for middle - aged patients , while , for patients aged 45 years or younger and those aged 80 years or older , there is no dramatic difference in ckd risk between those having large wc and those having small wc ( figure 2 ) . in this cross - sectional study , using data from 870 patients with t2d , we examined the association of obesity measures with the risk of ckd . we found that , as a central obesity measure , wc was associated with greater odds for ckd , independent of known ckd risk factors , and the association is age specific such that the effect of wc on ckd risk is most significant for middle - aged patients . our results contribute to the research on ckd among patients with t2d in several important ways . our findings lend empirical support that , in chinese patients with t2d , central obesity measure wc is more strongly associated with ckd than bmi . data from a clinical trial in the us supports that both bmi and waist circumference are associated with increased level of albuminuria . in contrast , a study of 1760 outpatients with t2d in italy found that bmi does not influence the progression rate of ckd in patients with t2d . a separate study in the us reported a similar finding where whr , but not bmi , is associated with incident ckd . another study reported that higher bmi reduced the deterioration of renal function in t2d patients with ckd stage 3 or 4 by reducing glomerular filtration rate decline . one possible reason for the inconsistency in the findings can be attributable to the obesity paradox where bmi , as a measure of overall level of adiposity or general obesity , does not distinguish fat and muscle mass , while more muscle mass results in higher serum creatinine concentration . on the other hand , central obesity measures , such as wc and whr , serve as better markers for visceral fat in ckd . separately , the difference in the patient populations may also contribute to the disparity of the findings . compared with the white population , asian population in general had a higher percentage of body fat at a lower bmi [ 22 , 23 ] . a recent study reported that , in korea , central obesity measures were associated with decline in renal function , but not bmi . however , data from the hong kong diabetes registry showed that higher bmi was protective against the risk of incident ckd in chinese patients with t2d . another study in turkey examined the relationship of four anthropometric measures ( bmi , wc , whr , and conicity index ) with 24-hour urinary albumin excretion rate and found that only wc exhibited independent association . a recent study in iran found that , in men , waist gain but not decrease in wc was associated with ckd risk , but no association of wc with ckd risk was found in women . these studies imply racial / ethnic disparities in the association of various obesity measures with risk of ckd . interestingly , we found higher proportion of ckd in patients with very low ( < 90 cm in men or < 85 cm in women ) or very high wc ( 110 cm in men or 105 cm in women ; figure 2 ) . this is consistent with findings from a recent study which reported higher odds of ckd in patients with low or high quintiles of wc . previous studies found a u - shape between bmi and all - cause mortality such that low or high bmi is associated with high mortality risk while the lowest mortality risk is reached for bmi between 22.5 and 25 kg / m . low anthropometry might represent an alteration of internal hemodynamics and patients with low wc may suffer damaged glomerulus and have different pathophysiology compared to obese patients [ 4 , 25 , 26 ] . previous researches imply that part of the reasons accounting for the discrepancy between the results regarding the relationship between central obesity and ckd risk might be due to different age distribution of the included participants . for example , a previous study in china that found wc was associated with increased ckd risk included subjects aged 3574 years , while two other studies in china that failed to find significant association included younger subjects ( 30 years old and 20 years old ) . few studies have examined the age - specific association of central obesity with ckd risk , and our results indeed indicate that the effect of wc varies by age . we speculate that the insignificant effect of wc on ckd risk in young and elder patients might be due to the fact that ( 1 ) young patients had relatively short duration of diabetes , which was a significant risk factor of ckd ( p = 0.01 ) , leading to less likelihood of developing ckd , and ( 2 ) older patients might suffer muscle loss and , as a result , the commonly used obesity measures , such as wc , might not be applicable to them . more longitudinal studies are needed to validate the age - specific effect of wc on ckd risk . first , although we have controlled potential confounders , the possibility of residual confounding by other unknown or unmeasured factors can not be ruled out . second , the cross - sectional nature of this study precludes analysis of longitudinal data and any causal inference . finally , participants of this study came from a single hospital , and our findings may not be generalizable to other populations . in summary , in this study , we examined the association of obesity measures with ckd risk in patients with t2d . we found that wc is significantly associated with ckd , independent of known ckd risk factors , but no association was found for bmi , indicating that central obesity , but not general obesity , might contribute to the pathogenesis of ckd . future studies are needed to validate our findings and to explore the mechanism underlying the observed association .	objective . to examine the association of three most common obesity measures including body mass index ( bmi ) , waist circumference ( wc ) , and waist - to - hip ratio ( whr ) with chronic kidney disease ( ckd ) risk in patients with type 2 diabetes mellitus ( t2d ) . design . cross - sectional evaluation of the effect of anthropometric measures on ckd risk . setting . outpatient department . subjects . t2d patients who were treated between october 2012 and may 2013 . intervention . none . main outcome measure . ckd risk . results . on average , the patients had a mean age of 60.2 years , and 40% were males . ckd was present in 46% of all the patients . in multivariate logistic regression using the imputed data , higher wc was associated with greater odds of ckd ( or = 1.019 , 95% ci = 1.0021.006 , p = 0.030 ) , but not bmi and whr . interestingly , we found that patients with very small wc seemed to have greater odds of ckd . we observed age - specific effect of wc such that the effect of wc on ckd risk is significant only in middle - aged t2d patients . conclusion . our study provides evidence for the association of wc with ckd in chinese patients with t2d . t2d patients , especially middle - aged t2d patients , should reduce their wc to decrease ckd risk .
over the past decades , superparamagnetic ( sp ) nanostructures of spinel ferrites ( mfe2o4 : m = fe , co , cu , zn , etc . ) have drawn intense scientific and technological interests because they possess a wide range of applications in magnetic fluid [ 1 - 3 ] , magnetic resonance imaging ( mri ) , and drug delivery technology [ 6 - 8 ] . recently , spinel cobalt ferrite ( cofe2o4 ) nanostructure materials have been extensively studied because they form a magnetic system which is an ideal candidate toward understanding and controlling magnetic properties at the atomic level through chemical manipulation . based on different techniques , sp cofe2o4 nano - scale particles have been synthesized by several research groups [ 10 - 13 ] . unfortunately , these small nanoparticles , especially those with sizes below 10 nm , have poor magnetic response abilities . the low magnetization properties caused by small size limit their usage in a number of practical applications since they can not be effectively manipulated by using moderate magnetic fields . in order to obtain a high saturation magnetization ( ms ) , simply making particles larger can not be an option at all , resulting in a strong aggregation due to the non - sp ferromagnetic attraction . recently , much effort has been focused on the preparation of large - size sp particles with high ms using simple composites . for example , lee et al . reported the fabrication of highly uniform sp mesoporous spheres with sub - micrometer scale , composed of silica and cofe2o4 achieved a high magnetization value . similar efforts were done by bao et al . in which controlled growth of sp nanostructures of spherical and rod - like cofe2o4 nanocrystals . however , their high ms required a high reaction temperature and expensive high - boiling point toxic solvents , which are disadvantages for their biological applicability . moreover , such composite nanostructures are incompatible with low expense of preparing procedure . herein , we report a single - step process for high - performance monodisperse sp cofe2o4 mss at low temperature ( at only 180c ) . as a consequence of the low - temperature synthesis , the as - obtained samples possess a high ms ( over 55 emu / g ) and biocompatibility . cobalt acetate [ co(ch3coo)2 4h2o ] , anhydrous ethanol(ch3ch2oh ) , iron(iii ) nitrate nonahydrate [ fe(no3)3 9h2o ] , and poly(vinyl pyrrolidone ) ( pvp , k30 ) are of analytic grade reagents and purchased without further treatment . in a typical reaction , 2 mmol co(ch3coo)2 4h2o and 4 mmol fe(no3)3 9h2o were dissolved in 35 ml ch3ch2oh forming a reddish brown solution , after which 0.2 g pvp was added . the mixture was stirred vigorously for 30 min and then sealed in a teflon - lined stainless steel autoclave ( 50 ml capacity ) . the autoclave was heated to and maintained at 180c for 1224 h and then was allowed to cool to room temperature . the dark products were subjected to magnetic decantation , followed by repeated washing with distilled water , ethanol , and acetone . the final products were dried in a vacuum oven at 60c for 8 h. phase and composition analyses of the products were performed by x - ray diffraction / photoelectron spectroscopy [ xrd / xps ( philips x pert prodiffractometer with cu k ( = 1.54056 ) radiation)/(axis ultra xps , al k ) ] . the size and morphology of the as - synthesized mss were investigated by using scanning / transmission electron microscopy ( sem / tem , jsm5600lv / jeol100cx - ii ) . figure 1a shows a typical xrd pattern of the as - fabricated sample ( reaction for 24 h ) . all diffraction peaks can be indexed to a simple cubic lattice ( fcc ) , and the positions along with relative intensity of peaks match well with standard cofe2o4 powder diffraction database ( jcpds file no . 22 - 1086 ) , indicating that the as - obtained products have an fd3 m cubic spinel structure . according to the scherrer equation , the average crystallite size which is calculated based on the xrd pattern ( 311 ) is approximately 8 nm . in fact , the xrd patterns of the others ( reaction for 12 and 36 h ) are similar to it ( not shown ) . figure 1b and 1c show the high - resolution xps spectra of 2p co and fe , respectively . the peak at 780.8 ev is from co2p3/2 , with a shake up satellite at 785.9 ev , while the peak at 797.2 ev is caused by co2p1/2 , with a satellite peak at 803.0 ev . the presence of those two peaks and the highly intense satellites near them is consistent with the presence of co in the high - spin state . all the fe2p spectra generally show a main peak at a binding energy ( be ) of around 710.3 ev , accompanied by a satellite line visible at a be of around 718.3 ev , only indicative of the presence of fe cations . further quantitative analysis finds that the atomic ratio between co and fe is about 1:2 , which is compatible with the data of xrd . the as - synthesized cofe2o4 mss for a xrd pattern , and xps spectra b co2p and c fe2p the sem images of the as - synthesized samples obtained at different reaction times are illustrated in fig . the average size of the cofe2o4 mss is ~220 nm [ solvothermal treated for 12 h ( fig . however , when extending the reaction time to 24 h , the average size increases to ~330 nm ( as revealed in fig . 2c and 2d , these micrometer - sized cofe2o4 spheres [ ~220 nm ( fig . 2c ) and ~330 nm ( fig . 2d ) in diameter , respectively ] can clearly be seen . additionally , an individual sphere is not a single microparticle but the assemblies of small cofe2o4 nanoparticles ( the diameter of ~8 nm ) , in which the size of primary nanoparticles is in excellent agreement with the xrd results . sem / tem images of products prepared at different reaction time : a / c 12 h , b / d 24 h to obtain a better understanding of the formation and evolution of cofe2o4 mss along with the reaction time , that the reaction duration was further extended to 36 h was carried out . the mss were evacuated resulting in octahedral - shaped cofe2o4 particles , which are confirmed by the sem image ( fig . 3b ) . as a consequence , a proposed mechanism of formation and evolution of the cofe2o4 mss it is well known that pvp can selectively absorb on a certain crystal facet of the as - prepared primary building blocks such as nanoparticles , nanosheets , nanoplates , nanorods , and so on [ 20 - 23 ] . in our experimental system , pvp surfactant contributes not only to preventing these primary building blocks from entropy - driven random aggregation but also to controlling the formation of the regular geometry . the formation and evolution of mss seems to be as follows : at first , this cofe2o4 phase undergoes consequent nucleation and growth around the entire surface stabilized by pvp . in the subsequent process , driven by the minimization of the total energy of the system , the small primary cofe2o4 nanoparticles aggregated together to form three - dimensional ( 3d ) spheres . ostwald ripening occurred under solvothermal conditions , resulting in the formation of cofe2o4 spheres from small grows into larger due to isotropic growth . the octahedral shape of the product obtained by reaction for 36 h may be explained by combination of thermodynamic aspects of crystal growth and the selective adsorption model of surfactants on different crystallographic facets . moreover , the structures may be deduced that development of the ( 111 ) facets was handicapped by the adsorbed pvp surfactant on these facets . a sem / b tem images of studied samples with 36 h reaction time schematic illustration of the formation and evolution of cofe2o4 mss along with the reaction time the magnetic behavior of cofe2o4 mss is very important for practical applications . 5a , 5b ) display the two relatively high ms values of 41.2 ( taken from 220 nm ) and 55.2 emu / g ( 330 nm ) , respectively . both values are , however , somewhat lower than the bulk value ( 71.2 emu / g ) . the hysteresis loop shows essentially no coercivity ( hc ) for the smaller size samples ( 220 nm ) and negligible value of 27 oe ( larger size specimens , a diameter of 330 nm ) , suggesting that improved coalescence of the crystallites in the nanostructures results in increased magnetic coupling and higher magnetization . according to the results of xrd and tem observations , the average size of primary crystals is about 8 nm , smaller than the sp critical size of cofe2o4 . so it is reasonable that the self - assembled cobalt ferrite mss reveal sp behavior even though their sphere size exceeds 200 nm . the stabilization of the mss in distilled water thanks to the surfaces capped of hydrophilic pvp . slight agitation can bring the mss to back into the aqueous solution when the magnet is removed ( fig . 5c ) although these magnetic mss can be completely separated from the solution when the solution is subjected to an external magnetic field within minutes , as shown in fig . it can be obviously seen that the cofe2o4 mss have rapid magnetic response ability at room temperature , as well as , highly monodispersed , and biocompatible . rt magnetization curves for the of cofe2o4 mss with different reaction time : a 12 h , b 24 h ( the inset shows enlarged magnetic hysteresis loops at low applied fields ) ; photographs of cofe2o4 mss dispersion in a vial , c without magnetic field , d with magnetic field for 1 min in summary , sp cofe2o4 mss have been synthesized through a simple surfactants - assisted solvothermal method at a relatively low temperature . all of the reactants are common reagents and inexpensive , as well as environment benign . the as - prepared mss , composed of about 8-nm cofe2o4 nanoparticles , have uniform sizes ( diameters up to several hundred nanometers ) , high ms and well water - dispersible make them good candidates for not only in advanced magnetic materials and ferrofluid technology , but also in biomedical fields such as biomolecular separations , targeted drug delivery , as well as magnetic resonance imaging . this work was partially supported by the program for science & technology innovation talents in universities of henan province ( no . 2008 hastit002 ) , innovation scientists and technicians troop construction projects of henan province ( no . 094100510015 ) , and by the natural science foundation of china under grant no . 20971036 . 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 . 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 .	based on a low - temperature route , monodispersed cofe2o4 microspheres ( mss ) were fabricated through aggregation of primary nanoparticles . the microstructural and magnetic characteristics of the as - prepared mss were characterized by x - ray diffraction / photoelectron spectroscopy , scanning / transmitting electron microscopy , and vibrating sample magnetometer . the results indicate that the diameters of cofe2o4 mss with narrow size distribution can be tuned from over 200 to ~330 nm . magnetic measurements reveal these mss exhibit superparamagnetic behavior at room temperature with high saturation magnetization . furthermore , the mechanism of formation of the monodispersed cofe2o4 mss was discussed on the basis of time - dependent experiments , in which hydrophilic pvp plays a crucial role .
maxillary protraction with a tooth - borne appliance and face mask is the conventional treatment in growing patients with class iii malocclusion caused by maxillary deficiency . however , it results in forward displacement of the maxilla , extrusion of maxillary molars , and clockwise rotation of the mandible.1234 several methods overcoming these drawbacks by skeletal anchorage have been reported.567 however , miniplate placement in the infrazygomatic crest and lateral nasal wall areas is an aggressive procedure as two surgical sites are required.89 several studies have evaluated the suitability of the palate as a skeletal anchorage site in the mixed dentition period.101112 favero et al.13 placed miniscrews in the palate of a patient with deciduous dentition for maxillary protraction . further , ludwig et al.14 used hybrid tooth- and bone - borne rapid palatal expanders for protraction in adolescents . recently , kook et al.1516 placed palatal plates for molar distalization in adolescents with almost no side effects on the palatal soft tissue . the palatal approach is more desirable than surgery for placing infrazygomatic crest miniplates because it involves a flapless procedure with a single plate that works bilaterally . the purpose of this report is to introduce an alternative modality of maxillary protraction in growing patients with dentoskeletal class iii malocclusion using a modified c - palatal plate and face mask . the appliance consists of a palatal miniplate with three hooks on each arm ( 0.8 mm thick , 2.0 mm wide , and 28.0 mm long ) . the plate is fixed to the palate with three miniscrews ( diameter , 2.0 mm ; length , 8.0 mm ) , driven into screw tubes 2 mm apart from the midpalatal suture ( figure 1a ) . a silicone jig is used during placement to guarantee a consistent distance between the plate arms and the palatal tissues . the plate is adapted to the contours of the cast with the arms extending to the gingival level in the canine areas while maintaining approximately 2 mm of space between the arms and the palatal surface . the fabrication of the jig has been described previously ( figure 1b).16 after administration of local anesthesia , the jig - plate assembly is placed on the palate guided by the occlusal surfaces of the jig . three self - drilling miniscrews are installed with a torque driver ( jeil medical corporation , seoul , korea ) at 30 rpm using less than 30 ncm ; then , the jig is removed and the miniscrews are tightened . the palatal plate functions as the point of force application and the face mask serves as extraoral anchorage . a female patient aged 9 years 8 months visited the clinic with the chief complaint of cross bite . intraoral examination revealed early mixed dentition and class iii molar and canine relationships with anterior cross bite ( figure 2a ) . to correct the cross bite , a c - palatal plate with hooks in the deciduous canine area elastics were connected to a face mask at approximately 20 downward to the occlusal plane for 14 hours daily to deliver a force of 500 g per side . a point as well as the maxillary molars were displaced anteroinferiorly , and the posterior nasal spine ( pns ) showed downward movement . cephalometric superimposition show improvement of the skeletal class iii relationship ( figures 2b , 3 , and 4 ; table 1 ) . the total treatment time was 18 months , and the patient and her parents were satisfied with the profile . the appliance consists of a palatal miniplate with three hooks on each arm ( 0.8 mm thick , 2.0 mm wide , and 28.0 mm long ) . the plate is fixed to the palate with three miniscrews ( diameter , 2.0 mm ; length , 8.0 mm ) , driven into screw tubes 2 mm apart from the midpalatal suture ( figure 1a ) . a silicone jig is used during placement to guarantee a consistent distance between the plate arms and the palatal tissues . the plate is adapted to the contours of the cast with the arms extending to the gingival level in the canine areas while maintaining approximately 2 mm of space between the arms and the palatal surface . the fabrication of the jig has been described previously ( figure 1b).16 after administration of local anesthesia , the jig - plate assembly is placed on the palate guided by the occlusal surfaces of the jig . three self - drilling miniscrews are installed with a torque driver ( jeil medical corporation , seoul , korea ) at 30 rpm using less than 30 ncm ; then , the jig is removed and the miniscrews are tightened . the palatal plate functions as the point of force application and the face mask serves as extraoral anchorage . a female patient aged 9 years 8 months visited the clinic with the chief complaint of cross bite . intraoral examination revealed early mixed dentition and class iii molar and canine relationships with anterior cross bite ( figure 2a ) . to correct the cross bite , a c - palatal plate with hooks in the deciduous canine area elastics were connected to a face mask at approximately 20 downward to the occlusal plane for 14 hours daily to deliver a force of 500 g per side . a point as well as the maxillary molars were displaced anteroinferiorly , and the posterior nasal spine ( pns ) showed downward movement . cephalometric superimposition show improvement of the skeletal class iii relationship ( figures 2b , 3 , and 4 ; table 1 ) . the total treatment time was 18 months , and the patient and her parents were satisfied with the profile . the application of skeletal anchorage is a viable modality to correct skeletal class iii malocclusion in growing patients . temporary skeletal anchorage devices are placed in the palate because of the ease of access , reduced susceptibility to inflammation , and good quantity of bone in the area.17 a palatal plate can be used as the point of force application to overcome dental side effects and maximize the skeletal gain of maxillary protraction . when compared with the aggressive surgical procedures required for miniplate placement in the infrazygomatic crest area , kim et al.18 evaluated stress distribution by three - dimensional finite element analysis of maxillary protraction with a palatal plate : they stated that the palatal approach is more efficient than traction from miniscrews placed buccally in the infrazygomatic area and reported forward displacement with counterclockwise rotation of the maxillofacial complex . in agreement , our patient showed anteroinferior displacement of a point with further downward movement of the pns , resulting in earlier contact between the posterior teeth and clockwise rotation of the mandible . this outcome may be explained by the lower point of force application to the center of resistance of the maxilla in the proposed method than in the buccal and conventional approaches . the profile improved because of the combination of forward maxillary displacement and clockwise mandibular rotation , causing backward displacement of the chin . further studies are recommended to evaluate the effectiveness of this treatment , especially by three - dimensional assessment using conebeam computed tomography ( cbct ) . in a meta - analysis , feng et al.19 showed that bone - anchored miniplates might result in maxillary advancement as well as reduction of mandibular rotation , extrusion of maxillary molars , and proclination of maxillary incisors . in addition , placement of a bonded transpalatal arch between maxillary first molars is recommended to decrease extrusion of maxillary posterior teeth , especially in patients with the hyperdivergent facial type . recently , nienkemper et al.20 demonstrated a 1.9 mm of forward movement of the maxilla using a bone - tooth - borne hybrid hyrax - facemask appliance in children with dentoskeletal class iii malocclusion . in addition , nguyen et al.21 reported 3.7 mm forward displacement of the maxilla using an intraoral skeletal anchorage appliance by three - dimensional superimposition of cbct - derived models . in our case , the difference in the amount of displacement might be primarily due to the aggressive protraction method in the previous study ; it required surgical placement of four miniplates , two in the infrazygomatic crests and two between the mandibular lateral incisors and canines . other reasons could be the differences between the evaluation methods and individual and ethnic variations of the patients . in our patient , the overjet was corrected and the profile slightly improved . however , these changes might have been supported by normal growth of the maxilla during the long treatment ( 18 months ) . therefore , the treatment effect might be smaller than 1.5 mm . the improvements due to growth and the treatment are not exactly distinguishable . in addition , some relapse of the corrected overjet is expected because of the chin cup effect of the face mask . moreover , the shape and depth of the palatal vault might have played a role in determining the relationship between the center of resistance and the force vector . a further study is warranted to evaluate the influence of different palatal vault configurations on the effects of a protraction palatal plate and growth . despite its advantages , a palatal plate might cause inflammation of the palatal mucosal and speech disturbance from decreased tongue space . therefore , several measures were taken to decrease the probability of inflammation , including the addition of screw tubes and accurate placement of the plate with a jig . the effect on speech has been evaluated using a different design : the previous study showed that the impact on speech articulation is reversible within two weeks due to high adaptability of the tongue.22 in conclusion , application of a modified c - palatal plate and face mask seems to be a suitable modality of maxillary protraction due to its maximal skeletal effects , minimal dental side effects , and nonsurgical approach . however , this appliance is not suitable for patients with maxillary transverse deficiency because it prevents opening of the midpalatal suture .	maxillary protraction is the conventional treatment for growing class iii patients with maxillary deficiency , but it has undesirable dental effects . the purpose of this report is to introduce an alternative modality of maxillary protraction in patients with dentoskeletal class iii malocclusion using a modified c - palatal plate connected with elastics to a face mask . this method improved skeletal measurements , corrected overjet , and slightly improved the profile . the patients may require definitive treatment in adolescence or adulthood . the modified c - palatal plate enables nonsurgical maxillary advancement with maximal skeletal effects and minimal dental side effects .
migration is a process of social change during which people move from one cultural setting to another in order to settle for a longer period of time or permanently . though migration has been occurring since the beginning of time , the advent of the era of globalization has resulted in the increased movement of people across and within national boundaries for various reasons . the social , cultural , financial , and other aspects of migration have been the focus of much research and many new policies initiated as a result . the relationship between migration and health has also been well explored . however , there is a lack of consistency among studies of migration and health with results indicating both higher and lower prevalence of diseases among migrants in relation to the residents [ 19 ] . the health status of return migrants , migrants , who have returned to their place of origin , has not been the focus of much research . a study from mexico had previously reported on the disadvantages faced by the return migrants . more research is needed in this area as the health status of the return migrants may be much different from that of the resident community . in this exploratory study , we address this information gap by studying a rural community in the south indian state of kerala which has a long history of migration . the state of kerala with a population of 34 million enjoys one of the best health indicators in india with an infant mortality rate of 12 and life expectancy at birth of 74 years . these levels are far better than the other indian states and are quite close to those of developed countries . kerala has also made significant progress economically with recent economic indicators showing that kerala is one of the indian states with the least reported levels of poverty . a significant proportion of the total receipts of kerala are contributed by remittances from emigrants . as of 2007 , 1.85 million keralites were living abroad ; of theses , 89% were residing in the persian gulf countries . surveys also indicate that one out of four households in kerala had at least one resident abroad . an interesting feature of migration in kerala is that a good proportion of the migrants return home after working for various periods in foreign countries . as in 2007 , there were 890,000 return emigrants , roughly one return emigrant for every two living abroad . another thing to note is the very high prevalence of chronic diseases in kerala , with the burden of deaths from cardiovascular diseases now exceeding that of industrialized countries . our own previous study has shown that the prevalence of self - reported diabetes was higher among the richer socioeconomic groups than the poorest group . with the kerala society at an advanced stage of demographic and epidemiological transition , research which focuses on it was with this view that the population registry for life style diseases ( prolife ) study was initiated . the initial goal was to assemble a cohort to study the epidemiology of chronic diseases in the selected rural community and to estimate the burden of risk factors . here we study the influence of migration on the health status of individuals by comparing the health status of return migrants with that of the residents who had never worked abroad . the present study explores the prevalence of selected chronic disease / risk factors among return migrants in comparison to age and sex matched local residents . we also explore the relationship between the duration of migration and prevalence of risk factors . prolife is a prospective cohort study initiated in 2001 involving the long time follow - up of the residents of varkala integrated child development services all residents were enrolled in the study ; 1 , 61,942 subjects living in 33,379 households were included in the baseline survey . separate adult questionnaires were administered to 78,173 subjects ( 33,978 men and 44,195 women ) who were 20 years as of january 1 , 2002 . the shortfall was primarily on account of some subjects being absent from home on three consecutive visits . other than deaths , migration would be the main reason for loss to follow - up . as of december 31 , 2004 , 721 subjects moved out of the study area and were lost to followup . 105 female healthly workers involved in routine mother and child care served as field investigators . a team of physician epidemiologists trained the workers in survey methods and in the recognition of common disease conditions . the workers were also trained on collecting cause of death information using a structured questionnaire . the baseline survey collected household level information capturing demographic , educational , and a host of socio economic attributes . additionally , information on deaths and births that occurred during the year previous to the survey was gathered . a pretested questionnaire was administered to every adult subject , ( aged 20 years or more on 1 january , 2001 ) . this questionnaire captured information on diagnosed diabetes , hypertension , and cardiac diseases , in addition to lifestyle attributes like physical activity , habits and migration history . diabetes , hypertension and cardiac diseases were reported by the subjects as diagnosed by a physician on the basis of clinical and biochemical parameters . information on the duration and destination of migration and type of employment during that time was ascertained . resource limitations prevented us from capturing dietary details and anthropological measurements in the present survey . the objectives of the study were explained to the head of each household who had the freedom to refuse . informed oral consent was obtained from every respondent and the study was overseen by an independent ethics committee . statistical analysis for differences between nonmigrants and migrants age - specific prevalence of diabetes , hypertension , and history of cardiac complaints among males and females was calculated . we analyzed data for possible relationship between the duration of migration and observed prevalence of the diseases by doing mantel test for trend . logistic regression analysis adjusting for confounders such as gender , education , and age was carried out to calculate the adjusted odds ratio . table 1 provides the salient features of the population and compares the characteristics of subjects with and without history of migration . the majority of subjects who reported a history of living outside kerala were male ( 86.3% ) . the mean age of subjects with a history of migration was higher than that of subjects with no history of migration ( 45.7 versus 41.4 years ) . return migrants had better educational levels with only 15.6% of subjects reporting low or less than 5 years of education , whereas 28% of subjects with no history of migration reported their educational status as low . prevalence of risk factors such as smoking ( 49.7% versus 17.6% ) and alcohol consumption ( 34.6% versus 11.6% ) were higher among return migrants . similarly , return migrants reported higher prevalence of diabetes ( 14.1% versus 6.0% ) , cardiac conditions ( 6.6% versus 3.7% ) , and hypertension ( 14.7% versus 9.9% ) . age - specific prevalence of diabetes , hypertension and history of cardiac complaints among males and females aged 3069 with a history of living outside kerala for more than 5 years and those with no history of migration is provided in figures 1(a ) and 1(b ) . figure 1(a ) presents data on age - specific prevalence of the diseases among males with a history of migration and nonmigrants . diabetes , hypertension , and cardiac complaints are more prevalent among those with duration of migration of over 5 years when compared with those with no history of migration . the age - specific increase in prevalence is substantially higher among migrants for diabetes and hypertension though the trend is unmistakable even for cardiac diseases . the same trend is seen for women aged 3069 as seen in figure 1(b ) . table 2 provides the odds ratio for the presence of physician diagnosed chronic diseases ( diabetes , hypertension , and cardiac conditions ) . after adjustment for age , gender , and education , subjects with a history of migration had higher odds of having chronic diseases ( or 2.2 , 95% ci 2.12.3 ) . the highest prevalence of all risk factors was noted in persons with a history of migration of more than 10 years . statistical analysis for differences between nonmigrants and migrants reveals high significance . we analyzed that data for possible relationship between the duration migration and observed prevalence of the diseases by doing mantel test for trend . table 3 shows that there is a clear trend of increased prevalence in relation to duration of migration . in our study , residents with a history of migration reported a higher prevalence of cardiac diseases , hypertension , and diabetes . the prevalence increased with the duration of migration with those residents who lived outside kerala for more than 5 years showing the highest prevalence . our finding that diabetes , hypertension , and cardiac diseases are substantially higher in migrants corroborates some of the previous publications in this regard . like similar studies , prevalence of chronic conditions has been estimated based on the self reporting of physician diagnosed conditions . also , our analysis is restricted to subjects who are currently residing in the study area . since these diseases tend to increase with age , the higher prevalence may be attributed to the age effect and not to the fact of a history of migration . however , logistic regression analysis showed that , after adjustment for age and gender , subjects with a history of migration had a higher prevalence of chronic diseases . the data on age - specific prevalence also disproves such an explanation ; between 30 and 70 years the prevalence is significantly higher among migrants in all four age groups . we can therefore conclude that the most important contributory factor to the observed increase in prevalence is history of migration . our analysis on the prevalence of 2 significant risk factors namely smoking and alcohol consumption for noncommunicable diseases in our study subjects clearly establishes that the prevalence for both risk factors is more than double that in those with a history of migration . also to be noted that there are the unusual high rates of drinking and smoking among women with a history of migration . this phenomenon requires further study and we believe that in the context of this study we explain the high prevalence of chronic diseases among migrant women in this rural community . infectious diseases like tb have been shown to be high among migrants than the resident population [ 18 , 19 ] . chronic diseases like diabetes and stroke have also been shown to be high among migrants [ 6 , 20 , 21 ] . higher incidence of mental disease and alcohol and drug abuse was also seen among migrants [ 22 , 23 ] . migrants from the indian subcontinent in many cases had higher incidence of disease than migrants from elsewhere [ 6 , 20 , 24 , 25 ] . however , some studies have shown that the mortality rate of migrants though higher than that of the residents are lower than that of their country of origin [ 2628 ] . this apparent paradox has been explained as due to reasons such as the healthy migrant effect , where by the persons who migrate are healthier than the nonmigrants and the salmon effect , where by elderly migrants return to their country of origin to die [ 2932 ] . it has been observed that the urbanization process associated with migration leads to the availability and abundance of calorie - dense / low - fiber foods and the adoption of sedentary lifestyles . this leads to increased risks of morbidity and mortality from diet and lifestyle - related chronic diseases . a recent report which focused on return migrants reported that the health of returning migrants is impacted by the cumulative exposure to social determinants and risk factors of health during the migration process , during the return movement , and following return . the remittances from migrants constitute a major part of the revenue for the state of kerala and the economy of kerala is much dependent on continuing remittances from the migrants . the government of kerala has initiated various programs which focus on the welfare of the nonresident keralites and has a dedicated department which looks after affairs of the nonresident keralites called norka . however , at present , most of the programmes concentrate on providing financial , labor , and rehabilitation assistance to return migrants . the inclusion of health as a major component in norka roots programme and similar programmes can have a significant effect on the health of migrants and return migrants . it is imperative that the state initiates programs that target the health and wellbeing of this very important segment of the population .	aim . to study the relationship between a personal history of migration and prevalence of chronic diseases and risk factors in a rural population . method . cross sectional survey data from prolife , a cohort study involving the long time follow - up of the residents of an administrative unit in kerala , india , was used . pre - tested questionnaire was administered to 78,173 adult residents . information on physician diagnosed diabetes , hypertension , and cardiac diseases and lifestyle attributes like physical activity , habits , and migration was captured . results . subjects with a history of migration had a higher prevalence of chronic disease when compared with those with no history of migration . diabetes ( 19.6% versus 4.1% ) , hypertension ( 18.8% versus 6.6% ) , and cardiac complaints ( 8.6% versus 4.1% ) are more prevalent among those with history of migration of over 5 years . after adjustment for age , gender , and education , we found that chronic diseases are higher among persons with a history of migration ( or 2.2 , 95% ci : 2.12.3 ) . age - specific increases in prevalence of chronic diseases are also substantially higher among migrants . conclusion . people with a history of migration have a higher prevalence of chronic diseases and risk factors .
incidence of obesity and overweight in children of some societies had increased dramatically ; this is one of the main factors seems related to the development of obesity . different studies in this area indicate that one in four children aged 614 years ( from 11% to 39% ) in developed and developing countries , is overweight or obese . on the other hand , low levels of physical activity are not only associated with the failure of health related physical fitness , but also inactivity correlates with the development of hypercholesterolemia , hypertension , metabolic syndrome , type 2 diabetes and cardiovascular diseases , in both adults and children . in addition , it seems plausible to assume that higher level of physical fitness in children results in a more favorable body composition . nonetheless , lack of ample data and applicable examinations , in regards to the relationship between aerobic fitness and body fatness , obscure the correlation between cardiovascular fitness and body composition in iran . moreover , little is known about the relationship between physical fitness and different body composition indicators such as body mass index ( bmi ) , and the percentage of body - fat in children . to the best of our knowledge , no information is available demonstrating interdependence between obesity and physical activity indicators in isfahan school children . therefore , this study was carried out to determine the relationship between cardiovascular fitness and body - composition in school children aged 1013 years in isfahan . this cross - sectional study comprised 12,946 students of middle schools from the city of isfahan . evaluations were performed during february to may 2010 school program , and stratified random sampling was used . at first , 30 schools were identified and randomly selected from the city of isfahan , and their pupils were become familiar with the aims of the study . not only , informed consents were obtained from guardians of students , but also medical history questionnaires were filled by parents . participants and their guardians furthermore , the aims of the study explained to both physical education teachers and school administrators . all of the participants , at the time of the project implementation , were in good health status comprising different aspects of locomotive , cardiovascular , and endocrine systems . height and weight were measured by using a stadiometer and calibrated balance beam scale ( novin , iran ) while scale intervals were 0.5 cm and 0.2 kg respectively . the percentage of body - fat was determined in accordance with gender - specific equations : ( if triceps and sub - scapular > 35 mm ; % fat = 0.783 sf + i males and % fat = 0.546 sf + 9.7 females ; and if triceps and subscapular < 35 mm ; % fat = 1.21 [ sf ] 0.008 [ sf ] + i males and % fat = 1.33 [ sf ] 0.013 [ sf ] + 2.5 females ) . both waist and hip circumferences were measured by a flexible tape at the level of the narrowest point between the lower costal border and the iliac crest , and the widest region of the hip , respectively . aerobic fitness or vo2 max of subjects subjects were required to run back and forth on a 20 m course and be on the 20 m line at the same time a beep is emitted from tape . the frequency of sound signals increased in such a way that running speed starts at 8.5 km / h and increased by 0.5 km / h each min . when the subjects could no longer follow the pace , the stage , the subjects were able to run for , was recorded and used for calculating the vo2 max . the relationships between body - fat , bmi , and waist - hip ratio ( whr)and aerobic fitness ( vo2 max ) were examined by pearson 's product - moment correlation coefficient , and p < 0.05 considered as statistically significant . data were analyzed using the spss - pc software ( version 20 , ibm spss inc . this cross - sectional study comprised 12,946 students of middle schools from the city of isfahan . evaluations were performed during february to may 2010 school program , and stratified random sampling was used . at first , 30 schools were identified and randomly selected from the city of isfahan , and their pupils were become familiar with the aims of the study . not only , informed consents were obtained from guardians of students , but also medical history questionnaires were filled by parents . participants and their guardians furthermore , the aims of the study explained to both physical education teachers and school administrators . all of the participants , at the time of the project implementation , were in good health status comprising different aspects of locomotive , cardiovascular , and endocrine systems . height and weight were measured by using a stadiometer and calibrated balance beam scale ( novin , iran ) while scale intervals were 0.5 cm and 0.2 kg respectively . the percentage of body - fat was determined in accordance with gender - specific equations : ( if triceps and sub - scapular > 35 mm ; % fat = 0.783 sf + i males and % fat = 0.546 sf + 9.7 females ; and if triceps and subscapular < 35 mm ; % fat = 1.21 [ sf ] 0.008 [ sf ] + i males and % fat = 1.33 [ sf ] 0.013 [ sf ] + 2.5 females ) . both waist and hip circumferences were measured by a flexible tape at the level of the narrowest point between the lower costal border and the iliac crest , and the widest region of the hip , respectively . aerobic fitness or vo2 max of subjects subjects were required to run back and forth on a 20 m course and be on the 20 m line at the same time a beep is emitted from tape . the frequency of sound signals increased in such a way that running speed starts at 8.5 km / h and increased by 0.5 km / h each min . when the subjects could no longer follow the pace , the stage , the subjects were able to run for , was recorded and used for calculating the vo2 max . the relationships between body - fat , bmi , and waist - hip ratio ( whr)and aerobic fitness ( vo2 max ) were examined by pearson 's product - moment correlation coefficient , and p < 0.05 considered as statistically significant . data were analyzed using the spss - pc software ( version 20 , ibm spss inc . the descriptive data ( mean standard deviation ) of the measured variables including height , weight , whr , bmi , body fat percent and cardiovascular fitness are shown in table 1 , and the results of statistical analysis and comparison of measured variables are shown in table 2 and figures 1 - 4 . there were significant differences between male and female students fat percentages ( 21.12% vs. 19.32% p < 0.05 , respectively ) . in comparison with girls , boys showed significantly lower bmi , whr , and body - fat percent ( p < 0.05 ) . moreover , boys attained better performance in aerobic fitness test and therefore had higher vo2 max ( p < 0.05 ) . relationship between body - fat and vo2 max for each gender is illustrated in table 2 . this inverse correlation was higher in boys ( r = 0.81 ; p < 0.01 ) , whereas girls demonstrated moderate correlation ( r = 0.77 ; p < 0.01 ) . descriptive data related to measured variables in students ( n=12,946 ) pearson correlation coefficient between aerobic fitness and body composition of subjects body composition distribustion of subjects comparison of body fat percent of students comparison of aerobic fitness of students comparison of body mass index of students this study demonstrated that aerobic fitness increases in inverse proportion to body - fat in isfahan school children aged 1114 years , also findings demonstrated that the prevalence of overweight differed between boys and girls . moreover , the latter ones had lower levels of aerobic fitness compared with their male counterparts . it has been reported that prevalence of overweight in mexican boys and girls using centers for disease control and prevention percentiles ranged from 10.8% to 16.1% and 14.3% to 19.1% respectively , whereas their obesity prevalence altered differently ( from 9.2 to 14.7% and 6.8 to 10.6% in boys and girls respectively ) . childhood obesity was emerging as a worldwide epidemic problem ( 38% for boys and 63% girls ) when children , who were obese and overweight by bmi criteria , were considered together . another study reported that over 37% of children from birmingham fell into overweight and obese classifications . indeed , one in five children ( 22% ) had more than 30% body fat , and obese group comprised almost girls ( 90% ) . this report also showed that 71% of children had a percentage of body - fat in excess of 20% , which was recognized as the upper end of the optimal range . in fact , the above - mentioned study indicates that boys tend to have a higher prevalence of obesity than girls ( 17% vs. 15% respectively ) . on the other hand , an interesting gender difference was suggested considering sexual maturation , with a positive association with overweight and obesity in girls but the associations were reverse for boys . one of the primary behavioral and environmental factors are food consumption patterns , sedentary behavior , and socioeconomic status , that led to gradual increase in body weight and obesity , that is also the consequence of a prolonged positive energy balance , that is , when energy intake exceeds energy loss . however , there are many factors that can influence energy balance and therefore , be identified as contributors to the current obesity epidemic in children , with biological , behavioral , environmental , and social being most cited . although the relative contribution of energy intake to the obesity epidemic is controversial , available data clearly indicate that physical activity plays an integral role in the prevention of obesity . obese children are at an increased risk of acute medical illnesses and chronic diseases , particularly osteoarthritis , diabetes mellitus , and cardiovascular diseases , which can lead to poor quality of life , an increased personal and financial burden to individuals , families , and society , as well as a shortened lifespan . aerobic fitness or vo2 max average for children assessed in the present study ( 32.9 ml / kg / min ) was lower than previous reports . for example , vo2 max for children aged 812 years ranged between 34 and 58 ml / kg / min , while boys had higher values of vo2 max . however , differences in body - composition and level of physical activity , can result in inconsistent reports . for example , lger and lambert reported aerobic capacity from 38 to 52 ml / kg / min for children aged 618 years . cardiovascular fitness or vo2 max changed by gender criteria ( 29.5 5.6 vs. 36.4 7.3 for girls and boys , respectively ) in accordance with the results of previous studies . in fact , aerobic capacity is consistently greater in boys throughout childhood and this difference exceeded at puberty . the observed difference has been ascribed to cumulative effects of different factors including body composition and cardiac size and function . regular physical activity plays an important role in the maintenance of body weight and composition as well as in the regulation of skeletal muscle and adipose tissue metabolism . precise assessment of habitual physical activity is critical for accurately descriptive epidemiology of the physical activity - obesity relationship for designing appropriate interventions aimed at modifying body composition and related risk factors and for promoting change in lifestyles . assessment of aerobic fitness and body fat are probably a more valid approach than measurement of self - reported physical activity and body - weight , particularly in children . it has been reported that overweight and obese children showed a lower physical fitness than normal children . however , studies analyzing association between aerobic fitness and different measures of body - composition in this population are scarce . indeed , results of the present study revealed a moderate - strong inverse relationship ( r = 0.81 for boys vs. r = 77 for girls ) between aerobic fitness and fat percentage of subjects . since people who have sedentary lifestyles also have low levels of fitness and excessive body fat , optimal altering of adiposity in children should begin with augmentation of physical activity and fitness , which leads to reductions in body - fat . moreover , children , who improve their aerobic fitness during childhood , have less overall adiposity and abdominal adiposity than their counterparts during adolescence and adulthood . due to the fact that bmi poorly correlates with aerobic fitness , other indicators of adiposity ( body fat percentage or whr ) rather than weight has been shown to be associated with aerobic fitness . one of the main limitations of the study was that the number of male subjects participated in this research , were about one - fifth of female subjects , because of educational curriculum end at may . it seems that more accurate and specific fitness tests are required to determine if there is association between fitness level of children and their body composition . moreover , analysis of the relationship between aerobic fitness and body - composition needs adjustments for pubertal development factors , which requires further investigation . one of the main limitations of the study was that the number of male subjects participated in this research , were about one - fifth of female subjects , because of educational curriculum end at may . it seems that more accurate and specific fitness tests are required to determine if there is association between fitness level of children and their body composition . moreover , analysis of the relationship between aerobic fitness and body - composition needs adjustments for pubertal development factors , which requires further investigation . our results suggest that cardiovascular fitness , as an indicator of physical activity , is linked to the increased level of adiposity in children . inactivity is interconnected with obesity ; however , it is likely to be as one of the easiest factors to be modified . although guidelines for physical activity in youths recommend involvement in moderate to vigorous physical activities for at least 60 min a day for health promotion and from a weight - control perspective . according to the results of the present study and the fact that academic curriculum of most countries of the world and iranian middle school students included only 90-min physical activity per week , initiatives should be put forward to promote physical activity in children in both school and out - of - school environment . certainly , diet modifications also have to be considered in implementation of school - based physical activity interventions , and it is vital that researchers and practitioners target alternative venues that still service a large amount of children , but are also accessible .	background : since correlations have been found between body composition and physical activity in different parts of the world , inactivity and poor physical condition likely contributes main factors in childhood obesity . this study was implemented to determine the relationship between cardiovascular fitness and body - composition in a group of iranian middle school students.methods:in this descriptive study , subjects comprised of 12,946 students ( 10,531 girls and 2,415 boys aged 1113 years ) in the city of isfahan . height , weight , body - fat percent , body mass index , and cardiovascular fitness of the aforesaid students were measured by valid tests.results:this study showed that fat percentage and aerobic fitness ( vo2 max ) of girls were 24.73% , and 29.5 ( ml / kg / min ) and boys19.32% and 36.4 ( ml / kg / min ) respectively . results also revealed that there was a negative significant correlation between fat percent and aerobic fitness of boys ( r = 0.81 ) , and girls ( r = 0.77 ) respectively.conclusions:to conclude , this study signifies that fat percentage augmentation leads to a decrease in aerobic fitness of children . thus , fat percentage can be associated with different chronic diseases such as cardiovascular ones .
infection with human cytomegalovirus ( cmv ) , a member of the herpesvirida family , is very common world - wide with seropositivity rates ranging from 40% in developed countries up to 100% in developing countries . severe scenarios occur when the immunocompetent host is critically ill and also in immunocompromised hosts . the epidemiology and pathogenesis of infection with cmv in pregnancy have been studied over the past decades . primary infection or reactivation of the previously acquired cmv can occur during pregnancy and can result in congenital cmv ; the most important cause of congenital viral infections . if the child survives , up to 90% of the cases will have complications such as hearing loss , vision impairment and varying degrees of mental retardation . whether primary infection or reactivation of cmv in a pregnant woman can cause pregnancy loss is still under discussion , results have been controversial , and underlying mechanisms are unclear . some studies found high presence of cmv antigens in tissues from abortion and others reported higher seropositivity . one prospective study also found a higher risk of pregnancy loss with cmv infection , though results of other prospective studies did not approved it . despite these reports on the role of cmv infection in spontaneous pregnancy loss , recurrent pregnancy loss ( rpl ) , usually referred to as three or more consecutive abortions prior to 22 gestational weeks , is one of the most frustrating and difficult areas in reproductive medicine . while about 15% of pregnant women experience sporadic pregnancy loss , about 2% and only 0.4 - 1% of them experience two and three consecutive abortions , respectively . the etiology of rpl is still unclear and few evidence - based diagnostic and treatment approaches are available . etiologic factors associated with rpl are suggested as anatomical , immunological , genetic , endocrine , infectious , thrombophilic , and environmental factors . while some authors reported higher prevalence and higher antibody titers to cmv in rpl cases , other studies showed comparable and even less prevalence of antibodies to cmv among women with rpl than normal pregnant women . accordingly , some investigators suggested that rpl cases might have some selective immunological unresponsiveness to cmv . previous studies on the association between cmv infection and rpl were mainly based on simple serological tests . considering the lack of data on the etiology of rpl , and also regarding the suggested role of altered immune response in this regard , we evaluated and compared humoral immunological response to cmv in women with and without rpl using igg and igm cmv antibody plus igg avidity index ( ai ) test which provide complementary data for diagnosis of and immune response to cmv infection . this case - control study was conducted on 43 women with rpl referred by gynecologists to a clinical immunology out - patient clinic in isfahan ( iran ) . inclusion criteria were age between 18 and 45 years , at least three recurrent spontaneous abortions before the 22 gestational week , and no evidence for organic or autoimmune diseases as the etiology for rpl confirmed by laboratory results . control subjects were randomly selected from healthy age - matched multiparous women without history of abortion referring to the gynecology clinic of the hospital . the study was approved by the ethics committee of isfahan university of medical sciences ( research project number : 389048 ) and informed consent was obtained from patients and controls . after evaluation of the inclusion criteria and interviewing with participants and gathering demographic data , blood samples were obtained from patients and controls . samples were centrifuged and stored in 70c and then were transferred to the infectious disease research center of isfahan , central laboratory . to evaluate cmv igg and igm antibodies the enzyme linked immunosorbant assay method ( elisa ) ai of anti - cmv igg was also studied by elisa method as proposed by the manufacturer ( focus diagnostics , california , usa ) . an ai of < 40% was considered as low , higher than 60% as high and between the 40% and 60% as moderate . according to serological tests , women were classified as follows : cmv seronegative : both cmv - igm and -igg negative.cmv seropositive : cmv - igm negative and cmv - igg positive.primary cmv infection : cmv - igm positive and cmv - igg negative . either a primary or a recurrent infection : both cmv - igm and cmv - igg positive . differentiation of primary and recurrent cmv infection in women with both cmv - igm and cmv - igg positive antibody was determined by the assessment of the anti - cmv igg ai at the same time . pregnant women with low ai were considered as having primary maternal infection , while those with high ai were considered as having recurrent maternal infection . quantitative data are expressed by mean standard deviation ( sd ) , and categorical variables are described by relative frequencies . for comparisons of the quantitative variables , we used the student 's t - test , and for comparisons between the categorical variables , we used the chi - square test with pearson modification . this case - control study was conducted on 43 women with rpl referred by gynecologists to a clinical immunology out - patient clinic in isfahan ( iran ) . inclusion criteria were age between 18 and 45 years , at least three recurrent spontaneous abortions before the 22 gestational week , and no evidence for organic or autoimmune diseases as the etiology for rpl confirmed by laboratory results . control subjects were randomly selected from healthy age - matched multiparous women without history of abortion referring to the gynecology clinic of the hospital . the study was approved by the ethics committee of isfahan university of medical sciences ( research project number : 389048 ) and informed consent was obtained from patients and controls . after evaluation of the inclusion criteria and interviewing with participants and gathering demographic data , blood samples were obtained from patients and controls . samples were centrifuged and stored in 70c and then were transferred to the infectious disease research center of isfahan , central laboratory . to evaluate cmv igg and igm antibodies the enzyme linked immunosorbant assay method ( elisa ) ai of anti - cmv igg was also studied by elisa method as proposed by the manufacturer ( focus diagnostics , california , usa ) . an ai of < 40% was considered as low , higher than 60% as high and between the 40% and 60% as moderate . according to serological tests , women were classified as follows : cmv seronegative : both cmv - igm and -igg negative.cmv seropositive : cmv - igm negative and cmv - igg positive.primary cmv infection : cmv - igm positive and cmv - igg negative . either a primary or a recurrent infection : both cmv - igm and cmv - igg positive . differentiation of primary and recurrent cmv infection in women with both cmv - igm and cmv - igg positive antibody was determined by the assessment of the anti - cmv igg ai at the same time . pregnant women with low ai were considered as having primary maternal infection , while those with high ai were considered as having recurrent maternal infection . data were analyzed with the spss software for windows version 16.0 ( spss inc . , chicago il . quantitative data are expressed by mean standard deviation ( sd ) , and categorical variables are described by relative frequencies . for comparisons of the quantitative variables , we used the student 's t - test , and for comparisons between the categorical variables , we used the chi - square test with pearson modification . forty three women with rpl ( mean sd age = 30.22 4.73 years , mean abortion number = 3.84 1.98 ) and 43 controls ( mean age = 29.87 5.32 years , p = 0.64 ) were included into the study . igm and igg titers and final results of the patients and controls are presented in table 1 . there was one ( 2.3% ) case of positive igm in each group of the rpl and controls . these patients had also positive igg and high ai and thus were considered as having recurrent or reactivated maternal infection . there were 39 ( 90.6% ) and 30 ( 69.8% ) cases of positive igg in the rpl and control groups , respectively ( p = 0.014 ) . no difference was found between the two groups in igg ai ; p = 0.781 [ table 1 ] . igm and igg titers and avidity index in patients and controls separate analysis of the igg titer below and above the cut - off ( according to the kit instruction : ratio > 0.8 or 16 ru / ml as positive ) in cases showed that igg titer was significantly higher in seropositive cases with rpl than seropositive controls ( 5.18 1.99 vs. 2.00 0.81 , p < 0.001 ) , and also in seronegative rpl cases than seronegative controls ( 0.82 0.28 vs. 0.53 0.20 , p = 0.015 ) [ table 2 ] . the role of infection in rpl has been intensely investigated during the past decades and viruses , in particular cmv , have been under more attention since they can produce chronic / recurrent intrauterine infections . possible mechanisms are suggested as production of toxic metabolites , fetal , placental , or chronic endometrial infection , and chorio - amnionitis . the aim of this study was to evaluate if exposure / infection to cmv and/or an altered immunological response to cmv is related to rpl in women of our population . for this aim , we assessed anti - cmv igg and igm specific antibodies along with igg ai , which is a good and reliable method to differentiate a recurrent / reactivated infection from primary infection . as the results showed , there was only one case with a recurrent or reactivated infection in each group of patients and controls . patients with rpl were significantly more seropositive than controls suggesting that previous exposure to cmv might be a risk factor for rpl . these patients had also higher igg titer which shows either hyper - response or more frequent exposure to cmv . because patients and controls were similar in igg ai , two possibilities are exist both indicating an altered immune response in rpl cases : that recurrent exposure to cmv is a risk factor for rpl , but patients with rpl have altered immune response to cmv exposure which does not lead to higher ai ; and that patients with rpl are hyper - responsive to cmv . few reports are available on the association between cmv infection and rpl and results have been controversial . the study by odland et al . on a large sample of rpl cases and controls found similar prevalence of seropositivity ( 78% vs. 81.1% ) . johnson et al . studied women with rpl of unknown etiology for immune responsiveness and found only 35% seropositive cases ( compared to 65% in controls ) . other investigators also found lower seropositivity in rpl women compared with age - matched female controls and an impaired lymphocyte proliferative response to cmv in seropositive rpl cases compared with seropositive controls . in contrast to these studies , and similar to our results , szkaradkiewicz et al . found more frequent seropositivity and higher levels of antibodies in women with rpl than controls and suggested that abortion might have resulted from fetal infection due to reactivation of chronic cmv infection in the course of pregnancy . however , other studies with using polymerase chain reaction method did not find cmv in gestational tissue of women with rpl suggesting that cmv infection of the gestational tissue is not a direct cause of abortions in rpl cases and highlights the role of immune response in rpl . differences between the studies results can be due to differences in the studied population and it is well - known that epidemiology of cmv infection is different among different populations . also , patients characteristics have been different among studies . for example , odland et al . found that seropositivity of cmv in rpl cases raised with increasing age ( from 76.5% in younger than 20 years to 91.4% in older than 34-year - old women ) , and previous studies were different regarding patients age distribution . future larger and using multi - socioeconomic cases of rsa , also evaluation of different components of their immune response to cmv need to be done to get more clear results . the results of the present study showed that previous exposure to cmv detected by a positive igg antibody is significantly higher in rpl . . we may conclude : ( 1 ) recurrent exposure to cmv is a risk factor for rpl , ( 2 ) other immunological processes like hyper - response ( higher igg titers ) to the similar number of exposure with cmv may be another risk factor , ( 3 ) recurrent exposure to cmv , but the inability to detect it by higher ai , mostly because of altered immune function might be other state , and ( 4 ) autoimmune etiologies and hypergammaglobulinemia in rpl cases may have caused false positivity in cmv antibody checking . further investigations evaluating these theories and also other mechanisms such as cell - mediated immunological response in rpl patients , with adequate sampling and sample size are required . ag , hz and mm collected the data and rs , mm , and ar did the analysis . all authors have assisted in preparation of the first draft of the manuscript or revising it critically for important intellectual content . all authors have read and approved the content of the manuscript and are accountable for all aspects of the work .	background : some evidence has shown a relationship between human cytomegalovirus ( cmv ) infection and pregnancy loss . however , whether recurrent or latent cmv infection or altered immune response to cmv is related to recurrent pregnancy loss ( rpl ) is unclear . we evaluated cmv infection and avidity of antibodies to cmv in women with rpl.materials and methods : this case - control study was conducted on 43 women with rpl referred to a clinical immunology out - patient clinic in isfahan ( iran ) , and 43 age - matched multiparous women without history of abortion as control subjects . patients and controls were evaluated for anti - cmv igg and igm antibodies and igg avidity index ( ai ) using the enzyme linked immunosorbent assay method . student 's t - test and chi - square test were used to analyze the data.results:one case ( 2.3% ) of positive anti - cmv igm was detected in each group . anti - cmv igg positivity was more frequent in patients than in controls ( 90.6% vs. 69.8% , p = 0.014 ) , but there was no difference between the two groups in anti - cmv igg ai ( 79.4 11.4 vs. 80.1 10.2 , p = 0.781 ) . igg titer was significantly higher in seropositive cases with rpl than seropositive controls ( 5.18 1.99 vs. 2.00 0.81 , p < 0.001).conclusion : we found that previous exposure to cmv was significantly higher in patients with rpl than the control group . however , no association was found between igg ai and rpl . further investigations are needed to find whether latent cmv infection starts an indirect process of autoimmune etiology in rpl or women with rpl have recurrent or reactivation of cmv infection .
it is impossible to imagine a modern socially active man who does not use mobile devices and/or computers with wi fi function . during the last two decades , there has been a significant increase in cell phone usage throughout the world . meanwhile , continuous electromagnetic radiation from mobile phones , through the development of oxidative stress and dna fragmentation , can obviously lead to the development of different pathologies including tumors , and also can violate spermatogenesis [ 1 , 2 ] . the effect of mobile device radiation on healthy male sperm parameters and fertility is the subject of recent interest and investigations . it is well known that infertile men are distinguished by abnormal semen characteristics . in general , the quality of sperm in recent years has worsened throughout the world . the majority of infertile or subfertile men are subjects with sperm motility violations and/or dna damage . the aim of this study was to investigate the direct in vitro influence of mobile phone radiation on sperm dna fragmentation and motility in healthy subjects with normozoospermia . 32 healthy middle aged men ( 27.5 3.5 years old ) with normal semen parameters ( normozoospermia ) were selected to study the direct influence of mobile phone radiation on sperm quality in vitro . the participants involved were chosen among normozoospermic men from childless couples with proved female infertility who were consulted at our clinics . all of them have given signed informed consent for permission to participate in the study . men were instructed to avoid pocket bearing of mobile phones for a 2 month period before examination . each sperm sample was diluted with isotonic solution at a temperature of 37c in a 1:1 ratio to prevent sperm agglutination ; the samples were then placed in an incubator for one hour . , each sperm sample was divided into two equal portions ( a and b ) . portions a of all involved men were placed for 5 hours in a thermostat , and portions b were placed in a second thermostat for the same period of time , where an enabled mobile phone in combined standby / talk mode was placed . the device was located 5 cm from the samples and was turned on in radiation frequency range 900/1800 mhz ( gsm standard ) . for authenticity of the experiment a call was carried out on the phone every 10 minutes , to imitate the transmission mode after 5 hours of incubation at a temperature of 27c , the sperm samples from both thermostats were re evaluated with respect to the basic semen parameters . sperm vitality and motility were evaluated according to the specifications of the world health organization . which classifies sperm movement accordingly as progressively motile , nonprogressively motile and immotile . the sperm portions for evaluation of dna damage were obtained from previously prepared and divided samples . a presence of dna fragmentation in both a and b portions of each sample was determined every hour using the sperm chromatin dispersion ( scd ) test with specific dna breakage kit , which allows the detection of dna breaks in lysed sperm nuclei . this test could be considered as simple , fast , versatile and reliable procedure to determine the frequency of sperm cells with fragmented dna . intact unfixed spermatozoa were immersed in an inert agarose microgel on a pretreated slide . an initial acid treatment denatures dna in those sperm cells with fragmented dna . following this , the lysing solution removes most of the nuclear proteins , and in the absence of massive dna breakage produces nucleotides with large halos of spreading dna loops emerging from a central core . however , the nucleotides from spermatozoa with fragmented dna either do not show a dispersion halo or the halo is minimal . we used halosperm in vitro diagnostic kit by halotech dna , sl that allows the measurement of sperm dna fragmentation quickly , easily and without the need of complex laboratory equipment . after sample processing with halosperm , spermatozoa without fragmentation were marked by dispersion halo , those with dna strand breaks were without halo . after checking the sample with a bright field microscope , we classified sperm cells as follows ( figure 1 ) . the results were expressed as percentage of sperm cells with fragmented dna in 500 sperm cells . for interpreting of results we calculated sperm dna fragmentation ( sdf ) index by formula : sdf index(%)=100number of spermatozoa with fragmented dnanumber of spermatozoa counted digital options of semen characteristics whitney test to establish differences in the performance of both groups : the difference was considered statistically significant at p < 0.05 . basic semen parameters before incubation sperm count in groups a&b during 5 hours did not change in general and was not statistically different from each other : 92.3 22.7 x 10 / ml vs 90.8 24.2 x 10/ml respectively . furthermore , the number of dead sperm in both study groups after 5 hours of incubation was not statistically different ( 9.1% 3.7% vs. 9.6% 4.1% ; p > 0.05 ) ( figure 2 ) . the dead sperm percentage in both groups a and b. however , the number of spermatozoa with progressive movement in group b , under the influence of electromagnetic radiation , is statistically lower than the number of spermatozoa with progressive movement in group a with no effect of a mobile phone ( 66.5% 6.3% vs 81.3% 7.2% , p < 0.05 ) ( figure 3 ) . medians of studied parameters in both groups a and b : the percentage of spermatozoa with progressive movement ( pm ) ; percentage of motionless sperm ( nm ) ; percentage of spermatozoa with non progressive movement ( i m ) . no differences between the number of spermatozoa with motionless sperm ( nm ) between groups a and b were reported ( 7.1% 2.5% vs. 7.4% 3.3% ; p > 0.05 ) , while the number of non progressive movement spermatozoa ( i m ) was significantly higher in group b , that was under the influence of cell phone radiation ( 25.3% 4.7% vs. 12.8% 5.8% , p < 0.05 ) ( figure 3 ) . figure 4 clearly shows a significant difference in the incidence of dna fragmentation between the studied groups . the samples for dna fragmentation were obtained from both thermostats each hour of incubation . finally the sperm samples exposed to mobile phone radiation after 5 hours are characterized by sperm dna fragmentation index of about 8.8% 2.2% , while this index in the control group samples after 5 hours incubation without electromagnetic fields influence was 4.2% 1.8% ( p < 0.05 ) . levels of dna fragmentation in sperm samples with ( b ) and without ( a ) electromagnetic exposure . at first after that this analysis was made every hour after placing semen samples into the irradiation zone . we aimed to establish the relationship between the duration of exposure of the semen samples and the level of dna fragmentation ( 5b ) . obtained results are presented in figure 5 . the level of dna fragmentation in semen samples exposed in the exposure area ( b ) and semen samples without electromagnetic field influence ( a ) . as shown in figure 5 , sperm dna fragmentation index before and during 5 hours of electromagnetic field exposure constantly increases ( 3.3 1.2% vs. 8.8 2.2% , p < 0.05 ) . however , the sdf index in the semen samples without electromagnetic influence did not statistically change during 5 hours of examination ( 3.3 1.2% vs. 4.2 1.8% , p > 0.05 ) . a positive correlation was noted between the duration of semen samples placement in the area of mobile phone electromagnetic radiation and the level of dna fragmentation : the increase of exposure term increases the frequency of dna damage . moreover the majority of dna damaging occurs during first 2 hours of exposure ( figure 5 ) . the individual influence of mobile phone electromagnetic waves in every semen sample was also determined . the percentage of spermatozoa with fragmented dna after 5 hours exposure to the radiation zone was statistically higher , by 65.3 12.4% on average , than this parameter in the same semen sample before irradiation . basic semen parameters after 5 hours of incubation in groups a and b sd statistical deviation thus , according to the presented data cell phone radiation decreases sperm motility , but only in previously motile sperm . overall reduction of the basic normal semen analysis parameters that characterize the sperm count , motility and morphology have been noted worldwide in recent decades . researchers are unanimous about the negative impact of certain environmental factors on sperm quality . the most well known are the harmful effects of smoking , the abuse of alcohol , intake of spermicidal food products , etc . likewise , the negative impact of increased local testicular temperature on spermatogenesis has been proven [ 7 , 8 , 9 ] . the reproductive toxicants like lead and cadmium should be considered the causes for the decline in semen quality . in idiopathic oligoasthenozoospermic males there were registered higher levels of lead and cadmium in their semen which correlated with impairment of sperm motility and vitality percentages and more importantly with higher sperm dna fragmentation and semen reactive oxygen species level . different harmful environmental influences have led to changes in semen analysis standards by reducing the lower limits of normal ranges , which were declared by the world health organization . the possible negative impact of mobile phone radiation on sperm quality has been established recently . while no certain conclusions can be drawn from current evidence , a growing number of studies indicate a decrease in male fertility associated with cellular phone usage . an excellent study , one of the first on this topic , was presented by agarwal a et al . in 2008 . the authors concluded that the use of cell phones by men is associated with a decrease in their semen quality . according to the researchers data the decrease in sperm count , motility , viability , and normal morphology v. boulos & h. hassan , in 2013 , concluded that cell phone use in men is associated with decreased semen quality in the form of decreased sperm count , motility , viability & normal morphology , which depend on the duration of cell phone exposure time . the authors found a significant positive correlation between the decrease in the different sperm parameters : the decrease in the value of one of these parameters is concomitant with other parameters changes . researchers compared sperm parameters in 4 groups from a total of 145 patients who completed the questionnaire : group 1 , varying mobile phone usage duration ( 030 min , 3060 min and over 60 min ) , group 2 , different mobile phone carriage mode ( in the pocket , on the belt and in the handbag ) , group 3 , wireless internet users ( duration : 030 min , 3060 min and over 60 min ) , group 4 , users of different types of internet connection ( wireless , wired ) . the authors have concluded that there was no significant difference between period of mobile phone usage and sperm count and motility ( p = 0.236 and p = 0.457 , respectively ) . similarly , they did not find a significant difference between the mobile phone carriage mode and sperm count and motility either ( p = 0.837 and p = 0.157 , respectively ) . however , according to their data , sperm motility decreased with the increased use of wireless internet ( p = 0.03 ) . similarly , spermatozoa motility was worse in the group of wireless internet users than that of the wired internet usage group ( p = 0.035 ) . furthermore , there are differences in the type of cellular device used , the transmission mode at which it operates ( talk vs. standby ) and also the distance between the sperm cells and phone . all of these variations contribute to the above mentioned ambiguity of the results presented in the different cell phone studies . original research conducted in 2009 , by subjecting in vitro samples of human spermatozoa to radio frequency radiation at 1.8 ghz and specific absorption rates ( sar ) of 0.4 to 27.5 w / kg , showed a correlation between increasing sar and decreased motility and vitality of sperm , increased oxidative stress and 8oxo2deoxyguanosine markers , stimulating dna base adduct formation and increased dna fragmentation . in contrast to de iuliis et al . , our results did not demonstrate a correlation between mobile phone radiation exposure and sperm vitality , compared to the control group without exposure ( figure 2 . ) . in our study we did not find differences between the percentages of dead and motionless sperm in samples with / without radiation exposure ( 9.1% 3.7% vs. 9.6% 4.1% ; p > 0.05 ; 7.1% 2.5% vs. 7.4% 3.3% ; p > 0.05 , respectively ) . however , like the above mentioned researcher 's results , our study also has shown a correlation between radiation exposure time and decreased sperm motility , but only in previously motile sperm ( figure 3 ) . compared to the control group , our results demonstrate a significant decrease in the number of sperm with progressive movement ( of 81.3% 7.2% vs. 66.5% 6.3% , p < 0.05 ) and a concurrent increase in the number of sperm with non progressive movement ( 12.8% 5.8% vs. 25.3% 4.7% , p < 0.05 ) when under electromagnetic radiation exposure for a 5 hour period . our presented data also suggests that electromagnetic radiation causes dna fragmentation of sperm . the sdf index before exposure in both groups was 3.3 1.2% , while after 5 hours of exposure the same index in the study group had increased to 8.8 2.2% ( p < 0.05 ) . however , the dna fragmentation levels in semen samples without electromagnetic influence did not statistically change during 5 hours of examination ( 3.3 1.2% vs. 4.2 1.8% , p > 0.05 ) ( figure 5 ) . their results demonstrate a significant decrease in sperm progressive motility and a significantly higher proportion of sperm with dna fragmentation when samples were incubated for 4 hours under the laptop . these differences were seen in comparison with aliquots of the same semen samples incubated under similar conditions , but outside the proximity of any computer or electronic device . the investigators speculated that the use of a laptop computer wirelessly connected to the internet and positioned near the male reproductive organs may decrease human sperm quality . thus , exposing the semen to radio frequency electromagnetic waves from laptops connected to the internet through wi besides affecting sperm motility , vitality and dna fragmentation , mobile phone radiofrequency electromagnetic fields ( rf emf ) leads to changes in morphological spermatozoa parameters . according to falzone n , et al , 2011 , a significant reduction in sperm head area ( 9.2 0.7 m vs. 18.8 1.4 m ) and acrosome percentage of the head area ( 21.5 4% vs. 35.5 11.4% ) was reported among exposed sperm compared with unexposed controls . also the mean number of zona bound sperm of the test hemizona and controls was 22.8 12.4 and 31.8 12.8 ( p < 0.05 ) , respectively . authors concluded that although radiofrequency electromagnetic fields exposure did not adversely affect the acrosome reaction , it had a significant effect on sperm morphometry . also , a significant decrease in sperm binding to the hemizona was observed . , after an investigation into the effects on rat testes of radiofrequency radiation emitted from indoor wi emf impact significant increases occur in serum 8hydroxy2deoxyguanosine levels and 8hydroxyguanosine staining in the testes of the experimental group . the authors also found decreased levels of catalase and glutathione peroxidase activity in the experimental group , which may have been due to radiofrequency effects on enzyme activity . the data obtained in this animal study raises questions about the safety of radiofrequency exposure from wi fi internet access devices for growing organisms of reproductive age , with a potential effect on both fertility and the integrity of their germ cells . neither we nor any of the above mentioned authors studied prospectively the influence of mobile phones irradiation on paternity . obviously , therefore , prolonged direct mobile phone radiation influence on sperm is able to progressively impair the basic semen motility parameters and bring about dna fragmentation similar to other well known causes of male infertility ( varicocele , inflammatory gonadal diseases , etc . ) . accordingly , it is likely that constant direct mobile phone radiation exposure on sperm ( testis ) could damage sperm and directly influence its motility . this is likely to affect the fertility of healthy men , possibly rendering them infertile in the future . it would be interesting to establish in the future if there is a decrease in fertility among heavy mobile phone users compared to men with short term device use . common metallic objects like coins , rings or zips , in pockets or on trousers and the body , may intensify rf emf impact . dr william whittow and colleagues , in 2008 , investigated specific absorption rates ( sar ) in the human body with a realistic mobile phone source positioned in a front trouser pocket of a truncated male heterogeneous anatomical body model . realistic everyday metallic objects , including a coin , ring and zip were added to the model . the authors concluded that these objects increased the sar in the body at different frequencies . the cumulative effect of these three objects generally increased the sar in the waist section over the maximum levels of frequency range and exposure permitted in the united kingdom . at the present time nobody knows the real intercommunication between the constant direct mobile phones irradiation affects on testes and developmental delays in the offspring . another interesting implication of the findings of our study is that women wishing to conceive a child should be careful about laptop and mobile phones usage . the fertile life of human sperm in the female reproductive tract may be 80 hours or more . spermatozoa can survive in the female cervix uteri and likely oviducts where they might be in danger from electromagnetic waves [ 20 , 21 ] . emr on granulosa cells , ovarian follicle numbers , endometrial tissue , quality of oocytes and embryos , and even alterations on fetal heart physiology during pregnancy . for example , ninety women with uncomplicated pregnancies , and 30 full term healthy newborn infants were included in the study by rezk ay et al , 2008 . the pregnant mothers were exposed to rf emr emitted by mobile phones while on dialing mode for 10 min during pregnancy and after birth . researchers found that the exposure of pregnant women to mobile phones significantly increased fetal and neonatal heart rate , and significantly decreased fetal cardiac output . that is why we consider that men readying themselves for fatherhood , as well as women wishing to conceive a child , especially when registered fertility problems are present , should be informed about the different risks and probably negative direct impact of long term mobile phone radiation on semen quality and embryo / fetus development . maybe person who could be selected for assisted reproduction techniques or even sperm donors should avoid this influence during some time before semen extraction too . besides the semen parameters rf - emr probably could negatively impact on sexual communication , fertility and quality of life by reducing the erectile function . men with erectile dysfunction ( ed ) use their cell phones longer those without ed . men who have ed carry their cell phones switched on much longer than men who do not have ed . currently , there is no consensus on mobile phone rf additional well designed investigations are needed to evaluate the real consequences of long term employment of these devices . emr leads to a significant decrease in the number of sperm with progressive movement and an increase in those with non progressive movement.prolonged direct mobile phone exposure may bring about sperm dna fragmentationfor men readying themselves for fatherhood , especially when registered fertility problems exist , it would be better to avoid holding a mobile phone in a trouser pocket for long periods of time . emr leads to a significant decrease in the number of sperm with progressive movement and an increase in those with non progressive movement . prolonged direct mobile phone exposure may bring about sperm dna fragmentation for men readying themselves for fatherhood , especially when registered fertility problems exist , it would be better to avoid holding a mobile phone in a trouser pocket for long periods of time .	introductionit is impossible to imagine a modern socially active man who does not use mobile devices and/or computers with wi fi function . the effect of mobile phone radiation on male fertility is the subject of recent interest and investigations . the aim of this study was to investigate the direct in vitro influence of mobile phone radiation on sperm dna fragmentation and motility parameters in healthy subjects with normozoospermia.material and methods32 healthy men with normal semen parameters were selected for the study . each sperm sample was divided into two equal portions ( a and b ) . portions a of all involved men were placed for 5 hours in a thermostat , and portions b were placed into a second thermostat for the same period of time , where a mobile phone in standby / talk mode was placed . after 5 hours of incubation the sperm samples from both thermostats were re evaluated regarding basic motility parameters . the presence of dna fragmentation in both a and b portions of each sample was determined each hour using a standard sperm chromatin dispersion test.resultsthe number of spermatozoa with progressive movement in the group , influenced by electromagnetic radiation , is statistically lower than the number of spermatozoa with progressive movement in the group under no effect of the mobile phone . the number of non progressive movement spermatozoa was significantly higher in the group , which was influenced by cell phone radiation . the dna fragmentation was also significantly higher in this group.conclusionsa correlation exists between mobile phone radiation exposure , dna fragmentation level and decreased sperm motility .
as the third most common urological disorder in men over the age of 50 following benign prostatic hyperplasia ( bph ) and prostate cancer , prostatitis represents 8% of all male urology office visits . it is also the most common urologic disorder in men under the age of 50 . the accumulative lifetime chance of receiving a diagnosis of acute or chronic prostatitis by 85 years of age is 26% . category iii prostatitis according to the national institutes of health ( nih ) classification system is chronic nonbacterial prostatitis / chronic pelvic pain syndrome ( cp / cpps ) . subgroups of this syndrome are categorized into category iiia , or inflammatory cpps , on the basis of the presence of excessive leukocytes in expressed prostatic secretions ( eps ) or post - prostatic massage urine or semen and category iiib , or non - inflammatory cpps , which is defined by the lack of significant leukocytes in similar specimens . patients with cp / cpps present with urological pain complaints as well as with voiding symptoms as a primary component of this syndrome . however , there is no universally successful therapy for cp / cpps . alternative therapeutic strategies and multimodal management may provide the best results at this time . among these therapies , heat therapy applied to the prostate gland may promote the biological process of fibrosis or scar formation in the chronic inflamed area and consequently could shorten the natural resolution of the inflammation . we performed transurethral thermotherapy for patients who were diagnosed with cp and were dissatisfied with standard medication and analyzed the results to evaluate the therapeutic efficacy before and after treatment . a retrospective study between october 2009 and september 2010 of 26 patients who had been diagnosed with cp was performed . the patients had been treated with conventional medical therapy such as -adrenergic blockers , antibiotics , and/or anti - inflammatory agents for at least 3 months without improvement of symptoms . twenty men were diagnosed with inflammatory cp ( nih - category iiia ) and the rest with non - inflammatory cp ( nih - category iiib ) . initially , the serum prostate - specific antigen ( psa ) level was tested to exclude prostate cancer . all patients also completed the nih - chronic prostatitis symptom index ( cpsi ) before and after treatment . this index consists of the three domains of pain ( location , frequency , and severity ) , voiding ( irritative and obstructive symptoms ) , and quality of life , including impact . the nih - cpsi has proven clinical importance in the evaluation and follow - up of patients in general urologic practice . we used the tempro system from direx - initia at an intraprostatic temperature of 55 for 50 minutes with a medium heating rate and a single treatment session . this system includes the 6-ring electrode mounted on a silicone - coated 16 fr latex foley catheter and incorporates a computer - controlled radiofrequency ( rf ) generator that enables bipolar treatment . the treatment area is chosen according to the prostate urethral length as measured by transrectal ultrasound ( trus ) for localized treatment , which is effected by applying energy to different electrodes of the catheter . patient follow - up was done by 3 months so that patients could complete the nih - cpsi after treatment . the baseline characteristics of the patients and their most bothersome symptoms can be seen in table 1 . for the subgroup analysis after treatment , we defined treatment success as a greater than 50% reduction in symptom scores . for statistical analysis , we used the t - test , analysis of variance , and chi - square test for comparison of the differences in averages depending on times and groups . for all analyses , spss ver . 12.0 ( spss inc . , chicago , il , usa ) was used , and p<0.05 was set for significance . a total of 26 patients were diagnosed with cp and treated with bipolar rf thermotherapy as an in - office procedure conducted in a single session . owing to the small diameter of the foley catheter , its insertion is smooth and painless , and there is no give during the procedure . the patients ' average age was 42.65.3 years ( range , 32 to 50 years ) , and their mean serum psa level was 0.90.3 ng / ml ( range , 0.29 to 1.39 ng / ml ) . mean prostate volume was 27.15.5 g ( range , 17 to 38 g ) , and mean prostate urethral length was 3.60.3 cm ( range , 2.8 to 4.2 cm ) . the most bothersome symptoms of the patients were pain and voiding symptoms ( table 1 ) . the average score for all 3 domains of the nih - cpsi significantly decreased after treatment . the total scores decreased from 19.87.1 to 11.17.0 , the pain domain decreased from 8.63.1 to 4.83.1 , the voiding symptoms domain decreased from 5.11.8 to 2.91.8 , and the effect on the quality of life decreased from 6.12.2 to 3.42.2 ( p < 0.05 ) ( fig . 1 ) . the average rate of improvement in the nih - cpsi score after treatment was 43.9% : 44.2% in men with nih - category iiia and 42.9% in men with nih - category iiib ; 7 of 26 men showed scores that markedly decreased by more than 70% after the thermotherapy ( fig . 2 ) . when we defined the criterion of treatment success as a greater than 50% improvement in symptoms , as mentioned above , the number of patients in the more successful group ( group a ) and the number of patients in the less successful group ( group b ) was both 13 . the baseline characteristics of the patients in group a and group b were not significantly different . additionally , when we performed age- and prostate volume - specific analysis according to the significant difference in rate of improvement between the two groups , 9 patients were aged more than 40 years old with a prostate volume of less than 30 g , and 6 of these 9 men were assigned to group a. in other words , 67% of the 9 men were in group a , which is higher than the 41% of group a in the other categories , even if this was not statistically significant ( p=0.465 ) . acute complications after the treatment were 4 urinary tract infections treated with antibiotics and 1 " de novo " lower urinary tract symptom treated with conservative management ( table 2 ) . prostatitis is a common urologic problem , and approximately 5% of visits to us urologists are reported to be for inflammatory diseases of the prostate . prostatitis also accounts for up to 15 to 25% of visits to urology clinics in south korea . although it is suggested that prostatitis affects men of all ages , approximately 5% of young men between 20 and 50 years of age have suffered from prostatitis . all men who participated in this study were less than 50 years old because of the differences in the prevalence of prostatitis and the symptomatic overlap between bph and prostatitis over the age of 50 . nickel found that approximately 5 to 20% of men diagnosed with bph have prostatitis - like symptoms , with over one third of men with bph also having prostatitis in the past . the disease is classified into four categories , including acute bacterial prostatitis , chronic bacterial prostatitis , cp / cpps , and asymptomatic inflammatory prostatitis . the third disease category , cp / cpps , accounts for approximately 90% of all chronic prostatitis cases and clinically presents with chronic pain in the perineum , rectum , penis , testicles , and abdomen . it is often accompanied by symptoms of obstruction or irritative symptoms such as weak stream , hesitancy , and frequency on voiding . the symptoms usually remain stable or improve slightly over time , but some symptoms wax and wane over months in cycles . cp / cpps is considered to be a consecutive process caused by inflammatory , immunologic , neuroendocrine , and neuropathic mechanisms that begin with an initiator in a genetically or anatomically vulnerable man . although cp / cpps is associated with bacteria in only about 8% of cases , infection has often been postulated to be an initiating factor . thus , medical therapy including antimicrobial or anti - inflammatory agents is started for patients diagnosed with cp / cpps . however , the pain is observed to persist even after the absence of evidence of infection during follow - up . urologists have difficulty in treating patients with cp / cpps that is peripherally and centrally sensitized , and therapeutic mechanisms directed at the initiators of the process may not work as well when the condition becomes chronic . thus far , treatment strategies have focused on symptomatic relief . among the various strategic managements advocated for cp / cpps , heat therapy in patients with poor response to standard therapies appears to be a promising therapeutic approach that has been reported to clinically reduce pain and voiding symptoms . until now , whichever device was used , overall improvements with heat therapy have been reported in regard to both objective and subjective measures . heat therapy may modify the afferent nerve fibers that convey the objective symptoms of pain from the inflammatory processes of the prostate gland . in addition , lower urinary tract symptoms have been consistently demonstrated to be associated with prostatic inflammation , and approximately 19% of men presenting with voiding symptoms as their chief complaint are diagnosed with prostatitis . brehmer and baba reported that urgency symptoms may be improved by decreased excitatory signals to the bladder with sensory denervation in the posterior urethra after the transurethral thermotherapy . coagulative necrosis with cell death therefore begins at temperatures in excess of 45 , with rapid thermoablation occurring at 60 or more . the term thermotherapy was coined to describe treatment temperatures above 45 and the term hyperthermia is used for those below this level . namely , the distinction between hyperthermia and thermotherapy is primarily their target temperature ranges , generally 42 to 44 for hyperthermia compared with temperatures of 45 to 50 for thermotherapy . the treatment outcome of heat therapy for cp / cpps shows inconsistent results according to the symptom measures used , temperature ranges , modes of treatment , and study design . the transrectal approaches report temperatures of 41 to 45 , as do the transurethral microwave hyperthermia procedures . documented symptom improvement in 50% of men with chronic abacterial prostatitis by transrectal microwave hyperthermia at the long - term follow - up . transurethral microwave thermotherapy ( tumt ) studies have reported temperatures of 45 to 60 , as have transurethral rf needle ablation ( tuna ) procedures . the clinical usefulness of tumt for the treatment of symptomatic bph has been reported in the urological literature , and tumt has been widely used throughout the world . because a rf electromagnetic wave has deeper and more even transmission of heat to tissues than does a microwave , transurethral rf thermotherapy for the treatment of bph has shown comparable results to other nonsurgical treatment modalities . the term radiofrequency refers not to the emitted wave but rather to the alternating electric current that oscillates in the high - frequency range ( 200 to 1,200 khz ) . the tempro system is a transurethral heating procedure with temperatures of 45 to 60 for thermotherapy . the transurethral approach is considered to be a more direct way of heating than the transrectal approach . bipolar technology enables concentrated rf transmission to the prostate with minimal energy delivery to the surrounding tissue . furthermore , a cooling system is not required in this manner , whereas a microwave heating system , whether through a transrectal or transurethral approach , requires a cooling system because the lower tissue penetration requires greater power . nickel and sorensen examined the safety and efficacy of tumt for nonbacterial prostatitis in 20 men randomly assigned to therapy or sham . at the 3-month follow - up , the patients treated by tumt had significantly improved symptom scores compared with sham - treated patients . seven of 10 men treated with tumt had a favorable result compared with 1 of 10 men treated with a sham therapy . aaltomaa and ala - opas reported that tuna relieved symptoms in cpps patients and the need for medication was reduced . in this study , 27% of patients treated with transurethral rf thermotherapy showed marked improvement ( symptom reduction of 70% or more ) , 27% showed moderate improvement ( symptom reduction of 30 to 70% ) , and 31% showed mild improvement ( symptom reduction 30% or less ) ; 15% were no better . whereas the mean rate of improvement in this study was 43.9% , kastner et al . reported the result of a pilot study in which 35 patients with cp showed improvement in the mean value of the nih - cpsi of 51% with cooled tumt through 12 months of follow - up . in this study , patients with cp / cpps were divided into two groups according to the rate of improvement after the treatment to analyze the factors that affect therapeutic efficacy , but we did not obtain significant results . in addition , considering that age and prostate volume can affect the rate of improvement , patients older than 40 years and with a prostate volume of less than 30 g showed a tendency for a better therapeutic effect , but the difference was not significant . although we showed a therapeutic effect on symptom improvement , study of the factors affecting therapeutic results has been left for future evaluations . long - term study of the therapeutic effect of transurethral rf thermotherapy is also needed . they evaluated 5 patients , and although there was some freedom from symptoms at 3 months , only 1 patient remained symptom - free for 6 months , and all patients were symptomatic at 9 months . considering the characteristics of cp / cpps , which has a chronic cyclic course , long - term follow - up will necessarily be needed . among the multimodal approaches for patients suffering from this difficult condition , bipolar rf thermotherapy is a simple and effective treatment that can be tried as a second - line management following standard medication . given that this study was conducted in patients who were unresponsive to conventional drugs , it is also fair to say that rf thermotherapy for cp / cpps has therapeutic efficacy . even though this was a short - term study with 3 months of follow - up , it showed significant improvement in pain and irritative voiding symptoms with a simple procedure and a minimal side effect profile , allowing that cp is a common male problem but is poorly managed . we suggest that transurethral rf thermal treatment could play a role in the management of refractory cp / cpps , although the long - term effects of the therapy remain unclear . cp / cpps is a poorly defined disease entity with no definite treatment modalities . of the multimodal strategies to improve pain and voiding symptom management for patients with cp / cpps intractable to conventional medication it can also be an efficacious and simple alternative to treating cp / cpps with minimal side - effects as an in - office procedure . because long - term data documenting the durability of this improvement are currently unavailable , we need to conduct long - term follow - up of large - scale studies .	purposechronic prostatitis ( cp ) does not yet have a universally successful therapy . alternative treatments including thermotherapy have been adopted in the multimodal management of pain and voiding dysfunction . we retrospectively analyzed the therapeutic efficacy of bipolar radiofrequency thermotherapy for patients who were unsatisfied with conventional medication for cp.materials and methodsa retrospective study between october 2009 and september 2010 of 26 patients who were under 50 years old and diagnosed with cp ( national institutes of health [ nih]-category iii ) was performed . twenty patients were diagnosed with inflammatory cp ( nih - category iiia ) and the rest with noninflammatory cp ( nih - category iiib ) . we used the tempro system at an intraprostatic temperature of 55 for 50 minutes with a medium heating rate . all patients also completed the nih - chronic prostatitis symptom index ( cpsi ) before and after treatment.resultsin the patients diagnosed with cp , the mean serum prostate - specific antigen ( psa ) level was 0.90.3 ng / ml , the prostate volume was 27.15.5 g , and the average score for all 3 domains on the nih - cpsi significantly decreased . the total scores decreased from 19.87.1 to 11.17.0 , the pain domain decreased from 8.63.1 to 4.83.1 , the voiding symptom domain decreased from 5.11.8 to 2.91.8 , and the effect on the quality of life decreased from 6.12.2 to 3.42.2 ( p<0.05).conclusionsbipolar radiofrequency thermotherapy for patients with cp intractable to conventional medication can provide significant improvement in the nih - cpsi . large , randomized controlled trials will also be required to confirm the efficacy of this therapy .
in humans , during the recent years , several factors have increased the risk of fungal infections , like broad spectrum antibiotics , immune - compromised patients , cytotoxic chemotherapy , and transplantations ( malani et al . , 2005 ; nucci & marr , 2005 ) . among all fungal pathogens , candida species remain the most common cause of invasive fungal infections ( pfaller & diekema , 2007 ) . although candida glabrata has been considered as a human commensal and can be often found in the oral cavity ( fidel et al . , 1999 ) , it has recently emerged as the second most prevalent cause of candida infections , just after candida albicans ( pfaller & diekema , 2004 ) . roughly , c. glabrata is responsible for 1525% of disseminated candidiasis ( perlroth et al . , 2007 ) . phylogenetically , c. glabrata is much closer to saccharomyces cerevisiae than to c. albicans and its sister species ( dujon et al . , 2004 ) . indeed , c. glabrata belongs to post - wgd ( whole genome duplication ) yeasts ( fig . candida glabrata with three sister species , kluyveromyces delphensis , candida castellii and kluyveromyces bacillisporus , have been classified to belong to the nakaseomyces genus ( kurtzman , 2003 ) . two additional clinical isolates , candida nivariensis and candida bracarensis , have later been added to the same genus ( alcoba - florez et al . , 2005 ; correia et al . glabrata group includes the three pathogenic species and n. delphensis , and the second group includes c. castellii and n. bacillisporus . a schematics phylogenetic tree ( adopted from gabaldn et al . , 2013 ) candida glabrata , c. nivariensis , c. bracarensis are pathogenic fungi within the glabrata group. the whole - genome duplication event ( wgd ) , which took place app . 100 million years ago , is arrowed . unlike s. cerevisiae , which is diploid and sexual , c. glabrata is believed to be obligate haploid and mating has never been observed ( kaur et al . , 2005 ; muller et al . , candida albicans can live in two different morphological forms ( sudbery , 2011 ) , but the switching between the yeast form and hyphae has not been observed in c. glabrata ( kaur et al . , 2005 ; fig . recently , there has been more focus on c. glabrata genome dynamics and several new sister species genomes have been reported , both providing additional insight into metabolism and virulence potential of the yeast species . dujon et al . ( 2004 ) have reported that c. glabrata cbs 138 has 13 chromosomes with the genome size of 12.3 mb . in comparison with s. cerevisiae , which has 5807 protein - coding genes ( dujon et al . , 2004 ) , c. glabrata has 5283 coding sequences , with an average size of 493 codons . thus , this yeast lineage has reduced its coding potential upon separation from other post - wgd yeasts . candida glabrata has two intrachromosomal rdna repeats loci in subtelomeric regions compared to s. cerevisiae , which has a single intrachromosomal rdna repeat locus . as s. cerevisiae , c. glabrata has 42 trna - encoding genes , which are scattered throughout the genome ( dujon et al . , 2004 ) . in c. albicans , the cug codon is translated as serine instead of leucine , but this genetic codon alteration is not found in c. glabrata . in general , most genes transcribed by rna polymerase iii and even other noncoding rna genes do not have well - conserved size among yeasts ( gabaldn et al . , 2013 ) . for example , in c. glabrata , rnase p rna ( rpr1 ) gene is 1149 nucleotides long , whereas in s. cerevisiae it is 568 nucleotides long ( kachouri et al . , 2005 ) . this large ncrna seems to be present in c. glabrata and sister species , and k. delphensis has a 1368 nucleotide long copy ( gabaldn et al . , 2013 ) . candida glabrata and other related species have a lower number of intron containing protein - coding genes than s. cerevisiae ( neuvglise et al . , 2011 ) . it has been found by conant & wolfe ( 2007 ) that 551 duplicated gene pairs are conserved between c. glabrata and s. cerevisiae , among them six gene pairs encoding glycolytic enzymes eno1/eno2 , pyc1/pyc2 , glk1/emi2 indeed , c. glabrata has recently been reported as an efficient alcohol producing yeast even under aerobic conditions ( hagman et al . , 2013 ) . however , it is so far unclear whether this trait plays an important role during yeast pathogenicity . for example , c. albicans and its sister species are crabtree - negative yeasts and do not produce ethanol under aerobic conditions ( hagman et al . , 2013 ) . all the species in nakaseomyces have transposon - free genomes that are smaller than s. cerevisiae and contain fewer genes ( gabaldn et al . , moreover , all species in this group have a haploid genome , the size of roughly 1012.5 mb , with the exception of n. bacillisporus which is a diploid . the chromosomal number ranges from 8 in c. castellii to 15 in n. bacillisporus , while the glabrata group yeasts have from 10 to 13 chromosomes . note that chromosome number in c. glabrata isolates varies ( ahmad et al . , 2013 ) . the gene - order conservation is very high between n. delphensis , c. bracarensis and c. nivariensis , but in general , it is low compared with s. cerevisiae ( gabaldn et al . , 2013 ) . the divergence between orthologous proteins varies from 53% between c. castellii and n. bacillisporus to higher identity level among , many genes have been lost in c. glabrata , for example , galactose metabolism ( five genes ) , phosphate metabolism ( four genes ) , cell defense and virulence ( three genes ) , and nitrogen and sulfur metabolism ( three genes ; dujon et al . , 2004 ) . candida glabrata genome has also lost genes involved in de novo biosynthesis of nicotinic acid ( bna ) , and this has been initially interpreted as a way to adapt to the host environment ( domergue et al . , 2005 ) . however , the species among this clade have different habitats , and the loss of bna has been observed in all of them and likely occurred in the common progenitor when it was still a free - living yeast organism . another example of gene loss in c. glabrata and glabrata group is loss of the six genes cluster involved in allantoin catabolism , dal , which is conserved in s. cerevisiae ( wong & wolfe , 2005 ) . on the other hand , the dal cluster is present in both c. castellii and n. bacillisporus ( gabaldn et al . , 2013 ) . apparently , similar to c. glabrata , the duplicated genes encoding glycolytic enzymes , for example eno1/eno2 and pyc1/pyc2 are conserved in nakaseomyces ( merico et al . , 2007 ) . in contrast to glabrata group where the glycolytic enzymes encoding genes are in pairs , pyruvate kinase , enolase , and glyceraldehyde-3-phosphate dehydrogenase encoding genes are present as a single copy in c. castellii and n. bacillisporus . compared to all nakaseomyces species that encode four hexokinase ( hxk ) genes , c. castellii genome encodes two hxk genes . moreover , all nakaseomyces genomes encode two copies of adh genes , which have orthologs in s. cerevisiae , adh1 and adh3 , and are involved in conversion of acetaldehyde into ethanol . saccharomyces cerevisiae genome also encodes adh2 , which involves in the conversion of the ethanol into acetaldehyde , and this gene has no ortholog in nakaseomyces . the genome of c. glabrata type strain ( cbs 138 ) contains a family of 18 epa genes ( homologues of the flo genes in s. cerevisiae ) , coding for glycosylphosphatidylinositol ( gpi)-anchored cell - wall proteins ( cwp ) , which are known to be adhered to human epithelium ( cormack et al . , 1999 ; similarly , c. bracarensis has 12 members of the epa family , while c. nivariensis genome harbors nine genes . on the other hand , the nonpathogenic yeasts in nakaseomyces , c. castellii , has three homologs of the epa genes , whereas n. bacillisporus has only one homolog , which is distant and clusters together with the pwp ( pa-14 containing wall protein adhesion gene ) and adhesion - like protein genes in c. glabrata . although the glabrata group has a common ancestor , the genome of n. delphensis has a single copy of the epa gene ( gabaldn et al . , 2013 ) . minisatellites are dna tandem repeats that show size variation among different yeast species ( richard et al . , 2008 ) . in s. cerevisiae , it has been found that several genes which are involved in cell wall formation contain minisatellites with variable sizes . the mannoprotein - encoding genes show a variable number of repeats among different yeast isolates and the length of the repeat is directly related to flocculation and cell adhesion . for example , strains with flo1 carrying a longer repeat show better adhesion and increased flocculation ( verstrepen et al . , 2005 ) . , the diversity of minisatellite sequences within the orthologous genes may indicate that the acquisition and loss of minisatellites have been fast during evolution ( richard & dujon , 2006 ) . despite similar genome size , the number of minisatellites in c. glabrata is significantly higher than in s. cerevisiae . in addition , c. glabrata has two more unusual minisatellites , namely , compound minisatellite , with different alternative repeats and megasatellites , with unusually long motifs ( thierry et al . , 2008 ) . haber & louis ( 1998 ) suggested that minisatellites originated by strand slippage during replication of two short ( 510 bp ) sequences or by unequal crossing over . this replication slippage has been reported in s. cerevisiae ( richard & dujon , 2006 ) but not observed in c. glabrata where other mechanisms might be involved ( thierry et al . , some authors proposed that the megasatellite motifs propagate by motif jumps , which may explain the presence of motifs between nonorthologous gene families ( rolland et al . , the genome of c. glabrata has 40 megasatellites mapped to 33 genes , which are distributed on 11 chromosomes with strong preference toward the subtelomeric region . the megasatellites have long motifs ranging from 135 up to 417 nucleotides and belong to two large families , shitt and sffit ( the names come from conservation of five amino acid in each motif in the megasatellite ; thierry et al . , 2008 ) . in a recent study , which covered 20 completely sequenced fungal genomes , 216 peptide motifs encoded by megasatellites and located in protein - coding genes the detected megasatellites have been characterized according to the motif sequence similarity and their amino acid composition . based on this analysis , there is a weak match between shitt and flo motifs despite their similar size ( tekaia et al . , 2013 ) . the originally sequenced c. glabrata has three epa genes containing megasatellites ( epa2 , epa11 , and epa13 ) , another three genes ( epa1 , epa3 , and epa15 ) contain minisatellites , but there are no tandem repeats in epa6 , epa7 , and epa8 ( thierry et al . , 2008 ) . a recent study has shown that the chromosomal translocations that occurred among different strains of c. glabrata are associated with deletions ranging from 130 bp up to 12 kb and two of them were located within megasatellites ( muller et al . , 2009 ) . even translocations and segmental duplications may operate through mini- and megasatellites ( polkov et al . , 2009 ) . candida glabrata isolates have been reported to show variable karyotypes ( shin et al . , 2007 ) . apparently , the genome of c. glabrata is frequently rearranged and some authors consider these rearrangements as an adaptive mechanism . 2009 ) have reported that the most frequent changes in karyotype belong to chromosomal length polymorphism ( clp ) , mainly due to chromosomal translocations and copy number variations within tandem gene repeats . these gene tandem repeats encode putative proteins like yapsins , cell wall aspartyl proteases ( kaur et al . , 2007 ) . 2009 ) have also shown that there is size variation in the subtelomeric epa genes , which is most likely due to the differences in the number of minisatellites within the genes , as proposed by thierry et al . ( 2009 ) , based on their french strain collection , claim that the c. glabrata genome is comparatively stable . this is in contrast to many other works , which propose a very dynamic nature of the c. glabrata genome . ( 2009 ) have found that among danish isolates , chromosomal size variation is associated with either gross intrachromosomal translocations or interchromosomal segmental duplications . these novel chromosomes carry duplicated genes , which are potentially involved in yeast host interaction , and they include the abc transporter family genes , which plays a role in multidrug resistance . the formation of new chromosomes has also been reported in a new study analyzing over 200 clinical isolates ; ahmad et al . ( 2013 ) have found several extra chromosomes originating through at least two different mechanisms ( fig . 3 ) . remarkably , even phylogenetically related isolates have shown rearranged chromosomes , implying that c. glabrata frequently remodels its genome . it has been speculated that in this way , the yeast can improve its fitness when exposed to new environmental conditions ( ahmad et al . , a striking observation of genome dynamics has been made on recent derivatives from a single laboratory strain . apparently , among these very closely related strains , different phenotypic groups correlated with specific karyotypic changes ( bader et al . , 2012 ) . thus , genome aberrations and functional adaptations may occur not only during infection , but also under laboratory conditions during a very short time span and without extreme selective pressures . two possible mechanisms behind the birth of small chromosomes are shown ( adapted from ahmad et al . , 2013 ) . ( 1 ) the small chromosome ( a ) originated by segmental duplication within chromosome a. ( 2 ) interchromosomal translocation , where a large segment of chromosome a is translocated to join the chromosome b leaving the rest of chromosome a as a small chromosome . three large gene families are associated with the cell wall , the yps gene cluster , consisting of six genes encoding the yapsins ( extracellular gpi - linked aspartyl proteinases ) , eight alpha-1,3-mannosyltransferase , and five beta - mannosyltransferase gene clusters ( dujon et al . , 2004 ; dujon , 2010 ; jawhara et al . , 2012 ) . apart from the chromosome remodeling , also gene duplications and cluster and gene family formation apparently play an important role in the specialization to specific environmental condition , virulence , and interaction with the host ( butler et al . , 2009 ; the genome of c. glabrata encodes sir complex including sir2p , sir3p , and sir4p . these proteins are responsible for subtelomeric silencing in c. glabrata ( castao et al . , 2005 ; maestre - reyna et al . , recently , it has been reported that c. glabrata isolates were more adherent to the epithelial cells with overexpression of the epa1 gene and , moreover , these strains contain several polymorphisms in sir3p , which might cause reduced silencing ( martinez - jimenez et al . , 2013 ) . on the other hand , it has been shown that the deletion of hst1 ( homologue of sir2 ) altered the c. glabrata response to stressful conditions with an increase in the fluconazole resistance and decrease in the susceptibility to hydrogen peroxide ( orta - zavalza et al . , 2013 ) . in short , recent studies on c. glabrata genome polymorphism have pointed out that this property is clearly connected to the virulence potential of this organism . future genome sequencing of different isolates will likely provide even further insights into the interplay between genome dynamics and virulence . the first genome structure has provided a background to develop different postgenomic tools and approaches . these could now in turn be used to analyze the connection between the genotype variability and different phenotypic traits . for example , the availability of commercial microarrays has provided a powerful tool to study global aspects of gene expression ( fukuda et al . , 2013 ) to identify potential virulence genes , as well as to determine the level of variation in gene expression among different clinical isolates ( o.p . ishchuk , j. pikur , unpublished data ) . unlike c. albicans , the mechanism of virulence at the molecular level has so far not been well understood in c. glabrata ( silva et al . , 2011 ) . the proteomic approach to study protein expression in c. glabrata has confirmed a previous observation that the virulence of c. glabrata increases when ace2p , transcriptional factor , is inactivated ( stead et al . , 2010 ) . in the same study , 32 of 123 proteins were overexpressed in the ace2 mutant ( stead et al . , 2010 ) . moreover , it has been found that the expression of proteins involved in glucose metabolism , the tca ( krebs ) cycle , respiration and protein synthesis is low when c. glabrata grows at ph 7.4 or 8.0 , whereas the expression of proteins involved in stress responses and protein catabolism was increased ( schmidt et al . , 2008 ) . the stress - response protein trx1p , a component of oxidative stress defenses of c. glabrata , was found to be upregulated ( seneviratne et al . , 2008 , 2010 ) . hsp12p is another protein , which has been highly expressed in c. glabrata biofilm . in s. cerevisiae , the expression of this protein is induced by different kinds of stress including heat shock , oxidative stress and osmotic stress ( perez - torrado et al . , 2005 ) , and it is involved in azole antifungal resistance in c. albicans ( karababa et al . , the proteomic studies have also shown that several glycolytic enzymes including fba1p , tdh3p , gpm1p , and eno1p are downregulated in c. glabrata biofilm ( seneviratne et al . , 2010 ) . it would also be interesting to apply the proteome approach on different clinical isolates to see whether the genome variation correlates with the proteome variation . a new analysis based on high - resolution mass spectrometry has been conducted to identify new peptides , which were previously unknown in c. glabrata ( prasad et al . , 2012 ) . by genome search for specific peptides ( gssp ) and other comparative genomic strategies , the conservation and absence of protein - coding genes in c. new postgenomic tools might also be helpful for early diagnosis and proper treatment , thereby reducing the mortality rate . to overcome the pathogen misdiagnosis , perl et al . ( 2011 ) have suggested a new sensitive approach to study the volatile organic compounds , multicapillary column ( mcc ) equipped ion mobility spectrometer ( mcc - ims ) , to differentiate between different pathogenic fungi and their strains . current sequencing projects of tens of different pathogenic isolates , for example at the cornell and lund universities , will provide further background for development of new postgenomic approaches and will help to reveal further genes behind c. glabrata virulence . the sequencing of their genomes will make these strains attractive models for postgenomic tool analysis .	the yeast pathogen candida glabrata is the second most frequent cause of candida infections . however , from the phylogenetic point of view , c. glabrata is much closer to saccharomyces cerevisiae than to candida albicans . apparently , this yeast has relatively recently changed its life style and become a successful opportunistic pathogen . recently , several c. glabrata sister species , among them clinical and environmental isolates , have had their genomes characterized . also , hundreds of c. glabrata clinical isolates have been characterized for their genomes . these isolates display enormous genomic plasticity . the number and size of chromosomes vary drastically , as well as intra- and interchromosomal segmental duplications occur frequently . the observed genome alterations could affect phenotypic properties and thus help to adapt to the highly variable and harsh habitats this yeast finds in different human patients and their tissues . further genome sequencing of pathogenic isolates will provide a valuable tool to understand the mechanisms behind genome dynamics and help to elucidate the genes contributing to the virulence potential .
congenital adrenal hyperplasia ( cah ) is an autosomal recessive disease caused by various genetic defects of the enzyme involved in cortisol biosynthesis in the adrenal glands and ranges from mild to severe according to the degree of enzyme dysfunction . twenty - one - hydroxylase deficiency , occurring in 1 of 16,000 births , is the most frequent type of cah . however , 17-hydroxylase/17,20-lyase deficiency of cah is very rare . loss - of - function mutation of cyp17a1 gene , which normally activates 17-hydroxylase/17,20-lyase , can result in defective p450c17 action and present as primary amenorrhea and abnormality of secondary sex characteristics in women and as pseudohermaphroditism in men . the deterioration producing cortisol in the adrenal cortex stimulates adrenocorticotropic hormone ( acth ) secretion in the pituitary gland , leading to hyperplasia of the adrenal cortex . long - term overproduction of acth due to inadequately treated or untreated cah rarely transforms to adrenal cortical adenoma or carcinoma . herein , we present a case of a huge adrenal mass confirmed as adrenal cortical adenoma in cah with 17 hydroxylase/17,20-lyase deficiency . a 36-year - old female visited the emergency department with pain in the left upper abdominal quadrant . she brought her abdominal computed tomography ( ct ) scan taken in another hospital showing a 106.38.6 cm - sized left adrenal mass with hemorrhagic necrosis ( fig . her systolic blood pressure was 150 to 170 mm hg and her diastolic blood pressure was 100 to 110 mm hg , higher than the standard values for her age . the routine biochemical study , random urine analysis , and blood coagulation test results were all normal , and the serum electrolytes showed na 141 mmol / l ( normal range [ nr ] , 135 to 145 ) , k 3.3 mmol / l ( nr , 3.5 to 5.5 ) , and cl 100 mmol / l ( nr , 98 to 120 ) . hormone tests showed cortisol was reduced to 0.9 g / dl ( nr , 6.7 to 22.6 ) and acth was elevated slightly , to 75.94 pg / ml ( nr , 7.2 to 63.6 ) . renin was reduced to 0.80 ng / ml / hr ( nr , 1.31 to 3.95 ) and aldosterone was elevated to 183.31 pg / ml ( nr , 29.9 to 158.8 ) . the results of 24-hour urine analysis were as follows : metanephrine 0.518 mg / day ( nr , 0 to 1.3 ) , epinephrine 2.3 g / day ( nr , 0 to 20 ) , and vanillylmandelic acid 4.04 mg / day ( nr , 0 to 8) ; all values were within the nr except those for 17-ketosteroids 2.77 mg / day ( nr , 7 to 20 ) and urinary free cortisol 37.6 ( nr , 55.5 to 286 ) . typical ct findings of a large adrenal mass with hemorrhagic necrosis led to the diagnosis of pheochromocytoma with biochemically burnt - out status . to further evaluate the characteristics of the adrenal mass , metaiodobenzylguanidine - single - photon emission computed tomography ( mibg - spect ) imaging was performed and showed no significant radiotracer uptake . next , 18f - fluorodeoxyglucose positron emission tomography - ct ( f - fdg pet - ct ) was performed , and the fdg uptake in the solid portion of the mass was elevated ( fig . the possibility of pheochromocytoma could not be excluded based on mibg - negative and pet - positive findings . after laparoscopic adrenalectomy , a left hemorrhagic adrenal mass was pathologically confirmed as adrenal cortical adenoma 1053 cm in size ( fig . after the operation , her acth was elevated to 406.9 pg / ml ( nr , 7.2 to 63.6 ) , and the low - dose acth stimulation test showed her cortisol was 1.0 g / dl ( nr , 6.7 to 22.6 ) at baseline , 1.6 g / dl after 30 minutes , and 1.0 g / dl after 1 hour showing blunted response . after reviewing her abdominal ct , we discovered that she had received plastic surgery on both breasts . we requested the patient regarding her medical history as she had failed to mention this procedure on the first visit . she also lacked secondary sexual characteristics such as pubic and axillary hair ( tanner stage i ) . the patient confessed she experienced no menarche in puberty and had kept these facts secret even from her family members . further hormone testing showed the followings : thyroid stimulating hormone 3.1 iu / ml ( nr , 0.4 to 4.9 ) , prolactin 27.2 ng / ml ( nr , 3.3 to 26.7 ) , luteininzing hormone 25.0 miu / ml ( nr , 1.2 to 10.9 ) , follicular stimulating hormone 51.5 miu / ml ( nr , 1.8 to 8.8 ) , estradiol < 20 pg / ml ( nr , 27 to 433 ) , progesterone 4.32 ng / ml ( nr , 0.2 to 23 ) , and 17-oh - progesterone 0.21 ng / ml ( nr , 0.1 to 5.0 ) . however , both dehydroepiandrosterone ( dhea ) and renin were reduced to 0.41 g / ml ( nr , 1.3 to 9.8 ) and 0.7 ng / ml / hr ( nr , 1.3 to 3.9 ) , respectively , whereas aldosterone was elevated to 171.9 pg / ml ( nr , 29.9 to 158.8 ) ( table 1 ) . furthermore , x - ray examination of the upper arm showed that the bone age was compatible with that of a 15-year - old female and bone mineral density was below the expected range for her actual age ( z - score -3.1 in the lumbar spine and z - score -3.4 in the femoral neck ) , probably due to sex hormone deficiency . chromosomal study of the patient was performed using peripheral blood leukocytes and showed a normal karyotype of 46 , xx , female . we also conducted polymerase chain reaction for all intron sequences in the neighborhood of the cyp17a1 gene using appropriate primers . according to mutation analysis , we found a compound heterozygous mutation for p.tyr329fs ( c.985_987deltacinsaa ) and missense mutation p.his373leu ( c.1118a > t ) in exon 6 . she was diagnosed with cah caused by 17-hydroxylase/17,20-lyase deficiency confirmed by the cyp17a1 gene mutation , presenting with lack of secondary sexual development , reduced production of cortisol and dhea , increased aldosterone and acth as well as atypical huge adrenal mass . the patient was started on oral prednisolone , 7.5 mg in the morning and 5 mg in the afternoon , combined with estrogen . finally , general weakness improved significantly , and the dose of oral prednisolone was reduced to 5 mg in the morning and 2.5 mg in the afternoon . to date , her hormone level has been monitored for 3 years . due to her poor compliance and concerns of weight gain ( although only 5 mg of prednisolone was prescribed ) , acth remained high . inadequate suppression of acth resulted in the progression of hyperplasia of the right adrenal gland observed on the 3-year follow - up ct scan ( fig . cah is characterized by adrenal enlargement resulting from excessive acth due to defective steroidogenesis . however , neoplastic transformation of the adrenal gland , such as adrenal cortical adenoma is a rare complication in cah . . reported on a 51-year - old female with a 7-cm virilizing adrenocortical tumor in cah due to 21-hydroxylase deficiency after reviewing nine cases including six adrenal adenomas and three carcinomas . . also reported on a 22-year - old patient with 11-hydroxylase deficiency having a 16-cm - sized adrenal cystic mass . to date , most adrenal cortical adenomas or carcinomas reported in cah have been due to 21-hydroxylase deficiency , and very few have been caused by 11-hydroxylase or 3-hydroxysteroid dehydrogenase deficiencies . although adrenal myelolipoma associated with cah has been reported , reports on 17-hydroxylase/17,20-lyase deficiency with a huge adrenal adenoma and degenerative cystic change in a patient have not yet been published . the case in the present study involved cah with 17-hydroxylase/17,20-lyase deficiency and a compound heterozygotic gene mutation of p.tyr329lysfs ( c.985_987deltacinsaa ) and p.his373leu ( c.1118a > t ) at different sites on the same exon ( exon 6 ) , indicating that each mutation exists in different allelic genes as a heterozygote type . cytochrome p450 17-hydroxylase enzyme , an important enzyme involved in cortisol biosynthesis , is encoded by the cyp17a1 , gene and 17-hydroxylase/17,20-lyase deficiency is caused by a mutation in the cyp17a1 gene , which was first reported by biglieri et al . in a single patient . cyp17a1 gene mutation resulting in 17-hydroxylase/17,20-lyase deficiency deteriorates the synthesis of cortisol , testosterone , and estrogen , leading to the stimulation of acth and gonadotrophin hormone manifesting as hypergonadotropic hypogonadism , with accompanying overproduction of mineralocorticoids . excessive mineralocorticoids may increase blood pressure or result in hypokalemia , as in this case . moreover , due to the sex hormone deficiency , a patient with chromosome 46,xx will not experience secondary sex development and menstruation , whereas a patient with 46,xy will show female external genitalia . primary amenorrhea , lack of secondary sexual development , and hypertension are the usual clinical presentations of 17-hydroxylase/17,20-lyase deficiency . atypically , the patient in this case came to the hospital at a comparatively old age complaining abdominal pain caused by the huge adrenal mass that had been suspected to be pheochromocytoma in the early stage , and this clinical manifestation was not the typical pattern compared with other cah cases with 17-hydroxylase/17,20-lyase deficiency . kim et al . reviewed the cyp17a1 mutations in adults with 17-hydroxylase/17,20-lyase deficiency in korea ; however , clinical presentation of adrenal cortical adenoma in a patient has not been reported . adrenal enlargement and neoplastic change could be due to inadequate glucocorticoid treatment or non - treatment . of the patients , seven who were treated properly showed no adrenal mass , but six , who were not treated properly showed gross enlargement of the adrenals . in our case , the patient had not been treated until her first visit at the age of 36 ; thus , the over 30-year continuous acth stimulation due to cortisol and sex hormone defects caused by 17-hydroxylase/17,20-lyase deficiency led to the large adrenal adenoma . whether the adrenal tumor was the result of long - term overstimulation of acth remains questionable . however , a correlation between adrenal hyperplasia and high acth levels is plausible given previous reports that adequate treatment that suppresses acth hypersecretion can reverse the adrenal enlargement . additionally , unlike the other cases of 17-hydroxylase/17,20-lyase deficiency , the aldosterone level was high in our patient . suggested the severity of 17-hydroxylase/17,20-lyase deficiency would be associated with the potency of cytochrome p450 corticosterone methyloxidase ( aldosterone synthase ) , a key enzyme in the conversion of corticosterone to aldosterone . in severe 17-hydroxylase/17,20-lyase deficiency , aldosterone synthase activity is more potentiated in outer zona fasciculata cells , leading to increased aldosterone . furthermore , chronically elevated acth may have affected the production of aldosterone in zona glomerulosa , which is also responsive to acth . of the three cah cases with 17-hydroxylase/17,20-lyase deficiency and a compound heterozygote gene mutation reported by kim et al . , one patient with a high aldosterone level presented with higher blood pressure and acth level and lower cortisol ( similar to our case ) compared with the other two cases . further studies of hyperaldosteronism - related phenotypes are necessary to explain the elevated aldosterone levels . in conclusion moreover , no huge adrenocortical adenoma has been reported in cah with 17- hydroxylase/17,20-lyase deficiency . in our case , a huge adrenal mass primary amenorrhea in female patients who present with both sexual infantilism and mineralocorticoid hypertension should be the focus of differential diagnosis . additionally , proper glucocorticoid replacement is important for preventing adrenal gland enlargement or reducing the possibility of transformation into a tumor .	congenital adrenal hyperplasia ( cah ) with 17-hydroxylase/17,20-lyase deficiency is usually characterized by hypertension and primary amenorrhea , sexual infantilism in women , and pseudohermaphroditism in men . hypertension , and sexual infantilism in women and pseudohermaphroditism in men . in rare cases , a huge adrenal gland tumor can present as a clinical manifestation in untreated cah . adrenal cortical adenoma is an even more rare phenotype in cah with 17-hydroxylase/17,20-lyase deficiency . a 36-year - old female presented with hypertension and abdominal pain caused by a huge adrenal mass . due to mass size and symptoms , left adrenalectomy was performed . after adrenalectomy , blood pressure remained high . based on hormonal and genetic evaluation , the patient was diagnosed as cah with 17-hydroxylase/17,20-lyase deficiency . the possibility of a tumorous change in the adrenal gland due to untreated cah should be considered . it is important that untreated cah not be misdiagnosed as primary adrenal tumor as these conditions require different treatments . adequate suppression of adrenocorticotropic hormone ( acth ) in cah is also important to treat and to prevent the tumorous changes in the adrenal gland . herein , we report a case of untreated cah with 17-hydroxylase/17,20-lyase deficiency presenting with large adrenal cortical adenoma and discuss the progression of adrenal gland hyperplasia due to inappropriate suppression of acth secretion .
negative social appraisal or diabetes - related social stigma is a major and potential consequence of living with diabetes . it is an important phenomenon in many countries , especially asian countries such as china , india , and iran . health - related stigma is based on an enduring feature of identity conferred by a health problem or a health - related condition . diabetes - related stigma is particularly severe since diabetes is a chronic and life - threatening condition . important life areas such as people 's dignity , social status , employment opportunities or job security , marriage , family relationships , and friendships are commonly affected by stigma . therefore , it is necessary to assess the stigmatizing effect of diabetes and to eliminate it for health promotion . in recent years , several experts and organizations have also emphasized on the necessity of efforts to reduce diabetes - related stigma . in fact , if we can reduce diabetes - related stigma , it would be possible to mitigate or prevent its negative consequences . thus , recently , taking efforts to overcome stigma and discrimination against people with diabetes has been proposed as a goal of the international diabetes federation ( idf ) . the healthcare systems are expected to try to overcome diabetes - related stigma . despite numerous calls for stigma reduction , so far , no study has been conducted to explore the useful strategies to overcome diabetes - related stigma in iran . literature reports have mentioned several approaches for other stigmatizing conditions , but these approaches are not specifically related to reduction of stigma on diabetes . on the other hand , it is impossible to develop generic stigma reduction strategies for all health conditions , given the specificity of these conditions and the complexity of the factors related to each person 's experience of stigma . this study explored the ways to overcome diabetes - related stigma at the community level in iran . this paper is the first part of the findings of an action research study which was designed in iran in 2012 to plan and implement a program for overcoming diabetes - related stigma . action research is a way for engaging people to share their experiences and to learn through collective reflection and analysis . action research studies consider local cultural beliefs and are an appropriate approach to address stigma . kemmis and mctaggart 's method guides the action research , which includes four phases : planning , action , observation , and reflection . qualitative content analysis was used to derive strategies to overcome the diabetes - related stigma in the planning phase . the study participants ( in the planning stage ) consisted of volunteered medical and nursing personnel working in a selected diabetes center , people with type 1 diabetes mellitus ( t1 dm ) , and people without diabetes living in isfahan , iran . the researchers contacted all people with type 1 diabetes referring to the selected diabetes center in isfahan . in addition , some people with diabetes introduced their family members who volunteered to participate in the study . also , the researchers chose volunteers without diabetes from public places . data collection was done through unstructured in - depth interviews , focus groups , e - mail , short message service ( sms ) , and telephone interview to extract strategies to overcome the diabetes - related stigma [ table 1 ] . participants were asked to reflect on the stigmatizing attitude and behaviors , and to respond to an open question , how can we reduce diabetes - related stigma in the community ? detailed questions were asked subsequently based on participants initial responses to encourage them to fully describe each mentioned strategy . she emphasized that the purpose of the meeting is not to reach consensus on strategies and the goal of the study is to have all possible strategies based on participants experiences and opinions . focus group meetings for people with t1 dm , people without diabetes , family members , and staff were held separately . finally , all participants were told if they remembered a different approach , they could give it via e - mail or sms . all interviews and focus groups were audio - recorded and transcribed verbatim . data collection continued until data saturation . due to the qualitative nature of the data in this phase of the action research this approach involves three steps including open coding , creating categories , and abstraction . for this purpose , after repeated listening , and reading the transcribed files , the text was reviewed for open coding . it was repeated several times , and as many headings as necessary were written down to describe all the mentioned strategies . finally , all the similar groups and classes were placed in larger classes , so that four categories were formed . moreover , peer debriefing ( all authors discussed the data analysis process ) and member checks ( all the extracted concepts were returned to the participants and examined ) were followed for enhancing credibility . inquiry audit by an independent qualitative researcher was used for enhancing dependability and confirmability . researchers selected the participants who volunteered to participate after introducing themselves and informing them about the research objectives . participants were assured that all stories would be confidential and they were free to quit at any time they wished . seventy - four people participated in extracting the anti - stigma strategies .. the participants consisted of 44 people with t1 dm ( 25 women , 19 men ; age 19 - 50 years ; a history of diabetes of about 2 - 35 years ; high school education up to phd ) , 12 family members ( 2 men , 10 women ; high school education up to bachelor 's degree ) , 15 non - diabetic patients ( 9 women , 6 men ; age 18 - 55 years ; with elementary education up to graduation ) , and 3 healthcare personnel in the selected endocrine center ( 3 women with bsc to msc degree ) . participants pointed to several strategies and stated that any efforts to overcome diabetes - related stigma can not be achieved without community - based strategies . proposed community - based strategies were education , community - based strategies to overcome diabetes - related stigma one of the main strategies mentioned was community education that should be done through the appropriate content and multiple channels . they believed that iranian society has little information on the nature of diabetes and its management and transmission . diabetes management , and myths about diabetes and people with diabetes was the main stigma reduction strategy . participants believed that education about the nature of diabetes included cause of diabetes , difference between type 1 and 2 diabetes , diabetes symptoms , transmission of diabetes , and controllable nature of diabetes . for example , most participants referred to the one of the challenges in dealing with others . they stated that diabetes is considered a disease of the elderly , and community members are surprised when dealing with a child with t1 dm . not only people with diabetes , but also the non - affected population mentioned that it is necessary to educate people about the difference between type 1 and 2 diabetes . two non - diabetic women of age 21 and 24 years , respectively , stated in a focused group thus : it is possible to explain it many people do not know that young teens may have type 1 diabetes . ability to use sweet food as possible , and recent news in the control and treatment of diabetes . insulin injection is one of the most important things considered strange and difficult by the society and sometimes results in misinterpretations . thus , participants believed that the content of education should show insulin , its advantages , and injection to eradicate fears and misinterpretations . participants referred to myths about the impact of diabetes on menstruation and fertility and diabetes and aggression that needed attention and correction . therefore , an educational program should be designed to replace these myths with true and scientific information . for example , some participants said that people around them believed that diabetes could prevent the development of reproductive system and result in infertility . a 27-year - old woman with diabetes stated : for example , it is a common belief that a diabetic girl could not have a baby , or it is difficult to marry her . it must be reformed . participants mentioned multiple channels which can be used to increase the community awareness and therefore they can reduce diabetes - related stigma . these included a variety of media sources including audio - visual media , visual media , and human resources . the audio - visual media sources mentioned were radio and television , internet , and mobile phone . radio and television was named one of the most influential media for community education . however , participants indicated that the media policy must be planned in such a way to attract the society 's attention and portray the empowerment and normal life of people with diabetes . they referred to a variety of programs , including the preparation of films on diabetes , to attract the audience 's attention . a 28-year - old woman with diabetes said : i think good movies on cinema or television ; i mean a well - made film is good idea . such as a film titled man in orange dress, that portrays a good picture of sweepers i do not know how , but i think it is necessary , since it is the most effective means to change culture and information . visual media can be utilized for community education , which includes the brochures , , newspapers , books , homework , and billboards . for example , billboards can be used as efficient tools to attract the audience 's attention . however , participants expressed the opinion that just the purposeful and planned use of billboards with simple visual message can be effective . attractive photos and caricature , as well as short phrases must accompany the visual messages . a 27-year - old man expressed : you can draw a young man with diabetes who holds insulin in a workplace . then write , diabetes does not limit to work. " human resources can also be used for community education . based on our data , human resources are divided into two groups . one of the main sources is the healthcare team working in the diabetes centers that can teach in different places such as schools ( courses for teachers , students , and parents ) , offices , cultural centers , parks , health centers , and mosques . according to the participants , religious leaders can educate the community on diabetes and fasting , and sin and disease efficiently . a woman said that since diabetes was diagnosed , my people do not get close to my kid . establishing contact between people with and without diabetes was another strategy that emerged , which contained two classes , indirect contact and direct contact . indirect contact through introducing successful people in the living with diabetes and introducing outstanding people with diabetes in athletic , scientific , family , and social fields was mentioned as a way for changing attitudes . in this way , people become familiar with people having a normal life and enjoying a fruitful and successful life despite having diabetes . actually , a real example can contribute to the strengthening of education . overall , community members should know that they are normal people like the rest and diabetes does not disrupt their life . one of the staff of a selected diabetes center said about the effectiveness of the introduction of empowerment people with diabetes thus : i think if we show people with type 1 diabetes in the tv show or at different places , it will be a condition that is more acceptable . the fear and stigma surrounding diabetes can be reduced through direct contact between people with and without diabetes . exhibition of achievements of people with diabetes and appreciation of successful diabetes control in public places were mentioned as a tool to boost self - confidence of people with diabetes and to change community 's attitude . a 25-year - old man without diabetes alternatively , you can have a station in the other exhibition and show achievement of people with diabetes . so , they never fear of disclosing their diabetes . " attempt to defend the rights of people with diabetes was the other proposed strategy by the participants . adjusting employment and labor laws and facilitating access to treatment and care . adjusting employment and labor laws all participants who had attempted for employment mentioned that there are some restrictions for employing people with diabetes . in fact , they spoke about the stigma of employment law . having no medical condition such as diabetes is a primary criterion in employment laws . a 22-year - old woman with diabetes believed that overcoming the stigma of laws is an effective way to reduce social stigma . when people see that we are educated and we can work , they will understand the correct facts . another measure to protect the rights of people with diabetes and to reduce its related stigma is to facilitate access to treatment and care . this would mean adjusting insurance laws , facilitating access to drugs and glucose test strips , facilitating dental services , and preparing national valid identification cards for people with diabetes . for example , they mentioned national valid i d containing information such as name , age , picture , diabetes duration , type of treatment , and physician 's name . preparing and providing information to the public on this card a 27-year - old woman with diabetes stated : make a special and national card for a person with diabetes , so that everybody knows it . if there is any diabetes - related stigma , people with diabetes do not carry card . i mean de - stigmatization must be your first step and making a card must be your next step . the other item frequently expressed by the participants was diabetes - related stigma imposed by the media . the statements of all the participants demonstrated that it is necessary to establish media watching teams to review stigmatizing issues appearing in the media ( such as newspapers , films , lyrics , television programs , etc . ) . protests can be done through publishing critical reviews in newspapers and writing letters to the editors and directors and meeting them . for example , participants pointed to the examples of the stigmatizing films and programs and stated that they can be criticized and published in newspapers . a 27-year - old woman with diabetes , referring to the bitter sugar , said : it was as a bad movie was about a little athletic boy who went to camps . it was about terrible times experienced by the boy and his mom . in my opinion they should not show this film again . on the other hand , it is possible to write a letter to the people who create the stigmatizing programs ( including the chief editor , program directors , etc . ) and meet them . a 27-year - old man with diabetes said about writing letters to editors and directors thus : we can write a letter to the editor , director , etc . , we can request for a meeting . then we can talk with him on the subject and convince him as much as possible . then he or she can correct the newspaper headline in the next issue , or the picture in the subsequent films . one of the main strategies mentioned was community education that should be done through the appropriate content and multiple channels . they believed that iranian society has little information on the nature of diabetes and its management and transmission . diabetes management , and myths about diabetes and people with diabetes was the main stigma reduction strategy . participants believed that education about the nature of diabetes included cause of diabetes , difference between type 1 and 2 diabetes , diabetes symptoms , transmission of diabetes , and controllable nature of diabetes . for example , most participants referred to the one of the challenges in dealing with others . they stated that diabetes is considered a disease of the elderly , and community members are surprised when dealing with a child with t1 dm . not only people with diabetes , but also the non - affected population mentioned that it is necessary to educate people about the difference between type 1 and 2 diabetes . two non - diabetic women of age 21 and 24 years , respectively , stated in a focused group thus : it is possible to explain it many people do not know that young teens may have type 1 diabetes . ability to use sweet food as possible , and recent news in the control and treatment of diabetes . insulin injection is one of the most important things considered strange and difficult by the society and sometimes results in misinterpretations . thus , participants believed that the content of education should show insulin , its advantages , and injection to eradicate fears and misinterpretations . participants referred to myths about the impact of diabetes on menstruation and fertility and diabetes and aggression that needed attention and correction . therefore , an educational program should be designed to replace these myths with true and scientific information . for example , some participants said that people around them believed that diabetes could prevent the development of reproductive system and result in infertility . a 27-year - old woman with diabetes stated : for example , it is a common belief that a diabetic girl could not have a baby , or it is difficult to marry her . it must be reformed . participants mentioned multiple channels which can be used to increase the community awareness and therefore they can reduce diabetes - related stigma . these included a variety of media sources including audio - visual media , visual media , and human resources . the audio - visual media sources mentioned were radio and television , internet , and mobile phone . radio and television was named one of the most influential media for community education . however , participants indicated that the media policy must be planned in such a way to attract the society 's attention and portray the empowerment and normal life of people with diabetes . they referred to a variety of programs , including the preparation of films on diabetes , to attract the audience 's attention . a 28-year - old woman with diabetes said : i think good movies on cinema or television ; i mean a well - made film is good idea . such as a film titled man in orange dress, that portrays a good picture of sweepers . such acceptable picture can portray people with diabetes i do not know how , but i think it is necessary , since it is the most effective means to change culture and information . visual media can be utilized for community education , which includes the brochures , , newspapers , books , homework , and billboards . for example , billboards can be used as efficient tools to attract the audience 's attention . however , participants expressed the opinion that just the purposeful and planned use of billboards with simple visual message can be effective . attractive photos and caricature , as well as short phrases must accompany the visual messages . a 27-year - old man expressed : you can draw a young man with diabetes who holds insulin in a workplace . then write , diabetes does not limit to work. " human resources can also be used for community education . based on our data , one of the main sources is the healthcare team working in the diabetes centers that can teach in different places such as schools ( courses for teachers , students , and parents ) , offices , cultural centers , parks , health centers , and mosques . according to the participants , religious leaders can educate the community on diabetes and fasting , and sin and disease efficiently . a woman said that since diabetes was diagnosed , my people do not get close to my kid . they believed that iranian society has little information on the nature of diabetes and its management and transmission . diabetes management , and myths about diabetes and people with diabetes was the main stigma reduction strategy . participants believed that education about the nature of diabetes included cause of diabetes , difference between type 1 and 2 diabetes , diabetes symptoms , transmission of diabetes , and controllable nature of diabetes . for example , most participants referred to the one of the challenges in dealing with others . they stated that diabetes is considered a disease of the elderly , and community members are surprised when dealing with a child with t1 dm . not only people with diabetes , but also the non - affected population mentioned that it is necessary to educate people about the difference between type 1 and 2 diabetes . two non - diabetic women of age 21 and 24 years , respectively , stated in a focused group thus : it is possible to explain it ability to use sweet food as possible , and recent news in the control and treatment of diabetes . insulin injection is one of the most important things considered strange and difficult by the society and sometimes results in misinterpretations . thus , participants believed that the content of education should show insulin , its advantages , and injection to eradicate fears and misinterpretations . participants referred to myths about the impact of diabetes on menstruation and fertility and diabetes and aggression that needed attention and correction . therefore , an educational program should be designed to replace these myths with true and scientific information . for example , some participants said that people around them believed that diabetes could prevent the development of reproductive system and result in infertility . a 27-year - old woman with diabetes stated : for example , it is a common belief that a diabetic girl could not have a baby , or it is difficult to marry her it must be said that they can get pregnant , they can get married . participants mentioned multiple channels which can be used to increase the community awareness and therefore they can reduce diabetes - related stigma . these included a variety of media sources including audio - visual media , visual media , and human resources . the audio - visual media sources mentioned were radio and television , internet , and mobile phone . radio and television was named one of the most influential media for community education . however , participants indicated that the media policy must be planned in such a way to attract the society 's attention and portray the empowerment and normal life of people with diabetes . they referred to a variety of programs , including the preparation of films on diabetes , to attract the audience 's attention . a 28-year - old woman with diabetes said : i think good movies on cinema or television ; i mean a well - made film is good idea . such as a film titled man in orange dress, that portrays a good picture of sweepers i do not know how , but i think it is necessary , since it is the most effective means to change culture and information . visual media can be utilized for community education , which includes the brochures , , newspapers , books , homework , and billboards . for example , billboards can be used as efficient tools to attract the audience 's attention . however , participants expressed the opinion that just the purposeful and planned use of billboards with simple visual message can be effective . attractive photos and caricature , as well as short phrases must accompany the visual messages . a 27-year - old man expressed : you can draw a young man with diabetes who holds insulin in a workplace . then write , diabetes does not limit to work. " human resources can also be used for community education . based on our data , human resources are divided into two groups . one of the main sources is the healthcare team working in the diabetes centers that can teach in different places such as schools ( courses for teachers , students , and parents ) , offices , cultural centers , parks , health centers , and mosques . according to the participants , religious leaders can educate the community on diabetes and fasting , and sin and disease efficiently . a woman said that since diabetes was diagnosed , my people do not get close to my kid . establishing contact between people with and without diabetes was another strategy that emerged , which contained two classes , indirect contact and direct contact . indirect contact through introducing successful people in the living with diabetes and introducing outstanding people with diabetes in athletic , scientific , family , and social fields , people become familiar with people having a normal life and enjoying a fruitful and successful life despite having diabetes . overall , community members should know that they are normal people like the rest and diabetes does not disrupt their life . one of the staff of a selected diabetes center said about the effectiveness of the introduction of empowerment people with diabetes thus : i think if we show people with type 1 diabetes in the tv show or at different places , it will be a condition that is more acceptable . the fear and stigma surrounding diabetes can be reduced through direct contact between people with and without diabetes . exhibition of achievements of people with diabetes and appreciation of successful diabetes control in public places were mentioned as a tool to boost self - confidence of people with diabetes and to change community 's attitude . a 25-year - old man without diabetes alternatively , you can have a station in the other exhibition and show achievement of people with diabetes . indirect contact through introducing successful people in the living with diabetes and introducing outstanding people with diabetes in athletic , scientific , family , and social fields was mentioned as a way for changing attitudes . in this way , people become familiar with people having a normal life and enjoying a fruitful and successful life despite having diabetes . overall , community members should know that they are normal people like the rest and diabetes does not disrupt their life . one of the staff of a selected diabetes center said about the effectiveness of the introduction of empowerment people with diabetes thus : i think if we show people with type 1 diabetes in the tv show or at different places , it will be a condition that is more acceptable . the fear and stigma surrounding diabetes can be reduced through direct contact between people with and without diabetes . exhibition of achievements of people with diabetes and appreciation of successful diabetes control in public places were mentioned as a tool to boost self - confidence of people with diabetes and to change community 's attitude . a 25-year - old man without diabetes alternatively , you can have a station in the other exhibition and show achievement of people with diabetes . attempt to defend the rights of people with diabetes was the other proposed strategy by the participants . these rights were their most important social challenges and were categorized as adjusting employment and labor laws and facilitating access to treatment and care . adjusting employment and labor laws all participants who had attempted for employment mentioned that there are some restrictions for employing people with diabetes . in fact , they spoke about the stigma of employment law . having no medical condition such as diabetes is a primary criterion in employment laws . a 22-year - old woman with diabetes believed that overcoming the stigma of laws is an effective way to reduce social stigma . when people see that we are educated and we can work , they will understand the correct facts . it takes a long time , but it is very effective . another measure to protect the rights of people with diabetes and to reduce its related stigma is to facilitate access to treatment and care . this would mean adjusting insurance laws , facilitating access to drugs and glucose test strips , facilitating dental services , and preparing national valid identification cards for people with diabetes . for example , they mentioned national valid i d containing information such as name , age , picture , diabetes duration , type of treatment , and physician 's name . preparing and providing information to the public on this card a 27-year - old woman with diabetes stated : make a special and national card for a person with diabetes , so that everybody knows it . if there is any diabetes - related stigma , people with diabetes do not carry card . i mean de - stigmatization must be your first step and making a card must be your next step . another measure to protect the rights of people with diabetes and to reduce its related stigma is to facilitate access to treatment and care . this would mean adjusting insurance laws , facilitating access to drugs and glucose test strips , facilitating dental services , and preparing national valid identification cards for people with diabetes . for example , they mentioned national valid i d containing information such as name , age , picture , diabetes duration , type of treatment , and physician 's name . preparing and providing information to the public on this card a 27-year - old woman with diabetes stated : make a special and national card for a person with diabetes , so that everybody knows it . if there is any diabetes - related stigma , people with diabetes do not carry card . i mean de - stigmatization must be your first step and making a card must be your next step . the other item frequently expressed by the participants was diabetes - related stigma imposed by the media . the statements of all the participants demonstrated that it is necessary to establish media watching teams to review stigmatizing issues appearing in the media ( such as newspapers , films , lyrics , television programs , etc . ) . protests can be done through publishing critical reviews in newspapers and writing letters to the editors and directors and meeting them . for example , participants pointed to the examples of the stigmatizing films and programs and stated that they can be criticized and published in newspapers . a 27-year - old woman with diabetes , referring to the bitter sugar , said : it was as a bad movie was about a little athletic boy who went to camps . it was about terrible times experienced by the boy and his mom . in my opinion they should not show this film again . on the other hand , it is possible to write a letter to the people who create the stigmatizing programs ( including the chief editor , program directors , etc . ) and meet them . a 27-year - old man with diabetes said about writing letters to editors and directors thus : we can write a letter to the editor , director , etc . , we can request for a meeting . then we can talk with him on the subject and convince him as much as possible . then he or she can correct the newspaper headline in the next issue , or the picture in the subsequent films . the results of the study showed that individuals with and without diabetes believed that overcoming diabetes - related stigma depends on reducing the social stigma . they believed that community - level strategies should be widely implemented in the long term . they mentioned several strategies including education , contacts , advocacy , and protest , which can improve the society 's attitude toward people with diabetes . since diabetes - related stigma is rooted in lack of information , all participants mentioned education as a core component in all destigmatizing programs and activities . participants statements and findings of abdoli et al. study ( 2013 ) indicated that iranian community is not familiar with diabetes , especially type 1 diabetes , and they are afraid of diabetes . what they know about diabetes complications and what they do not know about the controllable nature of diabetes result in fear of the condition therefore , participants mentioned that it is necessary to plan people 's education with appropriate training content via multiple channels . fukunaga et al . reported that participants experienced social stigma related to diabetes and felt that increasing the public 's understanding of the disease would alleviate the social stigma . developing information campaigns aimed at increasing public awareness of diabetes and correcting the myths and misconceptions surrounding diabetes education is effective even in the short term , and should include a definition of disease , prevalence , symptoms , course of disease , biology , assessment and treatment , superstition , and true facts . in most programs ( such as hiv / aids - related stigma ) , education is a major component aimed at increasing knowledge , replacing false assumptions , and correcting stigmatizing behavior and attitudes . the effectiveness of educational approaches can best be increased in combination with other approaches like contact and skills building . these strategies are found in several fields such as aids , leprosy , and epilepsy and share a common ground . their differences lie in the content of the information provided and the groups at which the messages are targeted . since stigmatization is a context - based phenomenon , the content of educational messages should be carefully considered and well adjusted to the context of stigmatized people . thus , although the literature on stigma mentioned education as a destigmatizing strategy , what distinguishes our finding is that it clearly expresses the effective available channels and appropriate educational content for the iranian community . participants cited a range of stigmatizing issues in diabetes that must be replaced with the facts , while so far no study has been done on this issue in diabetes . it has been reported that students form a good and easily accessible group and often have a flexible attitude and behavior . van der meij and heijnders stated that contact could be face to face or through the media . it has mixed results , but most studies on aids , epilepsy , and mental illness have shown positive results . according to white , one of the most effective strategies to reduce social stigma is to increase interpersonal contact between mainstream citizens and members of the stigmatized group . as a vehicle of stigma reduction , contact is most effective when it is between people of equal status ( mutual identification ) , is personal , voluntary , and cooperative , and is mutually judged to be a positive experience . advocacy is another strategy that was mentioned by the participants to reduce diabetes - related stigma . results indicate that advocacy through adjusting employment and labor laws and facilitating access to treatment and care can reduce the challenges of the patients in finding suitable jobs and can help in disease management and finally stigma . according to literature reports , advocacy is the way to change policies and discriminatory laws , and to better access to treatment and care . policies about how and where conditions are treated , divorce , immigration , employment policies , or banning people from public office , elections , or / and ownership can be stigmatizing . thornicroft also mentioned facilitating access to care and workplace adjustments as the necessary steps to overcome the stigma . however , different aspects of the findings of the present study showed that participants believed that establishing a comprehensive equipped diabetes clinic is the key component to facilitate access to treatment and , thus , to overcome the stigma . actually , they believed such a supportive source could reduce their problem , while this finding is contrary to the findings of other studies . for example , deacon et al . reported that assigning a special clinic for hiv / aids can be a barrier to receiving services , since some people may not feel comfortable about coming to hiv / aids clinics . protest highlights the injustice of specific stigmas and leads to a moral appeal for people to stop thinking that way . this approach may be effective in getting stigmatizing images removed from the advertisements , television , film , and other media outlets . there are several avenues available for protesting against public stigma , including writing campaigns , phone calls , public denunciation , marches and sit - ins , boycott , etc . some believe that it has only short - term positive effects on the attitude , and this issue needs further re search . so , as mentioned , although all the strategies are also used in other stigmatizing conditions , our findings are exclusively for diabetes and considering the iranian culture . these findings have been obtained from a wide range of people ( including people with and without diabetes ) . however , when planning to do these strategies , it should be noted that on one hand , the effectiveness of social strategies in different studies and different stigmatizing conditions is varied and many factors are involved . stigma research has shown that these strategies should be implemented simultaneously at different levels to see structural changes in the society . on the other hand , we must recognize that research on reducing health - related stigma has shown that changing attitudes and behavior has proved to be extraordinarily difficult . this is not good news , but it is a fact that must be acknowledged . evidence suggests that individuals and their families should be involved in all aspects of planning and programs , and plans should be made according to local conditions . in this study , the active participation of people with diabetes and their strategies were discussed . when the strategies are derived from individual experiences and are culturally fitted , the results of this study can help the healthcare teams to integrate anti - stigma strategies in their care plan at a community level to reduce the stigma . we are looking forward to observe the effectiveness of these strategies in this ongoing action research . limitations of this study are due to the inherent limitations of action research and qualitative studies . therefore , it must be cautioned when the findings are generalized to other communities which has different context and culture .	background : this study explored the strategies to overcome diabetes - related social stigma in iran.materials and methods : this paper is part of an action research study which was designed in iran in 2012 to plan and implement a program for overcoming diabetes - related stigma . participants were people with type 1 diabetes , their family members , people without diabetes , and care providers in a diabetes center . data collection was done through unstructured in - depth interviews , focus groups , e - mail , short message service ( sms ) , and telephone interview . data were analyzed using inductive content analysis approach.results:participants believed that it is impossible to overcome the stigma without community - based strategies . community - based strategies include education , advocacy , contact , and protest.conclusions:the anti - stigma strategies obtained in the study are based on the cultural context in iran . they are extracted from statements of a wide range of people ( with and without diabetes ) . however , during planning for stigma reduction , it is necessary to note that the effectiveness of social strategies varies in different studies and in different stigmatizing conditions and many factors are involved . these strategies should be implemented simultaneously at different levels to produce structural and social changes . it should be accepted that research on reducing health - related stigma has shown that it is very difficult to change beliefs and behavior . evidence suggests that individuals and their families should be involved in all aspects of the program , and plans should be made according to the local conditions .
extracellular matrix ( ecm ) proteins such as fibronectin induce resistance to chemotherapy via activation of intracellular survival pathways . we now show that the ecm protein tgfbi mediates specific sensitization to paclitaxel by inducing stabilization of microtubules via integrin - mediated signaling pathways . analysis of paclitaxel - treated ovarian cancers from a prospective clinical trial shows tgfbi protein expression in areas of paclitaxel - induced cytotoxicity . bioinformatic analysis of three microarray expression data sets from 223 ovarian and breast cancer samples show that tgfbi expression is tightly coregulated with other genes that induce paclitaxel sensitization such as thbs1 . these data show that paclitaxel response can be modulated by ecm proteins and raise the prospect of improving the therapeutic index of taxanes via manipulation of these proteins and their downstream signaling pathways . taxanes are microtubule - stabilizing drugs that have been extensively used as effective chemotherapeutic agents in the treatment of solid tumors ( mcguire et al . , 1996 ; sandler et al . , 2006 ) . however , taxane resistance limits clinical utility to approximately 50% of patients with breast or ovarian cancer . identification of mechanisms of taxane resistance that are therapeutically accessible is , therefore , required to improve treatment . paclitaxel , a prototype taxane , stabilizes microtubule polymers leading to mitotic arrest and apoptosis ( schiff et al . , 1979 ; general mechanisms of drug resistance , including overexpression of the abc / mdr transporter family of proteins ( peer et al . , 2004 ) , delayed g2/m transition ( tan et al . , 2002 ) , defective mitotic checkpoints ( anand et al . , 2003 ) , and alterations in apoptosis regulation ( huang et al . , 1997 , alterations of microtubules induce severe taxane resistance ( zhang et al . , 1998 ; gonalves et al . , 2001 ; alli et al . , 2002 ; these include -tubulin mutations which may decrease paclitaxel binding to microtubules ( giannakakou et al . , 1997 ) . alternatively , factors that increase the ratio of unstable to stable microtubules induce profound taxane resistance . this may occur by mutations in nonpaclitaxel binding sites , alterations in tubulin isoforms ( gonalves et al . , 2002 ; hari et al . , 2006 ) , overexpression of -iii tubulin ( mozzetti et al . , 2005 ) , and overexpression of the microtubule - associated protein stathmin ( alli et al . , 2002 ) . cells contain subsets of stable and dynamic microtubules that are functionally distinct ( gundersen et al . , 1984 ) , and there is strong evidence that extracellular stimuli regulate microtubule stability . serum starvation or loss of direct cell contact results in loss of microtubule stabilization , while fibronectin - mediated adhesion or treatment of cells with lysophosphatidic acid or tgf induce microtubule stability ( cook et al . , 1998 ; palazzo et al . , 2004 ; gundersen et al . , whether extracellular matrix ( ecm ) modulation of microtubule stability may alter paclitaxel sensitivity has been , to date , unknown . we describe here the identification of tgfbi ( transforming growth factor beta induced ) as an ecm protein that induces microtubule stabilization and modulates paclitaxel sensitivity in vitro and in patients receiving paclitaxel therapy . to identify genes associated with the acquisition of paclitaxel resistance , we studied the ovarian cancer cell line skov-3tr , which was derived from skov-3 by prolonged and repeated exposure to increasing doses of paclitaxel ( duan et al . , 1999 ) . we compared the expression profiles of the parent and resistant lines using cdna microarrays ( figure 1a ) after confirming that skov-3 and skov-3tr were isogenic by short tandem repeats genotyping ( data not shown ) . examination of the most differentially expressed genes showed several candidates previously identified in chemotherapy response including cp , sod2 ( ueta et al . , 2001 ) , hmga1 ( huang et al . , 1994 ; kasparkova et al . , 2003 ) , and cxcl1 ( taxman et al . , 2003 ) ( figure 1a ; table s1 in the supplemental data available with this article online ) . the most underexpressed gene in the taxane - resistant line was transforming growth factor beta induced ( tgfbi ; also known as big - h3 , -ig h3 , and keratoepithelin ) , which is an extracellular matrix protein whose secretion is induced by tgf1 stimulation ( skonier et al . , 1992 ) . its functions include cell adhesion to the ecm and integrin - mediated signaling ( jeong and kim , 2004 ; billings et al . , 2002 ) . quantitative real - time pcr confirmed striking underexpression ( > 1000 fold ) of tgfbi ( figure 1b ) . this underexpression was maintained after growing the cells without paclitaxel for several months ( data not shown ) . immunocytochemistry using anti - tgfbi antibody on paraffin - embedded cell blocks and immunoblotting of conditioned media from the two cell lines showed very low secretion of tgfbi protein ( figures 1c and 1d ) from the resistant skov-3tr cells . to examine the effect of loss of tgfbi expression on paclitaxel sensitivity , stable transfected cell lines expressing short interfering rnas ( sirna ) against tgfbi were generated from the parental skov-3 line . two vectors targeting 21 bp sequences at nucleotides 810 or 1318 of the tgfbi coding sequence were transfected independently or together to generate stable cell line pools skov3-a , skov3-k , and skov3-ak , respectively . effective knockdown ( kd ) of tgfbi mrna and protein was achieved in all three lines ( figures 1b and 1d and data not shown ) . early apoptosis was measured after 48 hr of paclitaxel exposure by flow cytometry of cells stained with fitc - annexin v and 7-aad ( wang et al . , 1998 ) . cell lines that lacked tgfbi ( skov-3tr and tgfbi - kd cells ) showed a significantly lower percentage of paclitaxel - induced apoptosis ( p < 0.001 , one - way anova ) in contrast to control skov-3 cells ( figure 1e ) . caspase 3/7 activation 48 hr following paclitaxel treatment was significantly reduced in skov-3tr and tgfbi - kd cells ( p < 0.001 , two - way anova ) ( figure 1f ) as was caspase 3 cleavage ( figure s1a ) . analysis of 9 ovarian and 11 breast cancer cell lines showed that tgfbi expression was significantly lower in resistant ( n = 7 ) cells ( p = 0.027 , two - way anova ) , and this was independent of cell type ( figures s1b and s1c ) . importantly , transient knockdown of tgfbi using a pool of four sirnas in four additional ovarian cancer cell lines , ovcar3 ( figure 1 g ) , tr175 ( figure 1h ) , 1847 ( figure s1d ) , and pe01 ( figure s1e ) , resulted in significant resistance to paclitaxel - induced caspase activation ( p < 0.001 , two - way anova ) . these results were confirmed using at least two individual sirnas in each cell line ( data not shown ) . skov-3tr cells have accumulated multiple paclitaxel resistance mechanisms during in vitro selection ( lamendola et al . , 2003 ) . for example , expression profiling of skov-3tr shows upregulation of mdr1 and abcb6 ( duan et al . , 2005 ; data not shown ) . the intensity values of a fluorescent derivative of paclitaxel were lower in skov-3tr compared to parental skov-3 and could be corrected using the mdr1 inhibitor verapamil ( figure s2a ) . however , induction of paclitaxel resistance in skov-3 cells by specific downregulation of tgfbi strongly suggested that tgfbi was an important component of the resistance shown in skov-3tr . to investigate how tgfbi modulated response to paclitaxel intracellular intensity of fluorescent paclitaxel was not different after tgfbi - kd ( figure s2a ) . in addition , apoptosis induction using uv , cisplatin , or nocodazole treatment was not significantly different between tgfbi - kd and parental cells ( figure s2b ) . these data show that paclitaxel resistance arising from specific loss of tgfbi is not caused by nonspecific alterations in apoptotic or multi - drug - resistance pathways . abnormal mitotic checkpoints have previously been shown to confer paclitaxel resistance ( anand et al . , 2003 ) . mitotic skov-3 cells lacking tgfbi showed a slight increase in prophase ( figure s2c ) , but no significant difference in the duration of mitotic progression was observed when measured by time - lapse imaging of individual cells ( p = 0.1 , one - way anova , figure s2d ) . a characteristic feature of overriding mitotic checkpoints is persistence of checkpoint proteins following metaphase ( anand et al . , figure s2e shows no significant difference ( p = 0.4 , chi square test ) between kd and control cell lines in the proportion of postmetaphase cells showing persistent cyclin b1 staining . bubr1 , a mitotic checkpoint protein expressed prior to anaphase , is thought to sense tension across the spindle when chromosomes are aligned in the metaphase plate ( nicklas , 1997 ) . spindle poisons such as nocodazole and paclitaxel abolish tension leading to persistent expression of bubr1 ( logarinho et al . , 2004 ) . cells with abnormal bubr1 function progress to anaphase in spite of exposure to spindle poisons . the expression of bubr1 in wild - type , kd , and skov-3tr cells was normal following nocodazole treatment indicating that sensing of spindle tension was not impaired ( figure s2f ) . delayed cell - cycle transition has been shown to confer paclitaxel resistance ( tan et al . , 2002 ) . there was no difference in cell - cycle profiles of nonsynchronized tgfbi - kd or skov-3tr cells as compared to mock - transfected skov-3 ( data not shown ) . to test cell - cycle profiles in synchronized cells , we used hela cells as they express comparable levels of tgfbi to those of skov-3 ( data not shown ) and are a well characterized cell - cycle model with intact mitotic checkpoints and are sensitive to paclitaxel ( anand et al . , 2003 ) . figure s2 g shows that transient knockdown of tgfbi had no effect on cell - cycle progression in hela cells synchronized in g1 by double thymidine block . microtubule bundle formation has been described as the hallmark of paclitaxel exposure in vivo ( schiff and horwitz , 1980 ) and is lost in resistant cells ( giannakakou et al . , 1997 ) . indeed , skov-3 cells treated with 2 m paclitaxel for 24 hr showed striking paclitaxel - induced bundles ( pibs ) in interphase cells ( figures 2a and 2b and figure s3a ) and severe failure of mitotic spindle organization . mitotic cells showed a prometaphase - like state where chromosomes formed ring - like structures around centrally radiating microtubule fibers , which we termed mitosis - like wheels ( mlws ) ( figure s3b ) . in contrast , a minority of skov-3tr cells showed pibs ( 2.4% versus 93% ) or mlws ( 1.7% versus 92% ) ( figures 2c and 2d and figure s3a ) . importantly , skov-3tr cells transfected with a myc - tagged tgfbi expression plasmid ( pcsmt - tgfbi ) showed a modest increase ( 12% versus 3.7% ) in the proportion of pibs and mlws following paclitaxel treatment ( p < 0.001 , two - sided t test ) ( figures 2e and 2f and figure s3a ) . paclitaxel induces microtubule stabilization , and posttranslational modifications of tubulin , such as detyrosination or acetylation , accumulate in these stable microtubules . detyrosination exposes glu residues at the carboxy terminus of alpha - tubulin that can be detected using antibodies and , therefore , used as a marker of microtubule stability ( gundersen et al . , 1984 ) . cells lacking tgfbi ( skov-3tr , skov3-k ) showed impaired paclitaxel - induced microtubule stabilization as evidenced by decreased glu - tubulin formation following paclitaxel treatment ( figure 2 g ) . similarly , transient tgfbi - kd of skov-3 using pooled sirnas resulted in intermediate reduction of paclitaxel induced glu - tubulin ( figure 2 g ) . these data suggest that selective loss of tgfbi induces paclitaxel resistance at the level of microtubules . alteration of microtubule function leading to paclitaxel resistance is associated with increased ratios of soluble to insoluble intracellular tubulin ( gonalves et al . , 2001 ; consistent with this , skov-3tr and skov3-k cells showed increased soluble tubulin compared to parental skov-3 cells ( figures 2h and 2i ) . tight regulation of tubulin dynamics is crucial for normal completion of mitosis , and alterations in microtubules that induce taxane resistance can cause geometric deformities in the mitotic spindle ( gonalves et al . mitotic cells from skov-3tr had a significantly higher proportion of abnormal mitosis compared to control skov-3 cells ( 14% versus 1.9% , respectively ; p < 0.001 , two - way anova ) ( figure 3c ) . to confirm that loss of tgfbi was sufficient to induce mitotic abnormalities , we examined the effects of tgfbi knockdown on mitosis in skov3-k and skov3-a cells . strikingly , abnormal mitotic figures were observed including monopolar spindle formation , multiple centrosomes with multipolar spindles ( figure 3a ) , and abnormal spindle architecture ( figure 3b ) in stably transfected cells 930 days following transfection . control cells transfected with empty vector and examined in parallel at the same time points did not show significant mitotic abnormalities ( figure 3c ) . in addition , interphase cells showed a significant increase in the proportion of cells with centrosome amplification ( figure 3d ) . these phenotypes were also observed 48 hr after transient tgfbi - kd of hela , 1847 , and ovcar3 cell lines and also in skov-3 using a810 and k1318 constructs , although lower proportions of abnormal mitoses were seen ( figure 3e and data not shown ) . to examine whether direct colocalization of tgfbi with microtubules or centrosomes could explain these findings , we characterized the subcellular localization of carboxy - terminal tagged tgfbi ( with green fluorescent protein or myc epitopes ) . tagged - tgfbi localized in the golgi apparatus and in intracellular vesicles along microtubule fibers and accumulated at cellular protrusions consistent with secretion ( figure s4 ) . no tagged - tgfbi was seen in the nucleus of interphase cells and there was no localization in mitosis to spindle poles , spindle fibers , or condensed chromosomes . integrin - mediated adhesion of cells to fibronectin induces microtubule stabilization ( palazzo et al . , 2004 ) , suggesting that ecm and paclitaxel might have additive effects on microtubules . the stabilizing effect of fibronectin requires integrin - mediated focal adhesion kinase ( fak ) activation and rho a ( palazzo et al . , 2004 ) . tgfbi is known to mediate adhesion also in an integrin - dependent manner ( billings et al . , 2002 ; nam , 2003 ; jeong and kim , 2004 ; park et al . , 2004 ) . this suggested a model where tgfbi could modulate paclitaxel sensitivity via microtubule stabilizing effects ( figure 4a ) . rtgfbi promoted adhesion of skov-3 cells , and this effect was partially antagonized by pretreating cells with a blocking antibody to alphavbeta3 integrin ( figures s5a and s5b ) ( p < figure 4b shows that cells plated on rtgfbi showed a significant increase in glu - tubulin formation indicating microtubule stabilization ( gundersen et al . , 1984 ; palazzo et al . , in addition , adhesion of cells to rtgfbi , or fibronectin , induced phosphorylation of fak ( figure 4c ) . attachment to fibronectin , but not to the integrin - independent adhesion peptide polylysine ( pl ) , also showed glu - tubulin formation ( figure 4d ) . to test whether fak was required for microtubule stabilization by rtgfbi , we knocked down fak in skov-3 cells using sirnas , and these cells showed a significant decrease in microtubule stabilization following adhesion to rtgfbi ( figure 4e ) ( p < 0.001 , two - sided t test ) . to test whether rho a was also required for rtgfbi microtubule stabilization , rho this resulted in a significant decrease in microtubule stabilization following adhesion to rtgfbi ( figure 4f ) ( p < 0.001 , two - sided t test ) . these data confirmed that rtgfbi induced microtubule stabilization and that this required intact fak and rho a signaling . extension of these experiments to nonmalignant cells using nih 3t3 fibroblasts also showed that rtgfbi induced glu - tubulin formation and fak phosphorylation ( figure s5c ) . in addition , inactivation of rho in nih 3t3 cells using a dominant - negative rho a expression construct significantly reduced the rtgfbi - mediated stabilization of microtubules ( figure s5d ) . to test whether extracellular tgfbi was able to sensitize cells to the effect of paclitaxel , skov3-a and skov3-k cells were plated on rtgfbi - coated wells . this significantly increased paclitaxel - induced apoptosis ( p < 0.001 , two - way anova ) ( figure 5a ) . importantly , kd of tgfbi significantly reduced the slope of the dose response curve for paclitaxel - induced glu - tubulin formation ( kd cells slope 1.3 , 95% c. i. 0.62.1 versus skov-3 control cells slope 2.8 , 95% c. i. 2.23.4 ) ( figures 5b and 5c ) . plating kd cells on rtgfbi restored the slope of glu - tubulin formation ( slope 2.4 , 95% c. i. 1.63.1 ) ( figures 5b and 5c and figure s5e ) . skov-3tr cells plated on rtgfbi and treated with paclitaxel showed only a modest increase in apoptosis ( data not shown ) , which we attributed to lower intracellular paclitaxel concentrations ( figure s2a ) . increasing the level of intracellular paclitaxel by inhibiting paclitaxel efflux using verapamil significantly increased the sensitizing effect of rtgfbi in skov-3tr ( figure 5d ) . verapamil also increased formation of stable microtubules in skov-3tr cells plated on rtgfbi following treatment with paclitaxel ( figure 5b ) . control skov-3 cells plated on rtgfbi without paclitaxel treatment did not show increased caspase 3/7 activity ( data not shown ) . we then tested whether rtgfbi was able to sensitize other paclitaxel - resistant ovarian cancer cells that had not been selected in vitro for resistance ( see also figure s1b ) . figures 5e and 5f show that rtgfbi significantly increased paclitaxel - induced caspase activation in a2780 and pe0188 cells ( p < 0.001 , two - way anova ) . incubation of tgfbi - kd cells in serum - free media conditioned with rtgfbi also resulted in a significant reversal of paclitaxel resistance ( p < 0.001 one - way anova ) ( figure 5 g and figure s5f ) . to test whether this sensitization was integrin dependent , we pretreated skov3-k cells with anti - alphavbeta3 blocking antibody before exposure to rtgfbi - conditioned media . this pretreatment significantly reduced paclitaxel - induced apoptosis ( p < 0.001 , one - way anova ) , confirming that rtgfbi binding to alphavbeta3 was required for paclitaxel sensitization ( figure 5 g and figure s5f ) . to examine whether microtubule stabilization was required for rtgfbi - mediated paclitaxel sensitization , we blocked rho and fak pathways downstream of rtgfbi - integrin binding . pretreatment with c3 toxin of skov3-kd cells for 4 hr before paclitaxel treatment decreased the sensitizing effect of rtgfbi ( figure 5 g and figure s5f ) . similarly , transient knockdown of fak using a single sirna blocked the sensitizing effect of rtgfbi in figure 5 g and figure s5f . fak induces proliferation and survival in cancer cells , and therefore , downregulation of fak was expected to sensitize cells to chemotherapy ( judson et al . , 1999 ; sood et al . , 2004 ) . however , fak is required for ecm - induced microtubule stabilization ( palazzo et al . , 2004 ) , and our data suggested that loss of fak might also induce paclitaxel resistance . we have recently shown using a rnai screen for all kinases ( in which , therefore , tgfbi was not included ) that loss of fak ( ptk2 ) and its family member ptk2b induced paclitaxel resistance in a549 non - small - cell lung carcinoma and hct116 colon cancer cells , respectively ( swanton et al . , 2007 ) . transient knockdown of fak in skov-3 and breast cancer mda - mb-231 cells ( defined as paclitaxel sensitive , figure s1b ) using independent sirnas also induced significant resistance to paclitaxel - mediated apoptosis ( p < 0.001 , two - way anova ) ( figure s5 g and data not shown ) . in addition , inhibition of rho a resulted in resistance to paclitaxel induced apoptosis ( figure s5h ) . these results show that perturbing components of pathways involved in microtubule stability is sufficient to induce paclitaxel resistance . in parallel with the in vitro work , we carried out a prospective randomized clinical trial ( ctcr ov01 ) specifically designed to examine the molecular response to carboplatin and paclitaxel monotherapy in patients with advanced ovarian cancer . tumor samples were collected prior to the start of neoadjuvant therapy and at subsequent interval debulking surgery ( supplemental data and figure s6a ) . as tgfbi is an integral component of the ecm , we investigated whether tgfbi expression correlated with the expression of other ecm transcripts that signal via integrins . we performed a correlation analysis between the expression of tgfbi and all other informative genes on the array ( n = 3426 ) , across all samples derived from patients treated with paclitaxel ( n = 20 ) , and ranked the genes according to their level of correlation with tgfbi . strikingly , 18 out of the 20 top ranked genes were ecm - related genes ( table s2 ) . notably , these genes included fibronectin ( fn1 ) ( r = 0.89 , pearson correlation ) , collagen 5a1 ( col5a1 ) ( r = 0.83 , pearson correlation ) , and thrombospondin-2 ( thbs2 ) ( r = 0.88 , pearson correlation ) . thbs1 is a homolog of thbs2 and was recently found to induce paclitaxel sensitivity through extracellular signaling ( lih et al . , 2006 ) . there was a significantly positive correlation between the probability of a gene being ecm - related and its degree of coexpression with tgfbi ( r = 0.89 , pearson correlation ; p < 0.001 , linear regression ) ( figure 6a ) . to confirm this finding in external data sets , we analyzed published expression microarray data from ovarian and breast cancers ( spentzos et al . , 2005 ; coregulation of tgfbi with ecm genes was reproduced with striking similarity ( figure 6b and data not shown ) . importantly , the top ranked coexpressed genes in these independent sets consistently included fibronectin and thbs2 . thbs1 was also highly ranked in the spentzos ovarian cancer data set ( data not shown ) . to determine whether tgfbi expression was associated with clinical paclitaxel resistance , we analyzed microarray expression data from twenty patients randomized to paclitaxel monotherapy in ctcr - ov01 . patients who showed paclitaxel resistance , as indicated by ca125 monitoring , had a significantly lower expression of tgfbi ( p = 0.046 , wilcoxon test ; 3.9% false discovery rate ) . patients who showed no paclitaxel response had a significantly lower expression of tgfbi ( p = 0.0087 , n = 16 , wilcoxon test ) in their pretreatment samples compared to those who responded ( figure 6c ) . as loss of tgfbi resulted in spindle deformities and centrosome amplification , we examined whether centrosome amplification correlated with paclitaxel resistance in clinical samples . to accurately count centrosomes in samples from the clinical study , we used three - dimensional reconstructions of confocal images from 20 m tissue sections . reconstructed images were scored by rotating the image to count the number of centrosomes associated with each nucleus ( figure 6d and movie s1 ) . using this method , a total number of 2376 nuclei were examined in 10 cancer samples , and counting was performed blind to the clinical outcome of patients . we found a significantly higher ( p = 0.019 , wilcoxon test ) proportion of cells with centrosome amplification in paclitaxel - resistant patients ( 7.6% ) compared to paclitaxel responders ( 3.9% ) ( figure 6e ) . we next examined ovarian cancer samples taken after paclitaxel treatment from the ctcr - ov01 study using immunohistochemistry for tgfbi . sections of ovarian cancer tissue revealed morphological changes typical of paclitaxel - induced cytotoxicity , including cytoplasmic vacuolation and multinucleation ( seiler et al . , 2004 ) , and these changes were localized to areas of high tgfbi staining ( figure 6f and figures s6b and s6c ) . tgfbi labeling was absent in the ecm around neighboring cells lacking paclitaxel - induced changes and in areas of necrosis ( figure 6f and figure s6d ) . positive tgfbi staining was significantly associated with evidence of paclitaxel - induced cytotoxicity ( n = 14 , p = 0.0035 , chi square test with monte - carlo simulation ) . importantly , focal tgfbi staining was present in pretreatment samples ; however cytoplasmic vacuolation and multinucleation were not seen ( figure s6e ) . our results suggest that the ecm protein tgfbi modulates paclitaxel response via regulation of microtubule stability . acetylated and detyrosinated microtubules define a stable subpopulation of microtubule polymers that resist depolymerization ( gundersen et al . , 1984 ) . this population is less dynamic compared to unstable microtubules and , therefore , less likely to contribute to the proportion of depolymerized tubulin in the cell ( figure 7 ) . as paclitaxel primarily targets polymerized microtubules , an increase in the proportion of unstable microtubules induces paclitaxel resistance ( gonalves et al . , 2001 ; barlow et al . , 2002 ; hari et al . , 2006 conversely , selective loss of tgfbi by kd resulted in mitotic spindle abnormalities and paclitaxel resistance . these findings mimic previous results from cell lines with intracellular alterations of tubulin that also caused paclitaxel resistance and mitotic instability ( gonalves et al . , 2001 ; martello et al . , 2003 ) . tgfbi was originally identified as induced by tgf stimulation in adenocarcinoma cells ( skonier et al . , 1992 ) , and our results may explain how tgf induces microtubule stabilization in serum - starved fibroblasts ( gundersen et al . , 1994 ) . recombinant tgfbi induced microtubule stabilization that was dependent on integrin - mediated fak and rho signaling . the exact mechanisms of microtubule stabilization downstream of fak and rho remain unknown but may include mdia1 or inactivation of the microtubule associated protein stathmin ( palazzo et al . previous studies have shown that ecm proteins induced resistance to dna - damaging drugs such as cisplatin and etoposide , and decreased apoptosis was associated with 1-integrin - mediated activation of pi3k and akt ( sethi et al . , 1999 ; hodkinson et al . , in contrast , the ecm protein thbs1 sensitizes pancreatic cancer cells specifically to taxane - induced apoptosis in a cd47-dependent manner ( lih et al . , 2006 ) . here , we show that tgfbi is tightly coexpressed with thbs1 in ovarian and breast cancers . it is possible that the two proteins may cooperate to induce taxane sensitization through activating cd47-driven pathways ( thbs1 ) and integrin - mediated microtubule stabilization ( tgfbi ) . the finding that the protein expression of tgfbi in tissue samples treated with paclitaxel monotherapy , specifically colocalized with areas of taxane - induced cytotoxicity suggests that tgfbi , and possibly other ecm proteins , modulate taxane effects in vivo . our data extend previous reports and suggests that the specific molecular targets of individual drugs are likely to determine whether the effect of the ecm is agonistic or antagonistic with chemotherapy . first , tgfbi protein expression is lost in a third of primary ovarian cancers ( a.a.a . , unpublished data ) and tgfbi has previously been shown to be methylated and downregulated in lung cancer ( shao et al . , 2006 ; zhao et al . , 2006 ) . future studies should correlate the expression of tgfbi with taxane response to test the value of tgfbi as a predictive biomarker . second , in ovarian cancer , fak is overexpressed , plays a role in regulating invasion and metastasis , and is associated with poor clinical outcome ( judson et al . , 1999 ; fak also regulates tumor growth either directly , through activation of erk - dependent pathways ( hecker et al . , 2002 ) , or indirectly through inducing angiogenesis ( sheta et al . , 2000 downregulation of fak using sirnas results in inhibited growth and metastasis of pancreatic cancer ( duxbury et al . , 2004 ) and could , therefore , be attractive therapeutically . however , previous studies have demonstrated that fak is required for adhesion - dependent microtubule stabilization ( palazzo et al . , 2004 ) . in the current study , importantly , and in contrast to previous publications ( halder et al . , 2005 ) , our results show that loss of fak results in increased resistance to taxanes , and this was confirmed using independent sirnas and experimental studies ( swanton et al . , 2007 ) . as fak is low or absent in one - third of patients with ovarian cancer ( sood et al . , 2004 ) , it could be used as a marker to identify patients who may not benefit from paclitaxel treatment . furthermore , although downregulation of fak may also induce growth inhibition , our data argue against combining fak inhibitors with paclitaxel treatment . we describe here a mechanism of specific paclitaxel sensitization via induction of microtubule stabilization by the ecm protein tgfbi . furthermore , we show that expression of tgfbi in ovarian and breast tumors is tightly coregulated with other ecm proteins that either induce microtubule stabilization or paclitaxel sensitization . the effectiveness of taxanes in improving survival in ovarian cancer remains controversial ( international collaborative ovarian neoplasm group , 2002 ) , and tgfbi could be used as a biomarker for selecting patients for taxane therapy . in addition , as tgfbi is an ecm protein , activating peptides or antibodies that mimic its action may be a strategy for modulation of response to taxane chemotherapy . cell lines were obtained from cell services ( cancer research uk london research institute ) and were maintained in rpmi 140 medium supplemented with 10% fetal bovine serum , penicillin , and streptomycin and incubated at 37c and 5% co2 . cells were grown in 6-well plates to 80% confluence before paclitaxel , nocodazole , cisplatin , or verapamil treatment ( sigma ) . for rtgfbi pretreatment experiments , adherent cells in 24-well tissue culture plates were incubated with rtgfbi at 50 g / ml in serum - free media ( sfm ) for 2 hr followed by paclitaxel treatment for 1 hr to make up the required concentrations . cells were then washed with pbs and incubated with normal media for 48 hr . for integrin - blocking experiments , attached cells were incubated with the mouse monoclonal antibody anti - alphavbeta3 ( chemicon ) for 1 hr ( 1 in 100 dilution ) in sfm . rho a inhibition in skov-3 cells was achieved by treating attached cells with 2 g / ml cell permeable c3 transferase recombinant protein ( cytoskeleton ) in sfm for 4 hr prior to paclitaxel treatment for 1 hr . early apoptosis was estimated using apoptosis detection kit ( r & d systems ) and 7-aad ( molecular probes ) following the manufacturers ' instructions and as previously described ( wang et al . , flow cytometric analysis was performed with a lsr ii flow cytometer ( bd bioscience ) and analyzed with the facsdiva software ( bd bioscience ) . we first confirmed the optimum number of cells and linear range for paclitaxel dose response for the assay . cells were plated at 1 10 cells per well in a 384-well plate in 20 l volume overnight before paclitaxel was added in 10 l volume to reach the final indicated concentrations . caspase 3/7 activity was estimated 48 hr following treatment by adding 30 l of the caspase - glo 3/7 assay reagent ( promega ) . luminescence was read following at least 1 hr of incubation on a luminescence plate reader ( infinite m200 , tecan ) using the i - control software ( tecan ) .	summarythe extracellular matrix ( ecm ) can induce chemotherapy resistance via akt - mediated inhibition of apoptosis . here , we show that loss of the ecm protein tgfbi ( transforming growth factor beta induced ) is sufficient to induce specific resistance to paclitaxel and mitotic spindle abnormalities in ovarian cancer cells . paclitaxel - resistant cells treated with recombinant tgfbi protein show integrin - dependent restoration of paclitaxel sensitivity via fak- and rho - dependent stabilization of microtubules . immunohistochemical staining for tgfbi in paclitaxel - treated ovarian cancers from a prospective clinical trial showed that morphological changes of paclitaxel - induced cytotoxicity were restricted to areas of strong expression of tgfbi . these data show that ecm can mediate taxane sensitivity by modulating microtubule stability .
nodular scleroderma is a rare variant of scleroderma which can occur in connection with systemic sclerosis or morphea . a biopsy from the lesion can demonstrate the scleroderma pattern , i.e. , keloid pattern or mixed type . we present a case of systemic sclerosis in a 50-year - old female who developed nodular scleroderma in the absence of deterioration of the scleroderma condition . although this condition is rare , it has been reported sporadically , and clinicians should be able to recognize this variant in cases of scleroderma presenting with firm nodules or plaques . a 50-year - old female presented with a 1-year history of systemic sclerosis and interstitial lung disease . her current medications were prednisolone 5 mg , cyclophosphamide 75 mg , amlodipine 20 mg , sildenafil 50 mg , and aspirin 80 mg daily . ten months later , she developed multiple asymptomatic papules on the neck , abdominal wall , and back ( fig 1 , fig 2 ) . the physical examinations demonstrated sclerotic skin changes on the face , trunk , and extremities with sclerodactyly , digital pitting scars , and raynaud 's phenomenon . there were multiple , nontender , firm skin - colored papules and plaques scattered on the neck , abdominal wall , and back . the laboratory tests revealed positive antinuclear antibody in a homogenous pattern ( titer 1 : 320 ) and positive anti - scl70 . the biopsy revealed thick sclerotic collagen fibers in the mid - dermis ( fig 3 ) . nodular or keloidal scleroderma is a rare variant of scleroderma [ 1 , 2 , 3 , 4 , 5 ] . the term nodular scleroderma is used when the pathological change resembles scleroderma , and the term keloidal scleroderma is used when the pathological change shows hyalinization of thick sclerotic collagens similar to keloid [ 1 , 6 , 7 ] . nodular or keloidal scleroderma have been reported in either systemic sclerosis or localized scleroderma , usually 6 months after the presentation of systemic sclerosis [ 1 , 2 , 3 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 ] . however , also the presence of nodular scleroderma before the onset of systemic sclerosis has been reported . this condition can be found in systemic sclerosis patients either with or without active systemic involvements [ 1 , 2 , 8 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 ] . the characteristic clinical presentations were firm nodules or plaques resembling keloid , distributed predominantly on the proximal extremities , i.e. , on the chest , back , and neck . nodular or keloidal scleroderma usually occur in middle - aged females ( table 1 ) [ 1 , 4 , 8 , 11 , 12 , 13 , 14 , 16 , 17 , 18 ] . variable histopathologic changes have been reported to include characteristics of keloid or hypertrophic scars , of morphea , or of both morphea and keloid [ 1 , 19 ] . complex interactions between cytokines , matricellular proteins , and local factors ( minor trauma and vascular insufficiency ) should be determined [ 2 , 19 , 20 ] . a role of pathogens , including acid - fast bacteria or chronic hcv infection , has been reported [ 11 , 14 , 21 ] . yamamoto et al . mentioned the role of increasing connective tissue growth factor ( ctgf ) in tissue fibrosis . nonetheless , an increase in ctgf was identified both in the nodular lesions and in skin with scleroderma . consequently , the increase in ctgf alone may not be the sole contributor to the pathogenesis of fibrosis in the lesions . . demonstrated increasing cartilage oligomeric matrix protein ( comp ) , collagen xii , and fibrillin-1 in nodular scleroderma lesions compared to perilesional skin and healthy skin . comp is induced by tgf- , which is an important cytokine in stimulating fibrosis in scleroderma and is involved in modulating the dermal collagen network as well as in sustaining fibroblast activation . our case presented with multiple skin - colored papules and plaques , a differential diagnosis that should be considered was localized cutaneous mucinosis , which can be found as a coexisting condition with systemic sclerosis or morphea [ 24 , 25 , 26 , 27 , 28 ] . however , the diagnosis of localized cutaneous mucinosis should be confirmed by mucin deposition in a skin biopsy . several treatment modalities are mentioned in the literature , including topical or intralesional steroids , systemic steroids , topical calcipotriene , psoralen photochemotherapy , cyclosporine , d - penicillamine , methotrexate , extracorporeal photochemotherapy , and excision [ 1 , 2 , 4 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 29 , 30 ] . however , these treatment modalities have shown unsatisfying results . a review of the reports on nodular or keloidal scleroderma in the past 10 years ( table 1 ) revealed that 1 case demonstrated stable nodular scleroderma without any specific treatment . interestingly , immunosuppressive agents that have previously been received can not prevent the development of nodular or keloidal scleroderma [ 11 , 12 , 13 , 16 ] . the condition of systemic sclerosis in our case has been stable , and the patient was not concerned about its appearance . therefore , we decided to closely observe and monitor her for any progression of the lesions . after 4 months , although this condition is rare , it has been reported sporadically , and it should be considered in scleroderma patients clinically presenting with firm nodules or plaques . this research did not receive any specific grant from funding agencies in the public , commercial , or not - for - profit sectors .	backgroundnodular scleroderma is a rare variant of scleroderma which can occur in connection with systemic sclerosis or morphea . a biopsy from the lesion can demonstrate the scleroderma pattern , i.e. , keloid pattern or mixed type . treatment is challenging , and several treatments modalities have been reported with unsatisfactory results.main observationswe present a case of systemic sclerosis in a 50-year - old female who developed nodular scleroderma in the absence of deterioration of the scleroderma condition . although no additional treatment was given , the lesions remained stable without progression.conclusionsalthough this condition is rare , it has been reported sporadically , and clinicians should be able to recognize this variant in cases of scleroderma presenting with firm nodules or plaques .
across a very broad taxonomic range animals frequently respond differentially to close kin , even if those kin were previously unfamiliar . logically , this differentiation between individuals according to kinship requires well - defined mechanisms to allow recognition . and whereas animals may learn the cues of familiar individual kin during rearing , recognition of unfamiliar kin must really be recognition of genetic similarity - either to self or to other known kin . a challenge in this area lies in discovering the cues that animals use for genetic recognition of kin , and the genetic encoding of such cues . in many vertebrates , odors are key to the recognition process , and have been widely implicated as cues that allow genetic kin recognition in many species of fish , reptiles and mammals ( figure 1 ) . however , vertebrate scents are generally complex , and there have been few attempts to identify the specific scent components used in kin recognition or their genetic basis . in work published recently in bmc evolutionary biology , boulet and colleagues have advanced this field by demonstrating a significant correlation between genetic similarity ( estimated from 11 - 14 microsatellite loci ) in a captive population of ring - tailed lemurs ( lemur catta ) and similarity of volatile chemicals in their genital gland secretions , as assessed by gas - chromatography mass - spectrometry . the genetic similarity of two individuals is thus manifest in the odor profile ( sometimes referred to as an ' odortype ' ) . even more intriguing , although the genital glands of the two sexes are anatomically distinct ( scrotal glands in the male , labial glands in the female ) , this covariance between genetic and chemical similarity is evident even between individuals of the opposite sex . while some components are expressed only by animals of one sex , more than half ( about 170 ) were expressed by individuals of both sexes . to provide a simple estimate of chemical distance between a pair of individuals , boulet et al . used the relative abundance of each of these shared compounds to calculate the euclidean distance between the pair ( derived from the pythagorean theorem , this sums the pairwise difference , , in abundance of all 170 compounds , such that chemical distance = sqrt(1 + 2 + 3 + .... 170 ) . while there was a broad spread of chemical distances between male - female dyads that had intermediate genetic distance , dyads with low genetic similarity had low chemical similarity whereas those with a high genetic similarity had a higher chemical similarity . this relationship is consistent with the hypothesis that odors from genital secretions can be used to assess genetic relatedness , and maybe close kinship . of particular interest , these relationships were significant both within and between the sexes during the breeding season , but were much weaker or nonsignificant during the non - breeding season . odortype may be particularly important during the competitive breeding season to prevent inbreeding and/or to direct nepotistic behavior towards more closely related individuals . however , this study is still only a first step in establishing whether such odor signals could offer a reliable means of recognizing kinship among ring - tailed lemurs and the genetic basis of the cues used . if lemurs used a measure of chemical distance based on all volatile compounds that are shared within or between the sexes , it would only be of very limited value in assessing kinship because of the considerable range in that measure between individuals of intermediate genetic relatedness . although very closely related animals have similar odortypes , so do many individuals that are much less closely related . if odortype were used to avoid inbreeding , for example , the consequence would be exclusion as mates of many individuals that are not closely related , reducing choice without gaining any genetic benefit . it is likely , therefore , that animals use more specific markers within the odortype to distinguish close relatives reliably ( figure 2 ) . chemical distance between pairs of animals based on all volatile compounds in a scent correlates with genetic distance ( a ) , but variance will be high for any particular genetic distance because some compounds are likely to be strongly influenced by non - genetic factors such as current hormone levels and bacterial flora . instead , selective assessment of specific semiochemicals within the scent that correlate strongly with genotype ( b ) will provide a much more reliable assessment of kinship . there has been surprisingly little progress in establishing the genetic and molecular markers used to recognize kin through scents in vertebrates . in part , this may be due to the molecular complexity of vertebrate scents , which are the product not only of an individual 's genes but also of hormonal and metabolic status , diet and microflora . for the past 30 years , the focus on genetic mechanisms underlying vertebrate kin recognition through odors has been on the major histocompatibility complex ( mhc ) , which is often held to be the major genetic component apparently determining an individual 's scent . inbred laboratory mice have been a key model organism for manipulating mhc genes on a constant genetic background as proof that animals can detect mhc type through scent . as mhc is so highly polymorphic in natural populations , those that share the same mhc type ( and mhc - based scent ) are very likely to be closely related - mhc odors could be used as a marker of genetic relatedness . yet , despite the precise genetic control offered by laboratory rodent strains , chemical analyses of volatile profiles have found correlations of some volatile components with mhc type but have not yet discovered consistent differences in compounds that are regulated by mhc type [ 3 - 6 ] . in reality , complex interactions are found with genetic background , microflora and diet , all of which alter the odor profile [ 3,5 - 7 ] . this plasticity of mhc - derived odortype creates a conundrum . to be useful in natural populations , kin markers must be stable and readily recognized against the variable genetic and environmental background of normal outbred animals . our own studies of wild - derived mice with normal genetic variation in semi - natural populations provided clear evidence that wild mice do not use mhc to avoid inbreeding . in fact , mice showed a very strong avoidance of inbreeding with those sharing another very highly polymorphic marker in mouse scent , the major urinary proteins ( mups ) , which have a strong influence on an individual 's scent profile regardless of other genetic and non - genetic variation . sharing of a single highly polymorphic marker , like mup or mhc type , can provide a reliable indicator of relatedness because only close relatives are likely to inherit both of the same alleles at a particular locus ( or both of the same haplotypes in the case of clusters of closely linked genes like mup or mhc ) . however , this type of mechanism can only be partially effective for kin recognition . for any single locus , the number of alleles shared between two relatives is a matter of chance ; even very close relatives such as full siblings are as likely to share no alleles as they are to share both alleles at a particular locus . modeling alternative genetic mechanisms that could be used to discriminate full sibs from unrelated animals reveals that reliance on a single genetic locus will either fail to identify many relatives ( if the requirement is that both alleles are shared ) or will mistake many unrelated animals as sibs ( if sharing of any allele is used ) . notwithstanding the theory , house mice do use sharing of mup type , encoded by a single tightly linked cluster of genes , to avoid inbreeding . this may be specific to house mice - there are insufficient data to assess whether such simple recognition systems are widespread . an alternative model is that instead of directly comparing the similarity of scents to self , imprinting on maternal scent encoded by several independent loci is employed to provide reliable recognition of all siblings and maternal half - sibs , because all offspring share with their mother one allele at every locus . laboratory cross - fostering studies in which newborn mouse pups were fostered onto a mother of different mhc type to their own have suggested that animals might imprint on the genotype of their mother and subsequently avoid ' inbreeding ' with those sharing the foster mother 's genotype rather than avoiding mates that match their own mhc type . however , maternal imprinting does not require recognition of the mother 's genotype for kin recognition ; instead animals must be able to recognize the separate haplotypes carried by the mother when these are combined with other unknown haplotypes . this recognition task is likely to be considerably more difficult given the complex effects that mhc type has on odors , particularly as the odors of mhc heterozygotes are not an additive combination of the two homozygous profiles . a key test would be whether mice ( or other animals ) can recognize the separate mhc haplotypes carried by a heterozygous animal when combined with other mhc haplotypes ( for example , animals imprinted on the mhchaplotype must be able to recognize mhcor mhc ) ; they also need to be able to do this on the randomly assorting genetic background of outbred animals . a recent study using inbred laboratory mice found that animals recognized non - genetic similarities in offspring from the same mother compared to those from another genetically identical female due to their shared maternal ( in utero and postnatal ) environment . the approach of relating genetic similarity to the global volatile profile of scent glands is a step towards the systems biology of complex behaviors . indeed , the application of global profiling methodologies to scents could be said to introduce the concept ( but preferably not the term ! ) of ' semiomics ' . as with many studies of this nature , the analyte mixtures are complex , and a major challenge is in unbundling the important semiochemicals from the entire volatile profile - although boulet and colleagues refer to a ' semiochemical profile ' , it is likely that many of the constituent compounds will be ' silent ' in kin recognition . an attractive way forward is to use the combined datasets to identify those chemicals that show the greatest correlation with relatedness , focusing on differences in relatedness that can be discriminated behaviorally . the candidates can be examined in ' kin - shifting ' experiments such that when they are spiked into a distant sample , they elicit a response more ' akin ' to a close relative . indeed , similar experiments could be conducted using humans to establish the extent to which we too can discriminate our own kin based on genetically determined scents . the development of these ideas was supported by research grants from bbsrc ( s19816 , bbc603897 ) and nerc ( neg018650 ) .	a recent study in bmc evolutionary biology has shown that genetically similar individual ring - tailed lemurs are also more similar in their scent composition , suggesting a possible mechanism of kin recognition . theoretical and experimental studies reveal challenges ahead in achieving a true systems - level understanding of this process and its outcomes.see research article http://www.biomedcentral.com/1471-2148/9/281 .
we enrolled 44 patients diagnosed with type 2 diabetes at the shaheed motahari hospital of fooladshahr , isfahan , during 2009 . on the basis of n = [ ( z1/2 + z1 ) s]/2 , we determined that 21 patients were needed for adequate power . a diagnosis of type 2 diabetes was confirmed if a patient either had a fasting plasma glucose 126 mg / dl or was taking oral glucose lowering agents or insulin ( 10 ) . exclusion criteria included any secondary cause of hyperglycemia , use of estrogen therapy , untreated hypothyroidism , smoking , and kidney or liver diseases . cardiovascular risks such as fasting blood glucose , a1c , weight , waist circumference , and lipid profiles were the primary outcomes . this study was approved by the research council and ethics committee of the isfahan university of medical sciences ( registered in http://www.clinicaltrials.gov ; i d number nct01049321 ) . after a run - in period of 3 weeks , patients were randomly assigned to a control diet or a dash diet for 8 weeks . the project dietitian enrolled participants and randomly allocated them to groups using random sequencing generated in spss at the end of the run - in period . because this was a dietary intervention , patients were not blinded . we prescribed two diets for each patient : the control diet and the dash diet . the control diet included a macronutrient composition of 5060% carbohydrates , 1520% protein , < 30% total fat , and < 5% of caloric intake from simple sugars ( 11 ) . the dash diet was rich in fruits , vegetables , whole grains , low - fat dairy products , and low in saturated fat , total fat , cholesterol , refined grains , and sweets . patient adherence was assessed in terms of attendance at monthly visits and through analysis of the 3-day food diaries . we used general linear models ( paired student t tests ) to globally compare means of the all variables at the end of the two different diet periods and the mean change for each variable in the two groups . statistical analyses were performed using spss for windows version 13.0 ( spss , chicago , il ) . we used a randomized crossover design . after a run - in period of 3 weeks , patients were randomly assigned to a control diet or a dash diet for 8 weeks . the project dietitian enrolled participants and randomly allocated them to groups using random sequencing generated in spss at the end of the run - in period . because this was a dietary intervention , patients were not blinded . we prescribed two diets for each patient : the control diet and the dash diet . the control diet included a macronutrient composition of 5060% carbohydrates , 1520% protein , < 30% total fat , and < 5% of caloric intake from simple sugars ( 11 ) . the dash diet was rich in fruits , vegetables , whole grains , low - fat dairy products , and low in saturated fat , total fat , cholesterol , refined grains , and sweets . patient adherence was assessed in terms of attendance at monthly visits and through analysis of the 3-day food diaries . we used general linear models ( paired student t tests ) to globally compare means of the all variables at the end of the two different diet periods and the mean change for each variable in the two groups . statistical analyses were performed using spss for windows version 13.0 ( spss , chicago , il ) . of the 44 participants , 31 type 2 diabetic patients ( 13 male and 18 female ) completed the entire crossover study ( one patient was diagnosed with cancer and one with anemia , and eleven patients did not follow the study protocol ) . analysis of the 3-day diet self - report showed that calorie intake of two the groups was not significantly different ( 2,165 29 vs. 2,189 35 kcal / day in the control and dash diets , respectively ; p = 0.62 ) . the results were the same regarding the actual protein intake ( 15 vs. 16% ) and total fat intake ( 28 vs. 29% ) as well as the percentage of the carbohydrate intake ( 57 vs. 55% ) in the control and dash diet groups , respectively . these two diets were different in sodium content ( 2,310 vs. 2,996 mg / day in the control and dash diets , respectively ) . the dash diet had higher amount of calcium ( 1,299 vs. 912 mg / day ) , potassium ( 4,399 vs. 3,219 mg / day ) and fiber ( 30 vs. 26 g / day ) . in the dash eating pattern versus the control diet , the number of servings of fruit ( 5 vs. 3 ) , vegetables ( 6.8 vs. 4 ) , dairy ( 3 vs. 2 ) , and whole grains ( 4.5 vs. 2.5 ) was higher . effects of the two diets on cardiometabolic risks are shown in table 1 , indicating a significant reduction in most risk factors from the dash diet . means of the cardiometabolic variables among type 2 diabetic patients after consumption of the dash or control diet * the control diet was a designed to control diabetes . the general recommendation for macronutrient composition of the diet was 5060% carbohydrates ; 1520% protein and < 30% total fat . the dash diet was rich in fruits , vegetables , whole grains , and low - fat dairy products , and low in saturated fat , total fat , cholesterol , refined grains , and sweets . p values are for comparisons between the two diet periods ( general linear model ) . dbp , diastolic blood pressure ; fbg , fasting blood glucose ; hdl - c , hdl cholesterol ; ldl - c , ldl cholesterol ; sbp , systolic blood pressure ; tg , triglyceride . we found that the dash - eating pattern had beneficial effects on type 2 diabetic patients ' cardiometabolic parameters . the prescribed caloric intake of both diets was the same , but the calorie density of food in the dash diet was lower than that in the control diet . a long - term weight - loss trial over 18 months also indicated beneficial effects of using low - calorie dense diets for weight loss ( 12 ) . furthermore , the dairy content , which might be related to weight reduction ( 13 ) , was higher in the dash diet was higher than the control diet . the dash eating pattern also had a more beneficial impact on the patient 's glycemic control . more fiber , phytoestrogen , and isoflavone intake due to higher fruit and vegetable consumption , along with more weight reduction might be responsible for these effects ( 4 ) . the present study suggests the dash diet plan could reduce ldl and increase hdl cholesterol . our previous research on patients with metabolic syndrome also indicates a beneficial effect of this type of diet on lipid profiles ( 4 ) . there was no difference in the serum triglyceride levels when we compared the effects of the two diets . however , dash was compared with the control diet , which also had beneficial effects on lowering the serum triglyceride level . higher intake of legumes such as soy in the dash diet might also be responsible for its beneficial effects on metabolic parameters ( 14 ) . consuming higher amounts of nonhydrogenated vegetable oil with the dash diet might be related to its more favorable effects . because nonadherent participants did not participate in all phases of the study , we could not use intention - to - treat analysis . dietary intake in the present study was self - reported , and patients were given recommendations to follow a particular diet ( rather than receiving prepared foods ) , likely resulting in possible imperfect adherence to the diets . the omniheart ( optimal macro - nutrient intake heart ) study ( 15 ) has expanded the macronutrient variability of the dash dietary pattern , which will be interesting to explore in future studies . the dash eating pattern may play an important role in managing cardiometabolic risks among type 2 diabetic patients .	objectiveto determine the effects of the dietary approaches to stop hypertension ( dash ) eating pattern on cardiometabolic risks in type 2 diabetic patients.research design and methodsa randomized crossover clinical trial was undertaken in 31 type 2 diabetic patients . for 8 weeks , participants were randomly assigned to a control diet or the dash eating pattern.resultsafter following the dash eating pattern , body weight ( p = 0.007 ) and waist circumference ( p = 0.002 ) reduced significantly . fasting blood glucose levels and a1c decreased after adoption of the dash diet ( 29.4 6.3 mg / dl ; p = 0.04 and 1.7 0.1% ; p = 0.04 , respectively ) . after the dash diet , the mean change for hdl cholesterol levels was higher ( 4.3 0.9 mg / dl ; p = 0.001 ) and ldl cholesterol was reduced ( 17.2 3.5 mg / dl ; p = 0.02 ) . additionally , dash had beneficial effects on systolic ( 13.6 3.5 vs. 3.1 2.7 mmhg ; p = 0.02 ) and diastolic blood pressure ( 9.5 2.6 vs. 0.7 3.3 mmhg ; p = 0.04).conclusionsamong diabetic patients , the dash diet had beneficial effects on cardiometabolic risks .
critical care is about specialized and dedicated care given by a team to a patient who is seriously ill . it not only improves the chances of patients with desperate illness but also likes to promote an improvement in the general level of medical and nursing care . critically ill patients are treated with a large number of different drugs that have significant alterations in their pharmacokinetics and pharmacodynamics and great potential for drug misadventure . mortality , morbidity , and economic devastations are the most important consequence of medication errors . adverse drug events ( ades ) occur in approximately 30% of hospitalized patients , and the patients at intensive care units ( icus ) settings are at greater risk of developing ades . the complexity of icus , combined with the high acuity of the patients treated in these areas , creates an environment that is more vulnerable to harmful patient 's outcomes when medication errors occur . undoubtedly , drug - related problems ( drps ) have to be controlled or prevented to provide better patient care . although few drps are not preventable as they are nonpredictable because of their idiosyncratic nature , the majority of the interventions are predictable and therefore be preventable . the idea of a specialized intensive care pharmacist was first established in the world of pharmacy in 1982 . the increasing use of high - tech drugs and sophisticated drug delivery methods would seem to make the icu a prime area for specialist pharmacy practitioners . the involvement of a critical care pharmacist has been shown to decrease drug - related costs , prevent ade , and reduce the morbidity and today they are recognized as an integral member of critical care team along with the other health care professionals . in a developing country like india increased expenditure for health care is not affordable by the majority of the patients . hence , this study was indented to assess the impact of clinical pharmacist recommendations on the cost of drug therapy in icu patients of a tertiary care teaching hospital . this was a prospective interventional study , conducted in the icus of jss hospital , mysore , over a period of 7 months from july 2007 to january 2008 . patients of either sex aged 18 years who stayed at least overnight in surgical intensive care units ( sicu ) , intensive cardiac care units ( iccu ) , and medical intensive care units ( micu ) were included in our study . the institutional human ethical committee of jss college of pharmacy , mysore , approved the study . an intervening pharmacist reviewed the patient 's progress charts and drug profile on day - to - day basis . this study considered the intervention as any action by a pharmacist that directly resulted in a change to patient management or therapy . where a drp was identified , it was discussed with the academic clinical pharmacist for further review and to seek his / her opinion . the drps identified during the study were categorized based on categories as described by hepler and strand . each drp was assessed for its level of significance and was graded either as minor , moderate or as major based on the expected clinical outcome . an independent clinical panel was convened to review and assess the impact of clinical pharmacist interventions on cost savings . the three - member panel consisted of a consultant physician , a medical doctor , and an academic clinical pharmacy practitioner . an intervening pharmacist reviewed and assessed all those interventions that were accepted and changed by the physician , for any possible impact on length of stay ( los ) , readmission probability , medical procedures , laboratory monitoring , and probability of mortality . the assessment and quantification of percentage probability was based on a review of the individual case , and the decision by the panel was taken on the basis of their expertise knowledge and referral of appropriate literatures and websites such as www.medal.org.com . those interventions perceived to have had impact only on the drug cost were not assessed by the panel but instead the intervening pharmacist calculated the impact of cost savings arising directly from such interventions . cost analysis was made only for those interventions that were accepted and changed , cost savings was considered if the cost of therapy was reduced after the intervention , compared to the cost of therapy prior to the interventions . the cost analysis was made based on each intervention rather than each patient . the net cost saving was calculated by subtraction of the cost increment of any from the total savings made from that intervention . the probabilities of readmission were estimated based on the probability , expressed as a percentage likelihood , of a readmission event occurring without the intervention compared with the probability of a readmission after the intervention has occurred , as assessed by the independent clinical panel . costs were then calculated by multiplying this probability with the average cost of the treatment for specific disease costing at our hospital . the panel quantified the impact of each intervention on los by estimating the change in the number of days in either a general medical ward or icus . the change in los was on the basis of likelihood of changes in los occurring if the intervention had not done . the local independent clinical panel decided as to subclassification of the wards based on the individual case . the cost impact of changes in los was then calculated based on jss hospital 's average treatment cost for the specific condition at that particular ward . the independent clinical panel examined the changes to laboratory monitoring or medical procedures and allocated a probability of the event being changed as a result of the intervention . the cost impact was then calculated by multiplying this probability by the jss hospital 's costs for the particular medical procedure or the laboratory test . direct drug cost saving arising from interventions consisted of discontinuation of medication due to inappropriate use ( e.g. duplicate therapy , lack of indication , and drug interactions ) ; substitution of less expensive alternative ( e.g. oral vs. intravenous route of administrations ) ; dosage adjustment in elderly patients or patients with hepatic and renal impairment . daily drug cost savings were calculated based on the frequency of drug administration and the total drug cost savings were calculated by multiplying daily cost savings and number of days of prescription from the day of intervention till the last day of therapy or day of discharge from the intensive care settings whichever was earlier . annualized cost savings were calculated by extrapolating the net cost savings made during the 7 months to over a period of 1 year . an independent clinical panel was convened to review and assess the impact of clinical pharmacist interventions on cost savings . the three - member panel consisted of a consultant physician , a medical doctor , and an academic clinical pharmacy practitioner . an intervening pharmacist reviewed and assessed all those interventions that were accepted and changed by the physician , for any possible impact on length of stay ( los ) , readmission probability , medical procedures , laboratory monitoring , and probability of mortality . the assessment and quantification of percentage probability was based on a review of the individual case , and the decision by the panel was taken on the basis of their expertise knowledge and referral of appropriate literatures and websites such as www.medal.org.com . those interventions perceived to have had impact only on the drug cost were not assessed by the panel but instead the intervening pharmacist calculated the impact of cost savings arising directly from such interventions . cost analysis was made only for those interventions that were accepted and changed , cost savings was considered if the cost of therapy was reduced after the intervention , compared to the cost of therapy prior to the interventions . the cost analysis was made based on each intervention rather than each patient . the net cost saving was calculated by subtraction of the cost increment of any from the total savings made from that intervention . the probabilities of readmission were estimated based on the probability , expressed as a percentage likelihood , of a readmission event occurring without the intervention compared with the probability of a readmission after the intervention has occurred , as assessed by the independent clinical panel . costs were then calculated by multiplying this probability with the average cost of the treatment for specific disease costing at our hospital . the panel quantified the impact of each intervention on los by estimating the change in the number of days in either a general medical ward or icus . the change in los was on the basis of likelihood of changes in los occurring if the intervention had not done . the local independent clinical panel decided as to subclassification of the wards based on the individual case . the cost impact of changes in los was then calculated based on jss hospital 's average treatment cost for the specific condition at that particular ward . the independent clinical panel examined the changes to laboratory monitoring or medical procedures and allocated a probability of the event being changed as a result of the intervention . the cost impact was then calculated by multiplying this probability by the jss hospital 's costs for the particular medical procedure or the laboratory test . direct drug cost saving arising from interventions consisted of discontinuation of medication due to inappropriate use ( e.g. duplicate therapy , lack of indication , and drug interactions ) ; substitution of less expensive alternative ( e.g. oral vs. intravenous route of administrations ) ; dosage adjustment in elderly patients or patients with hepatic and renal impairment . daily drug cost savings were calculated based on the frequency of drug administration and the total drug cost savings were calculated by multiplying daily cost savings and number of days of prescription from the day of intervention till the last day of therapy or day of discharge from the intensive care settings whichever was earlier . annualized cost savings were calculated by extrapolating the net cost savings made during the 7 months to over a period of 1 year . the probabilities of readmission were estimated based on the probability , expressed as a percentage likelihood , of a readmission event occurring without the intervention compared with the probability of a readmission after the intervention has occurred , as assessed by the independent clinical panel . costs were then calculated by multiplying this probability with the average cost of the treatment for specific disease costing at our hospital . the panel quantified the impact of each intervention on los by estimating the change in the number of days in either a general medical ward or icus . the change in los was on the basis of likelihood of changes in los occurring if the intervention had not done . the local independent clinical panel decided as to subclassification of the wards based on the individual case . the cost impact of changes in los was then calculated based on jss hospital 's average treatment cost for the specific condition at that particular ward . the independent clinical panel examined the changes to laboratory monitoring or medical procedures and allocated a probability of the event being changed as a result of the intervention . the cost impact was then calculated by multiplying this probability by the jss hospital 's costs for the particular medical procedure or the laboratory test . direct drug cost saving arising from interventions consisted of discontinuation of medication due to inappropriate use ( e.g. duplicate therapy , lack of indication , and drug interactions ) ; substitution of less expensive alternative ( e.g. oral vs. intravenous route of administrations ) ; dosage adjustment in elderly patients or patients with hepatic and renal impairment . daily drug cost savings were calculated based on the frequency of drug administration and the total drug cost savings were calculated by multiplying daily cost savings and number of days of prescription from the day of intervention till the last day of therapy or day of discharge from the intensive care settings whichever was earlier . annualized cost savings were calculated by extrapolating the net cost savings made during the 7 months to over a period of 1 year . a total of 895 patients were admitted to three icus during the 7-month study period from july 2007 to january 2008 . of the total cases admitted , out of 805 cases reviewed , 67.2% ( n = 541 ) of patients were male and the average age of the patients admitted to the icus was 51.21 + 17.23 ( mean + sd ) years . demographic details of the patients admitted to intensive care units the most frequent drp detected was overdose [ ( 25% ) n = 28 ] followed by drug interactions [ 17% ( n = 19 ) ] [ figure 1 ] . of the 94 interventions accepted and changed , 60 interventions resulted in cost saving amounting to rs . 4840.00 and rs . 2.00 , and the average cost savings per intervention were rs . however , the average net cost savings per intervention were higher with drug interaction ( rs . class duplication ( n = 9 ) , drug duplication ( n = 5 ) , dispensing errors ( n = 1 ) , drug use without prescription ( n = 2 ) , sound - like errors ( 6 ) , contraindications ( 3 ) , wrong time of administration ( n = 2 ) . a dose of drug was prescribed with multiple number of lower strengths ( 2 ) impact of pharmacist - initiated changes to the drug therapy on cost the majority [ ( 25.6% ) n = 21 ] of the interventions were associated with antibiotics followed by antihypertensive [ ( 12.2% ) n = 14 ] and lipid lowering drugs [ ( 13.4 % ) n = 11 ] . class of drugs intervened and associated cost savings cessation of drugs was the most frequent [ ( 31.9% ) n = 30 ] suggestion provided by the intervening pharmacist resulting in net cost savings of rs . 14018.07 ) [ table 4 ] . intervening pharmacist suggestions and associated cost savings during the 7-month study period , the study pharmacist spent a total of 1018.18 h in different icus . the total time spent for interventions was 21.35 h while the average time spent for each intervention was 14.17 min . the maximum and minimum time spent for although many studies were conducted to evaluate the value of addition of clinical pharmacy services into the patient care in critical care settings , its economic impact was very rarely studied . therefore this study aimed to demonstrate the economic impact of clinical pharmacist services in critical care settings of a tertiary care teaching hospital . of the 895 patients admitted to the various intensive care settings , 805 patients were reviewed . in our study , we could not review 90 patients as they were either shifted from the icu settings within few hours or within a day of admission , death occurred or due to admission and discharge of patients during the weekend holidays . of the total ( 161 ) drps remaining 44 interventions were not considered for resolutions as they were considered to be minor drps and did not have any clinical significance in terms of patient 's outcome . of the 99 patients in whom drps identified , 48 patients received 610 drugs / patient / day and 32 patients received 1115 drugs / patient / day . an increase in number of medication consumption would have resulted in the increased occurrence of drps . other reasons for the increased occurrence of drps could be increased workload of doctors / medicine pgs and lack of adequate knowledge especially about the newly marketed drugs , urgency in providing the treatment , and possible stress on health care professionals . among the 94% of interventions accepted , 85% of interventions led to the change in drug therapy , while there was no change in drug therapy in 10% of interventions . this observation correlates with other studies wherein the acceptance rate was reported to be 85% and 90% , respectively . however , in 10% of interventions there was no change in drug therapy as in few cases physicians were hesitant / reluctant to change the prescription immediately while in other cases suggestions provided were considered as either to be insignificant or with inadequate evidences . in seven cases , one of the major reasons for this could be that the pharmacist failed to understand the sophisticated prescribing behavior , i.e. , prescribing decisions governed by the clinical experiences of the physicians . in few cases , experienced physicians did not change their routine prescribing pattern especially in the case of interventions of , it could be due to the lack of convincible answer provided by the pharmacist . in four cases , it was unclear whether or not there was change in drug therapy as patients were not follow - up owing to their discharge or death of the patients . only those interventions that were accepted and resulted in change in drug therapy were considered for the cost implications . in our study , the annualized cost savings due to pharmacist - initiated changes to drug therapy was found to be rs.135205.22 . dooley et al . reported the annualized cost savings of rs : 21,33,50,112 [ us$4444794 ] . the difference in the annualized cost savings between these two studies is due to the fact that difference in the study population and number of hospitals included in the study . study was conducted at eight major acute care hospitals with a well - trained and experienced pharmacists . however , our study was conducted in a single teaching hospital . moreover , in our study , the intervening pharmacist is a postgraduate student with minimal experience on drug therapy reviewing and managing drps . in addition , there could be differences in the charges of laboratory tests , hospital stay charges , drug cost , etc . the overall observation made from this study was that pharmacists have greater responsibility preventing and/or minimizing drps occurring in intensive care settings . in conclusion , clinical pharmacists not only have greater potential in preventing and/or minimizing the drps , but also have potential to reduce the unnecessary healthcare expenditures arising from drps occurring in patients treated in intensive care settings . identifying drps and making patient - specific recommendations by a trained clinical pharmacist may reduce the unnecessary healthcare expenditures . active participation of a clinical pharmacist in the intensive care settings may greatly improve the patient outcome . the clinical pharmacist involvement in the drug therapy decision may help / assist healthcare professionals in the overall management of patient .	objective : to analyze clinical pharmacist interventions in the intensive care units ( icus ) setting of a tertiary care indian hospital and to assess the pharmacoeconomic impact on drug - related problems ( drps).materials and methods : a postgraduate clinical pharmacist reviewed drug prescriptions over a period of 7 months . whenever a drp is identified , it was discussed with a physician and appropriate suggestions were provided , later it was documented on a preprepared form . clinical significance of each intervention was graded based on the predicted clinical outcome . acceptance of the interventions is entirely at the discretion of the medical staff . each intervention was analyzed with respect to potential cost saving and/or additional cost incurred to existing drug therapy . an independent clinical panel was convened , and all the interventions made by the intervening pharmacist were critically reviewed for potential cost savings.results:the intervening pharmacist made 117 recommendations , of which 94% was accepted by the medical professionals . the most frequent drp identified was overdose ( 24% ) . the total net cost savings made was rs . 77260.13 ( usd 1796.73 ) . this corresponds with rs . 965.75 per patient and an annualized savings of rs . 135205.22.conclusion:clinical pharmacist interventions had a significant impact on the cost of drug therapy and the patient outcome in intensive care settings of our hospital .
psoriasis is a common chronic skin disease mediated by cellular immune mechanisms and characterized by an intense neutrophile cell infiltrate and proliferative activation of epidermal keratinocytes . it is generally assumed that unbalanced immune responses contribute to the pathogenesis [ 1 , 2 ] . the exact sequence of events , as well as the molecular mediators that lead to hyperproliferative responses , is yet to be defined . as a potent regulator of keratinocyte growth and differentiation , the multifunctional signaling molecule nitric oxide ( no ) has been considered to be a strong candidate in the pathogenesis of psoriasis [ 35 ] . this heat - labile and unstable compound is synthesized in endothelial cells as well as neurons by constitutive nos synthase ( cnos ) , while inducible no synthase ( inos ) is found in leucocytes , macrophages , and mesengial cells . a small amount of no produced by cnos in endothelium is responsible for the relaxation of adjacent smooth muscles and prevents adhesion of platelets and leucocytes to the endothelium . however , when produced in large amounts no can destroy tissues and impair immune response . high levels are demonstrated in immunological disorders like systemic lupus erythematosus or rheumatoid arthritis . hence , inhibition of inos is an effective modality of treatment in these conditions . the production of nitric oxide is 10-fold higher in nonlesional skin of psoriatics and 10-fold higher again in the plaques themselves . although studies showed the elevated no levels in psoriatic tissue samples , as far as we know there are very few studies exploring serum no levels in psoriatic patients [ 914 ] . methotrexate ( mtx ) is an analogue of folic acid and an antiproliferative agent through its competitive binding to dihydrofolate reductase . although the effectiveness of mtx in the treatment of psoriasis is very well established , the mechanism of action is poorly understood [ 15 , 16 ] . there are some studies suggesting that mtx decreases the no levels in psoriasis . in our study , the role of no in the pathogenesis of psoriatic inflammation , the relation between no and pasi and the effect of methotrexate on no in psoriatic patients serum were investigated . twenty - two patients ( sixteen males and six females ) with clinically active psoriasis vulgaris and twenty - one healthy controls ( fourteen males and seven females ) were selected from the outpatient clinic of department of dermatology , gazi university hospital . active psoriasis was defined as plaques increasing in size and number at the time of research . the ages of patients and controls ranged from 18 to 59 years ( mean 35.00 11.80 ) and 17 to 46 years ( mean 29.48 7.54 ) , respectively . the duration of psoriasis ( years ) ranged from 1 to 20 ( mean 8.80 6.52 ) . the assessment of the severity and extent of disease was done by pasi score . the exclusion criteria were coexisting inflammatory skin disease , topical therapy within 4 weeks , systemic therapy or photochemotherapy within 3 months . five ml of venous blood was collected using vacutainer tubes with ethylenediaminetetraacetic acid ( edta ) as anticoagulant and centrifuged at 1000 g within 30 min of collection . serum samples of patient and control groups were stored at 70c until they were assayed in one run . the dose of methotrexate was 20 mg per week ( given in three divided doses with a 12-hour interval between doses ) . clinical response was usually evident in 714 days , but maximal response was taken between 4th8th weeks . as soon as acceptable control was achieved , the dosage was lowered or the interval between doses was extended . patients who were treated with methotrexate underwent the following laboratory tests every two weeks during the first month and monthly thereafter , a complete blood count and assays of electrolytes , serum creatinine , blood urea nitrogen , aspartate aminotransferase , alanine aminotransferase , alkaline phosphatase , and bilirubine . serum samples were obtained before mtx therapy , and at the time the lesions completely disappeared ( pasi = 0 ) . the length of the therapy and the cumulative mtx dose were different for each psoriasis patient . the results were compared with 21 healthy volunteers . the relation of the results with the clinical severity and the cumulative mtx dose was also evaluated . as no is an unstable molecule , it is rapidly converted to nitrates and nitrites in the body ; hence their concentration is parallel to no levels . nitrate determination nitrate was measured using the enzymatic one - step assay with nitrate reductase . this method is based on the reduction of nitrate to nitrite by nitrate reductase in the presence of -nadph . tubes containing 250 l of 100 mmol / l potassium phosphate buffer ( ph 7.5 ) , 50 l of 12 mmol / l -nadph , and 100 l sample were equilibrated at 25c . to start the enzymatic reaction , 40 l of 500 u / l nitrate reductase was added . the oxidation of -nadph was monitored in terms of the decrease in absorbency at 340 nm . the method of standard addition was used to minimize the effect of interfering substances from the serum . samples with internal standard , and serum and reagent blanks were also analyzed . to compare differences in nitrite - nitrate levels , results ( before and after therapy , and nonpsoriatic patients ) were assessed using wilcoxons test and mann - whitney u test . correlations between pasi score , cumulative mtx dose , and nitrite - nitrate serum levels were analyzed with the spearman rho correlation . all values were expressed as mean standard of means ( sem ) unless stated otherwise . statistical significance level was set to 0.05 for all calculations . to compare differences in nitrite - nitrate levels , results ( before and after therapy , and nonpsoriatic patients ) were assessed using wilcoxons test and mann - whitney u test . correlations between pasi score , cumulative mtx dose , and nitrite - nitrate serum levels were analyzed with the spearman rho correlation . all values were expressed as mean standard of means ( sem ) unless stated otherwise . the mean pasi score was 36.10 1.87 at the baseline of therapy and the mean cumulative mtx dose was 235 22.34 mg at the time the lesions completely disappear ( pasi = 0 ) . the patients tolerated the therapy well . and there were no significant adverse effects . the mean baseline values of nitrite - nitrate concentrations in the sera of psoriasis patients were 27.98 7.40 mol / l , while that in the sera of patients after treatment were 10.028 2.05 the mean values of serum nitrite - nitrate concentrations of healthy controls were 2.98 0.77 mol / l and 19.08 5.0 mol / l . serum levels of nitrite - nitrate were both significantly higher in patients with psoriasis before treatment than in controls and patients after treatment ( p < no correlation was found between pasi score and serum nitrite - nitrate levels before therapy in the patients ( r = 0.085 and p = 0.708 for nitrite ; r = 0.368 and p = 0.092 for nitrate ) . we found no correlation between cumulative mtx dosage serum nitrite - nitrate levels in patients after treatment . ( r = 0.262 and p = 0.239 for nitrite ; r = 0.094 and p = 0.677 for nitrate ) . in recent years , the clonality of lymphocytes in psoriatic lesions and the increased cytokine release in that region are well established . cytokines , such as ifn , tnf , il-8 , il-1 , and il-6 , are most important ones [ 9 , 19 , 20 ] . no is a labile mediator that can be detected in high levels in the existence of these cytokines . no is released continuously in low concentration while for high concentrations a stimulation is needed [ 21 , 22 ] . it has been shown that no is increased in a number of skin disorders with a stimulation . in the lesional tissue samples of contact dermatitis , atopic dermatitis , systemic lupus erythematosus , and psoriasis , no is significantly higher than controls [ 2326 ] . increased no synthesis suggests a role for the pathogenesis of psoriasis . no may trigger the psoriatic disease process at least partly through increment of the release and actions of calcitonin gene - related peptide and substance p , which are considered to play important roles in the pathomechanism of psoriasis by inducing the production of adhesion molecules , keratinocyte hyperproliferation , mast cell degranulation , vasodilatation , and chemotaxis of neutrophils . cals - gierson and ormerod have suggested that no stimulates epithelial cells to release chemokines and growth mediators which appear to be important for keratinocyte proliferation and angiogenesis . in previous studies , no levels have been measured in tissue samples from psoriasis patients [ 8 , 9 , 11 , 13 , 14 ] . however , we could find very few similar research investigating nitrite and nitrate levels in blood samples of psoriatic patients [ 10 , 12 ] . and there has been no study showing the effects of the mtx upon no levels in human sera . the labile nature of no makes it impossible to analyze serum and tissue levels . the nonfunctional metabolites of no ; nitrite and nitrate are helpful to search the amount of no . nitrite levels are considered the marker of no in tissue and other body fluids . it is important to study nitrate levels as the nitrite is reduced into nitrate by oxyhemoglobine , in blood [ 1113 , 17 ] . orem et al studied no levels in 17 patients of psoriasis during active and inactive phases . in that study the baseline serum nitrite - nitrate levels of severe psoriatic patients ( the mean pasi score was 16.5 ) compared with after topical treatment results and found only the nitrite levels are significantly different . gokhale et al found significantly high no levels in patients with active disease as compared to normal individuals . in that study there was significant positive correlation between the severity and duration of disease and no levels in patients with chronic plaque - type psoriasis . in our study all the patients had the pasi score above 15 before treatment . the baseline serum nitrite - nitrate levels were significantly higher than the levels in nonlesional ( pasi = 0 ) period . serum nitrite and nitrate levels of the control group were significantly lower than those of the psoriatic patients during the inflammatory stage . these findings suggest a possible role of no in the pathogenesis of psoriasis during the inflammatory phase . the results of our study are consistent with the findings of previous studies in which no and inos levels in the tissue samples were investigated [ 11 , 14 , 18 , 25 , 29 ] . the nitrite plasma levels in the control group were significantly lower than in the psoriasis patients both before and after treatment in our study ( p = 0.0004 ) . the nitrate levels after treatment were higher than the control group but the difference was not statistically significant ( p > .05 ) . we suggest that serum nitrate level may be an important indicator for the lesional period . the finding of higher serum nitrite - nitrate levels than the healthy controls supports the idea that the inflammation in psoriasis does not descend the normal levels in clinically healed psoriatic patients . this opinion is supported by the results of studies in the literature showing the microscopic inflammation in nonlesional tissue samples [ 1 , 8 , 13 , 14 ] . as far as we know there are only two studies exploring the correlation between psoriasis severity and no levels in the literature . gokhale et al found significant positive correlation between the severity of disease and no levels in patients with chronic plaque - type psoriasis . but we found no correlation between serum nitrite - nitrate levels and pasi in our study . our results support the theory that the serum nitrite - nitrate levels indicate the systemic inflammation and establish a relation between no and psoriasis activity that is possible in laboratory conditions . however , it seems that the nitrite - nitrate concentration is not a biomarker of psoriasis severity according to our findings . methotrexate was thought to act primarily on the rapidly dividing basal keratinocytes of the psoriatic lesion . the group of weinstein demonstrated that proliferating lymphoid cells in psoriatic lesions are over 1000 times more sensitive to the cytotoxic effects of mtx than primary human keratinocytes . it also has been shown that mtx exerts anti - inflammatory effects mediated through intracellular accumulation of 5-aminoimidazole-4-carboxyamide ribonucleotide , thereby increasing the release of adenosine . adenosine exerts anti - inflammatory effects mainly on neutrophils , where an inhibition of adhesion and reactive oxygen intermediate production has been demonstrated [ 31 ] . the basic studies exploring the effect of mtx on no levels are in vitro . these studies emphasize that mtx inhibits no by means of constitutive and/or inducible no synthases inhibition [ 3235 ] . there are some studies suggesting that mtx decreases the no levels in psoriasis although there is no relation between the cumulative dosage and nitrite - nitrate levels in noninflammatory period in our study , nitrite - nitrate levels after treatment were significantly lower than the levels before . this result may show that mtx decreases no levels and makes an anti - inflammatory effect independent of the cumulative dosage . this finding is similar to other reports in the literature [ 13 , 35 , 36 ] . we can suggest that immunological and inflammatory mechanisms are important in the etiopathogenesis of psoriasis and that no plays a role in both mechanisms . the results in our study are comparable with the studies investigating the lesional psoriatic tissue inos and no levels [ 9 , 11 , 14 ] . this relationship between psoriasis and no may pave the way for novel therapeutic approaches such as selective inos inhibitors in the management of this difficult disease . serum levels of nitrite were significantly higher in patients with psoriasis before treatment than in controls and patients after treatment ( p < .05 ) . serum levels of nitrate were significantly higher in patients with psoriasis before treatment than in controls and patients after treatment ( p < .05 ) .	psoriasis is a chronic , recurrent , inflammatory , and hyperproliferative disease . recently there have been studies regarding increases in the levels of no in inflammatory dermatoses including psoriasis . in this study , 22 patients with psoriasis were scored with pasi ( psoriasis area and severity index ) and the levels of serum nitrite - nitrate were evaluated before and after therapy with methotrexate ( mtx ) . the results were compared with age- and sex - matched healthy volunteers . the relation of the results with the clinical severity and the cumulative mtx dose were also evaluated . the serum levels of nitrite - nitrate of the psoriatic patients with active lesions were found to be significantly higher than the levels of the healthy volunteers and the patients after therapy . the elevated nitrite - nitrate serum levels in the inflammatory period may suggest the possible role of this mediator in the etiopathogenesis of psoriasis and the potential future use of no inhibitors in the treatment of psoriasis .
osteoporotic fractures diminish the quality of life and are a major cause of hospitalization and medical expenses in elderly patients . the incidence , mortality and morbidity of hip and spine osteoporotic fractures have been reported ( 1 - 5 ) . distal radius fractures occur in middle - aged women and men ( 6,7 ) and are a risk predictor of subsequent osteoporotic fractures ( 8) . a better understanding of the incidence , mortality and excess mortality after distal radius fracture is needed for prevention of further osteoporotic fractures . distal radius fractures are the second most common type of osteoporotic fracture including spine , humerus , and hip fractures . yet , few epidemiologic studies of distal radius fractures have been published and involved western developed countries including sweden , norway and other european countries ( 6,9,10 ) . only two asian countries have reported on the incidence of distal radius fractures ( 7,11 ) . the purpose of this study was to determine the incidence , mortality and excessive mortality after distal radius fracture in koreans among those 50 years of age and older using recent 5-year national claims data and to compare the findings with other studies . korean national health insurance ( knhi ) covers 100% of the population including 97% of health insurance and 3% of medical aid ( 2 ) . all information about the volume and burden of disease can be obtained from this centralized database , with the exception of procedures that are not covered by insurance , such as cosmetic surgery or traffic accidents , which are covered by traffic insurance companies . all clinics and hospitals submit claims data on inpatients and outpatients including data on diagnosis and medical costs . knhi data include information on date of discharge and discharge diagnoses ( both principal diagnoses and additional diagnoses ) , assigned exclusively by the physician at discharge according to the international classification of diseases , 10th revision ( icd-10 ) . the advantage of an osteoporotic fracture study using knhi data is that the high - energy trauma that led to the fracture is spontaneously excluded because traffic accidents and industrial accidents are covered by different insurance systems . complete paid claims data and eligibility files were merged to create a database consisting of data for all filled prescriptions , procedures , outpatient physician encounters and hospitalizations . using knhi data , we identified all claims records of outpatient visits or hospital admissions of patients among those 50 years of age and older between january 1 , 2008 and december 31 , 2012 . one or more claims listing an international classification of disease , tenth revision diagnosis codes s52.5 ( fracture of lower end of radius ) and s52.6 ( fracture of lower end of both ulna and radius ) and treatment codes n0607 ( open reduction of ulnar or radius ) , n0603 ( open reduction of ulnar and radius ) , n0993 ( closed pinning of ulnar or radius ) , n0994 ( closed pinning of unlar and radius ) , n0983 ( external fixation of forearm bone ) , n0643 ( closed reduction of forearm bone ) , t6020 ( long arm cast application ) , and t6030 ( short arm cast application ) were required for inclusion . age standardized incidence rates of people in the corresponding age groups in a standard population were also determined . unique personal identifiers permitted the tracking of individuals for multiple visits or admissions . where an individual had more than three outpatient visits or one admission for distal radius fracture , the patient was followed from the first event and recounted if a further event occurred 6 months or longer after the original visit or admission ( 2 ) . we followed each patient by code to identify the death date . using the poisson model , mortality at 1 year after distal radius fracture was compared to that of the general korean population among those 50 years of age and older . the mortality of the general population was obtained from the web site of the korean statistical information service . to determine the excess mortality associated with distal radius fracture , the standardized mortality ratio ( smr ) was calculated as the observed mortality divided by the expected mortality for each age and gender group . all data of korean men and women over 50-years - of - age was based on the web site of the korean statistical information service . the 95% confidence intervals for the smr of each group were calculated by the poisson method . all database management and analysis were performed using sas statistical package version 9.1.3 ( sas institute , cary , nc , usa ) . the study protocol was approved by the nhis institutional review board ( number : nhis-2015 - 4 - 001 ) . the study protocol was approved by the nhis institutional review board ( number : nhis-2015 - 4 - 001 ) . the total number of distal radius fractures increased by 54.2% over the 5-year study ( 48,145 in 2008 and 74,240 in 2012 ) , whereas the number of individuals among those 50 years of age and older in the general population increased by 19.5% ( 13,103,814 in 2008 and 15,657,674 in 2012 ) ( table 1 ) . the incidence of distal radius fracture increased from 367.4/100,000 in 2008 to 474.1/100,000 in 2012 . in terms of the gender - specific distribution of distal radius fracture from 2008 to 2012 , the incidence of distal radius fracture was 134.8/100,000 for men and 566.1/100,000 for women in 2008 , and 162.9/100,000 for men and 744.3/100,000 for women in 2012 . during the 5-year period , the incidence of distal radius fractures increased by 20.8% in men and 31.5% in women . * numbers in parenthesis are patients who passed away at 12 months follow - up . the mean age - specific incidence by 5-year age groups increased in all age groups ( fig . 1 ) . in terms of age - specific distribution of distal radius fracture from 2008 to 2012 , the incidence of distal radius fractures steadily increased more in men and women in their 60s ( fig . age - specific incidence of distal radius fractures in patients aged 50 years and older . gender - specific incidence of distal radius fractures in patients aged 50 years and older . the cumulative mortality rate over the first 12 months after distal radius fracture was decreased from 2.0% ( 968/48,145 ) in 2008 to 1.4% ( 1,045/74,240 ) in 2012 . 1.8% ( 734/40,006 ) for women in 2008 , and 2.0% ( 257/11,855 ) for men and 1.3% ( 788/62,385 ) for women in 2012 . during the 5-year study periods , the mean year mortality over 5 years in men ( 2.6% , 1,279/50,128 ) over the first 12 months was 1.7-times higher in men than in women ( 1.5% , 3,952/257,045 ) ( table 1 ) . age- and gender - adjusted standardized mortality ratios for different study periods are shown in table 2 . the mean of smr of distal radius fracture at 1 year post - fracture was 1.45 in men and 1.17 in women . total smr of distal radius fracture at the 1-year follow - up was slightly higher than the general population ( table 2 ) . in men , higher smr was observed for subjects aged 50 - 89 years , with no difference of smr of 90 - 100 age group at 1 year post - fracture . in women , the higher smr was only observed for subjects 80 - 89-years - of - age , with no difference of smr other age groups at 1 year post - fracture ( table 2 ) . of osteoporotic fractures including spine , hip , humerus , and distal radius fractures , in terms of morbidity and mortality , distal radius fractures tend to be less important than hip and spine fractures . however , distal radius fractures in elderly patients can be a significant risk predictor of subsequent osteoporotic fractures ( 8) . the present nationwide , claims database based , observational study of distal radius fracture in korea determined incidence , mortality , and excess mortality . distal radius fractures in subjects among those 50 years of age and older increased in incidence during the study period , with mortality was decreasing . distal radius fractures represent the second most common type of osteoporotic fracture in korea ( 12 ) . the total number of distal radius fracture increased by 50.1% , from 48,145 in 2008 to 74,240 in 2012 , and the annual incidence of distal radius fracture increased by 21.3% , from 367.4/100,000 in 2008 to 474.1/100,000 in 2012 . the incidence of distal radius fracture in this study is one of the highest among data of reported countries ( table 3 ) . in this study , this finding is slightly lower than norway ( 525.7/100,000 ) ( 6 ) and finland ( 494.4/100,000 ) ( 10 ) . however , lower incidence has been reported in the same region including taiwan ( 144.9/100,000 in 2007 ) ( 7 ) and japan ( 111.1/100,000 in 2006 ) ( 11 ) . t , total ; m , male ; f , female ; nr , not reported ; drf , distal radius fracture . this finding is similar to a population - based study using claims data that investigated the trend in incidence of distal radial fractures in taiwan from 2000 to 2007 ( 7 ) . however , studies in the developed industrialized countries of japan and the netherlands reported steady or decreasing trend of distal radius fracture ( 9,11 ) . the reasons for the differences from this study might be related to the proportion of the elderly population , the health care system , and preventive programs of osteoporotic fractures . of these , the proportion of elderly population is an important factor for the increasing rate of distal radius fracture . in korea korea became an aging society ( elderly population 7% of the total population ) in 2000 . in 2018 , korea will become an aged society ( defined as an elderly population 14% of the total population ) , and by 2026 will be a super - aged society ( elderly population 20% of the total population ) . total annual mortality rates in this study decreased from 2.0% in 2008 to 1.4% in 2012 . although the incidence of distal radius fracture in men was lower than in women , the mortality rate in men was 1.7-times higher than in women . however , the mortality rate of distal radius fracture was lower than in other studies ( 13,14 ) . endres et al . ( 13 ) observed that 3% of the distal radius fracture patients from germany died during a 1.5-year follow - up period . ( 14 ) reported a 1- and 5-year mortality rate of 6% and 26% , respectively , in a swedish study . the reasons for these differences may involve cohort characteristics , geographic difference , and the proportion of older patients ( table 3 ) . the mortality risk after distal radius fracture was similar to that of general populations ( 14 - 16 ) . however , smrs of both gender in this study were slightly higher than the general population until 1 year after distal radius fractures . therefore , distal radius fractures might reflect frailty and poorer comorbidity , resulting in high mortality in these groups . for example , distal radius fractures may not be diagnosed or treated by health care institutions not necessary to require hospitalization . second , reliance on icd-10 diagnostic codes to identify incident fractures may have caused incident distal radius fracture to be misclassified . to minimize this problem , we analyzed icd-10 diagnostic codes with treatment codes such as operation and cast or splint application codes . third , there was not considered medical comorbidity to analysis of mortality , which influence mortality in older patients . thus , the confounding effect on results imposed by medical comorbidity is inevitable in this study . fourth , patients with multiple traumas such as hip fracture with other sites of fracture could not be detected . such patients are more susceptible to mortality and poor morbidity . finally , whereas incidence of distal radius fracture in this study increased , mortality rate after distal radius fracture decreased . the possible reason of decreasing mortality after distal radius fracture could be evaluated through further study such as cohort based study . in conclusion , this study using a nationwide database demonstrates that the distal radius fractures are increasing with a decreasing mortality in korea . considering the increasing incidence rate of distal radius fractures , we can expect an increased risk of associated osteoporotic fractures . therefore , a public health strategy to prevent distal radius fractures is prudent to minimize secondary osteoporotic fractures , economic burden , and improve public health .	the purpose of this study was to assess the incidence and mortality of distal radius fracture among patients 50 years of age and older with diagnosis code ( icd10 ; s52.5 , s52.6 ) and treatment code using a nationwide claims database from 2008 to 2012 . all patients were followed using patient identification code to identify deaths . standardized mortality ratios ( smrs ) of distal radius fracture were calculated based on age and gender - specific rates in the entire korean population . the number of distal radius fractures increased by 54.2% over the 5-year study ( 48,145 in 2008 and 74,240 in 2012 ) . the incidence of distal radius fracture increased from 367.4/100,000 in 2008 to 474.1/100,000 in 2012 . the cumulative mortality rate over the first 12 months after distal radius fracture was decreased from 2.0% ( 968/48,145 ) in 2008 to 1.4% ( 1,045/74,240 ) in 2012 . the mean year mortality over 5 years in men ( 2.6% , 1,279/50,128 ) over the first 12 months was 1.7-times higher than in women ( 1.5% , 3,952/257,045 ) . the mean of smr of distal radius fracture at 1 year post - fracture was 1.45 in men and 1.17 in women . this study using a nationwide database demonstrates that the distal radius fractures are increasing with a decreasing mortality in korea .
diabetes mellitus ( dm ) , also known as simply diabetes , is a chronic and heterogeneous metabolic disorder that affects millions of people worldwide . the previous study showed that the increasing global prevalence of type 2 diabetes mellitus ( t2 dm ) is associated with the rising obesity rates , and the obesity is a key factor in the development of t2 dm . reported that , for the uygur and kazak ethnic groups ( i.e. , two major ethnic minorities in xinjiang ) at the equal body mass index ( bmi ) level , more kazak people developed hypertension , whereas more uygur people developed diabetes . in addition , the uygur subjects had significantly greater waist - hip ratio ( whr ) than the kazak subjects , and the uygur subjects had increased fat distribution in the abdominal viscera , whereas the kazak subjects had more subcutaneous fat . additionally , ibrahim reported that abdominal obesity imparts a greater risk of developing diabetes and future cardiovascular events than peripheral or gluteofemoral obesity , and visceral adipose tissue has a higher rate of insulin - stimulated glucose uptake compared with subcutaneous obesity . furthermore , perrini et al . reported that cytokine release profiles were distinct in the subcutaneous and visceral adipose tissue . although uygurs and kazaks have essentially the same eating habits and living environments , their body fat distributions are different . in the current study , to further explore the correlation of obesity position and t2 dm , we examined the characteristic of fat tissues traits and gene expression in visceral adipose tissues of the normal , obesity , and t2 dm individuals in uygurs population . our study consisted of 980 uygur participants ( 580 males and 400 females ) and 1122 kazak participants ( 415 males and 707 females ) from yili and kashi in xinjiang province of china , and the subjects used in our study are new and different individuals from those in . all patients completed a series of conventional questionnaires , including disease history and daily living and eating habits . in addition , all patients underwent the following measurements : blood biochemical analysis and blood pressure , height , weight , waist , and hip circumference measurements ; and for the blood pressure measurement the systolic pressure and diastolic pressure were detected and recorded by measuring 3 tests . subsequently , 18 uygur subjects ( 9 males and 9 females ) and 18 kazak subjects ( 9 males and 9 females ) aged from 40 to 60 years were randomly selected from the 980 uygur participants and 1122 kazak participants , respectively , to have body composition analysis , including determining their overall fat content using the underwater weighing test and mrc measurements of subcutaneous abdominal fat weight and visceral fat . we label - grouped all examined samples into 3 groups ( normal , obesity , and diabetes , resp . ) . our definition of obesity was determined using the 1999 diagnostic criteria from the world health organization and the international obesity task force asian adult standard from 2000 . specifically , a bmi 25 kg / m was defined as obese or overweight , and a bmi < 25 kg / m was normal . additionally , all patients with t2 dm ( 2-hour postprandial glucose 11.1 mmol / l , fasting glucose 7.0 mmol / l ; world health organization in 1999 ) were confirmed by the xinjiang uygur autonomous region people 's hospital department . the intra - abdominal adipose tissues of another 124 uygur participants , which were contained in the 980 uygur participants , were collected from kashi city in xinjiang province of china : 50 samples for normal control ( normal ) group , 48 samples for obesity group , and 26 samples for t2 dm group . the biopsy of abdominal adipose tissues undergoing surgery was on the protocol to perform pathological analysis , and the mrna sample taken had been informed before surgery , and every subject accepted to participate by signing a written informed consent . one day before elective abdominal operation , the weight , waist , hip , and glycemic index were detected and bmi and whr were calculated . t2 dm was diagnosed according to t2 dm diagnostic criteria ( 2-hour postprandial glucose 11.1 mmol / l , fasting glucose 7.0 mmol / l ; world health organization in 1999 ) . the patients suffering from cancer , acute inflammation , liver and kidney disease , patients with type 1 diabetes , and patients recently taking drugs that may interfere with glucose and lipid metabolism were not included in the present study . the fasting plasma glucose ( fpg ) was detected using the glucose oxidase method , and total cholesterol ( tc ) , triglyceride ( tg ) , high - density lipoprotein cholesterol ( hdl - c ) , and low density lipoprotein cholesterol ( ldl - c ) were all detected using automatic biochemistry analyzer . waist and hip circumferences were measured using a flexible tape with tension calipers at the extremity ( gulick - creative health product , inc . , plymouth , mi ) , midway between the xiphoid and umbilicus during the midexpiratory phase and at the maximum circumference in the hip area , respectively . skinfold thickness was measured at 5 different anatomical sites ( i.e. , subscapular diagonal and vertical , chest , midaxillary , abdominal horizontal and vertical , and suprailiac diagonal and vertical ) using lange skinfold callipers ( cambridge scientific instruments inc . truncal skin folds were computed as the sum of the skin folds at these 5 anatomic sites . mri was used to measure intra - abdominal ( visceral ) and abdominal subcutaneous adipose tissue volume , as previously described . in brief , mri studies were performed using a 1.5 t imaging device ( philips gyroscan intera , holland ) . fat volume was measured in each slide by mapping subcutaneous and intra - abdominal adipose tissue compartments using computerized images . volume was converted into adipose tissue mass , assuming an adipose tissue density of 0.9196 the abdominal omental adipose tissues were collected on the day of surgery , and about 3 cm 3 cm tissues were collected from each individual , immediately washed with 0.75% nacl solution , and snap - frozen and stored in liquid nitrogen until rna extraction . total rna was isolated from the visceral adipose tissue ( abdominal omental adipose tissue ) in the 124 uygur subjects ( 50 samples for normal group ; 48 samples for obesity group ; and 26 samples for t2 dm group ) by using trizol reagent ( cat#15596 - 026 , life technologies , carlsbad , ca , usa ) and purified by using an rneasy minikit ( cat#74106 , qiagen , gmbh , germany ) . rna integrity was checked on an agilent 2100 bioanalyzer ( agilent technologies , santa clara , ca , usa ) . reverse transcription conditions for each cdna amplification were 25c for 5 min , 42c for 60 min , and 70c for 15 min . real - time pcr was used to detect gene expression using the sybr premix ex taq ( takara ) on a 7500 real - time pcr system ( applied biosystems , foster city , ca ) with the primers shown in table 1 . part ( 1 l ) of each rt reaction product was amplified in a 20 l pcr reaction system . reaction mixtures were incubated in an abi prism 7500 sequence detection system ( applied biosystems ) , programmed to conduct 1 cycle at 95c for 30 s and 40 cycles at 95c for 5 s and 60c for 34 s. moreover , dissociation curves were analyzed using the dissociation curve 1.0 software ( applied biosystems ) for each pcr reaction to detect and eliminate possible primer - dimer artifacts . the relative amounts of target gene transcripts were calculated using the comparative cycle - time method . all participants were informed of the usage of their basic and clinical information prior to the sample collection , and all participants provided written informed consent for study participation . the consent form and ethical approval were provided by the medical ethics committee at first affiliated hospital , shihezi university school of medicine ( reference number 2014ll22 ) . all data are presented as mean plus standard deviation ( mean sd ) . the shapiro - wilk test was used to test the normality of data . if the data follow the normality role , differences between groups were analyzed using unpaired student 's t - test ; else the differences between groups were analyzed using rank sum test . the data in table 2 shows that at similar bmi values , compared with the kazak subjects , the uygur subjects had increased values in weight , waist circumference , hip circumference , and whr . in addition , the values of hdl - c in uygur male and female subjects were significantly lower than those in kazak male and female subjects , and the values of creatinine , uric acid , diastolic blood pressure , blood glucose , and fructosamine in uygur male subjects were significantly lower than those in kazak male subjects . in contrast , the systolic blood pressure values in uygur male and female subjects were significantly greater than those in kazak male and female subjects , and blood glucose values were significantly greater in uygur female subjects than in kazak female subjects . additionally , consistent with the previous report , our results also indicated that the uygurs had a significantly greater risk of diabetes than the kazaks for both males and females . no significant difference was detected in the values of triglycerides , cholesterol , and ldl - c between both male and female uygur sbujects and kazak subjects . both 18 individuals from the uygur and kazak ethnic subjects ( 980 and 1122 , resp . ) and 6 individuals ( 3 male and 3 female ) aged from 40 to 60 in each group ( including normal , obesity , and t2 dm ) were randomly selcected for body composition analysis , and the baseline characteristics for 36 subjects are shown in table 3 . there were no significant differences observed in the ages of the uygur and kazak participants . in both uygur and kazak ethnic groups , the body weight , bmi , and whr values of the normal control group were significantly lower than those of the obesity and t2 dm groups ( p < 0.05 ) . notably , although there was no significant difference in total lipid content between subjects from the two ethnic groups , the ratio of vf / saf in uygurs was significantly higher than that of the kazaks in both obesity and t2 dm groups ( p < 0.05 ) . however , there was no difference between the two ethnic groups regarding fasting blood glucose levels . to reveal the function change of the adipose tissues among normal , obesity , and t2 dm individuals , five candidate genes ' expressions in visceral adipose tissue of normal , obesity , and t2 dm individual in uygur population the result showed that five candidate genes , including t - box protein 1 ( tbx1 ) , transcription factor 21 ( tcf21 ) , adiponectin ( apn ) , tumor necrosis factor - alpha ( tnf- ) , and monocyte chemotactic protein 1 ( mcp-1 ) , were all expressed in the visceral adipose tissue of normal , obesity , and t2 dm individuals in uygur population . the tbx1 and tcf21 expression levels in the obesity group were significantly higher than those of normal and t2 dm groups ( p < 0.01 ) ; and the tbx1 and tcf21 expression levels in the t2 dm group were significantly lower than those of normal and obesity groups ( figure 1 , p < 0.01 ) . in addition , statistical analysis showed that the ratio of expression level of tbx1 to tcf21 ( tbx1/tcf21 ) in obesity group was significantly greater than those in normal and t2 dm groups ( figure 1 , p < 0.01 ) . additionally , the expression of apn in the normal individual was significantly higher than those in obesity and t2 dm groups , the expression levels of tnf- and mcp-1 in normal group were significantly lower than those in obesity and t2 dm groups ( table 4 , p < 0.05 ) , and no significant differences of apn , tnf- , or mcp-1 expression were detected between obesity and t2 dm groups ( table 4 , p > 0.05 ) . the previous report showed that visceral and subcutaneous adipocytes may exhibit different properties in the production of bioactive molecules . in the current study , the results showed that , at the same bmi level , both male and female uygur subjects have significantly greater values in whr , abdominal visceral fat content , and diabetes risks , compared with kazak subjects . in addition , the values of hdl - c in uygur male and female subjects were significantly lower than those in kazak male and female subjects , and the values of creatinine , uric acid , diastolic blood pressure , blood glucose , and fructosamine in uygur male subjects were significantly lower than those in kazak male subjects . in contrast , the systolic blood pressure values in uygur male and female subjects were significantly greater than those in kazak male and female subjects , and blood glucose values were significantly greater in uygur female subjects than in kazak female subjects . these results showed that the metabolic difference result from obesity exists between the uygurs and kazaks populations . toyoda et al . reported that visceral adipose tissue is a better predictor for mortality than subcutaneous tissue . our results showed that gene expression in the visceral adipose tissue of normal , obesity , and t2 dm subjects in the uygurs was significantly different , and the expression levels of tbx1 and tcf21 , the marker of white adipocytes and brown - like white adipocytes , respectively , were significantly greater in the obesity group than in normal and t2 dm groups and significantly lower in the t2 dm group than in normal group ( p < 0.01 ) . these results suggested that the lipid metabolic activity of visceral adipose among normal , obesity , and t2 dm subjects was different in the uygurs population . the ratio of expression level of tbx1 to tcf21 ( tbx1/tcf21 ) in obesity group was significantly greater than those in normal and t2 dm groups ( p < 0.01 ) , suggesting that the lipid metabolic activity of visceral adipose tissue in obesity individuals was lower than those in normal and t2 dm individuals . apn is an adipocytokine produced by adipose tissue and plays a protective role in vascular injury and insulin resistance ( ir ) . the previous study in other ethnic groups showed that the subjects with diabetes mellitus ( dm ) had greater insulin and lower apn level , and asian participants , who suffered from higher chronic disease risk for obesity , had lower serum levels of apn than caucasian participants across all levels of bmi . our result showed that the visceral adipose tissue expression levels of apn were lower in obesity and t2 dm groups than in normal group ; this is consistent with the previous study and suggested that downregulation of apn might be a reason for the obesity and t2 dm . tnf- and mcp-1 are two proinflammatory cytokines that can be released by adipose tissue . in the current study , the results showed that the visceral adipose tissue expression levels of tnf- and mcp-1 in normal group were significantly lower than those in obesity and t2 dm groups ( p < 0.01 ) , which were consistent with the previous study where tnf- and mcp-1 were elevated in poor glycemic control and good glycemic control overweight and obese patients , suggesting that the immune response of visceral adipose tissue was changed during the occurrence of obesity and t2 dm . collectively , our result indicated that the t2 dm in the uygurs population was mainly associated with not only distribution of adipose tissue in the body , but also the change in metabolic activity and adipocytokines secretion of adipose tissue .	our results showed that , at the same bmi level , uygurs have greater whr values , abdominal visceral fat content , and diabetes risks than kazaks . in addition , values of hdl - c in uygur subjects were lower than those in kazak subjects , and values of creatinine , uric acid , diastolic blood pressure , blood glucose , and fructosamine in uygur male subjects were lower than those in kazak male subjects . in contrast , systolic blood pressure values in uygur subjects were greater than those in kazak subjects , and blood glucose values were greater in uygur female subjects than in kazak female subjects . additionally , in uygurs , visceral adipose tissue expression levels of tbx1 and tcf21 were greater in obesity group than in normal and t2 dm groups and lower in t2 dm group than in normal group ( p < 0.01 ) . the visceral adipose tissue expression levels of apn in normal group was greater than those in obesity and t2 dm groups , and visceral adipose tissue expression levels of tnf- and mcp-1 in normal group were lower than those in obesity and t2 dm groups ( p < 0.01 ) . in conclusion , t2 dm in uygurs was mainly associated with not only distribution of adipose tissue in body , but also change in metabolic activity and adipocytokines secretion of adipose tissue .
type 1 diabetes ( t1d ) is a t - cell - mediated autoimmune disease that involves the progressive destruction of pancreatic cells resulting in complete loss of insulin secretion . while a combination of genetic predisposition and autoimmune processes contributes to its development , inflammatory mediators and innate immunity play a key role in the induction and amplification of the immune assault as well as in the maintenance of insulitis [ 2 , 3 ] . based on studies using animal models of autoimmune diabetes such as the biobreeding ( bb ) rat and the nonobese diabetic ( nod ) mouse , the involvement of -cell autoantigens , macrophages , dendritic cells ( dcs ) , and t and b lymphocytes in the development of t1d has been established [ 46 ] . antigen - presenting cells ( apcs ) such as macrophages and dcs are the first to infiltrate islets followed by cd4 + and cd8 + t lymphocytes , natural killer ( nk ) cells , and b lymphocytes . macrophages play an essential role in the development and activation of cytotoxic cd8 + t cells , the final effectors of -cell destruction . studies indicate that il-12 secreted by macrophages may activate th1-type cd4 + t cells and subsequently il-2 as well as other proinflammatory cytokines released by the activated cd4 + t cells ( for e.g. , interferon - gamma ifn- , tumor necrosis factor tnf- and tnf- ) contribute to maximizing the activation of cytotoxic cd8 + t cells . activated cd4 + and cd8 + t cells act in unison to activate -cell death via apoptosis . additionally , ifn- released by cd4 + t cells may in turn activate macrophages to release proinflammatory cytokines and reactive oxygen species ( ros , e.g. , superoxide , hydrogen peroxide , nitric oxide ( no ) , etc . ) . ultimately , t - cell - mediated -cell destruction is effected overall by the interplay between receptor - mediated interactions ( e.g. , fas - fas ligand , cd40 ligand , tnf - tnf receptor ) , secretion of proinflammatory cytokines and ros as well as the release of granzymes and perforin from cytotoxic effector t cells . furthermore , dna damage in -cells leads to the activation of poly ( adp - ribose ) polymerase which rapidly depletes nad that has been shown to be protective against radical - induced necrosis . interestingly , among the regulators of apoptosis , nuclear factor - kappa b ( nf-b ) has emerged as a master switch of cytokine - induced -cell dysfunction and death . recognition of ligands by pattern - recognition receptors ( prrs , e.g. , toll - like receptor tlr3/4 , rigi , mda5 ) on -cells leads to the activation of key transcription factors such as nf-b and signal transducer and activator of transcription 1 ( stat-1 ) , inducing the release of chemokines and cytokines that recruit and activate immune cells , increase expression of mhc class i antigens , and activate proapoptotic signals resulting in -cell destruction . proinflammatory cytokines further induce stat-1 , nf-b , and interferon regulatory factor 3 ( irf-3 ) in -cells , contributing to the maintenance and amplification of the immune processes that result in the progressive and selective destruction of pancreatic -cells . several studies indicate the potential role of cytokines in modulating the immune response towards cytotoxic effector cell or suppressor cell dominance . t1d is thought to be a th1-dominance disease with type 1 cytokines such as ifn- , tnf- , il-2 , il-12 , and il-18 actively involved in -cell destruction . on the other hand , cytokines such as il-4 , il-5 , il-10 as well as tgf- secreted by th2/th3 cells are thought to inhibit -cell destruction . of tremendous interest is the recent discovery that th17 cells are potent inducers of tissue inflammation and autoimmunity and may have a role in t1d as indicated in a study using nod mice [ 8 , 9 ] . a humanized t1d model has now been established in nod - scid il2r ( null ) mice to study the selective destruction of mouse islet -cells by a human t - cell - mediated immune response , providing a valuable tool for translational research into t1d . data from large epidemiologic studies indicate that t1d accounts for 5% to 10% of the total cases of diabetes worldwide , with its incidence increasing by 2% to 5% . in the united states , the prevalence of t1d by 18 years of age is approximately 1 in 300 [ 11 , 12 ] . strategies to prevent or reverse t1d are broadly based on the concepts of -cell regeneration , -cell replacement , or protection of islets from immune destruction . while regenerative strategies broadly involve -cell regeneration from progenitors / stem cells ( e.g. , putative -cell progenitors / pancreatic stem cells ; embryonic , mesenchymal , hematopoietic , and umbilical cord blood - derived stem cells ( ucb - scs ) , induced pluripotent stem cells ( ipscs ) , etc . ) or neogenesis from ductal and non--cell progenitors , -cell replacement strategies include transplantation of whole pancreas or islets of langerhans or genetically engineered insulin - secreting cells . the current standard of treatment for t1d requires lifelong exogenous insulin administration by either insulin pump or multiple daily injections which although successful by no means represents a cure , often resulting in hypoglycemic episodes and in microvascular complications in a high percentage of patients . in the year 2000 edmonton protocol that consisted of induction therapy with a monoclonal antibody ( mab ) against the interleukin-2 ( il-2 ) receptor ( daclizumab ) , and maintenance therapy with a calcineurin inhibitor ( cni , tacrolimus ) and a mammalian target of rapamycin ( mtor ) inhibitor ( sirolimus ) . over a followup period of approximately 11.9 months , all patients were insulin - free . for instance , in 2006 an international multicenter trial demonstrated successful restoration of endogenous insulin producion and glycemic stability in 44% of transplant recipients at one year following the final islet transplantation . persistent graft function defined by detectable c - peptide levels and improved glycemic control was observed in 70% of the recipients at the two - year followup although the insulin independence rate was disappointingly low ( 14% ) . the 2008 update from the collaborative islet transplant registry ( citr ) summarizing the results from 279 recipients of one or more islet alone allotransplants between 1999 and 2007 also reported achievement of insulin independence in 23% of islet - alone recipients at 3-year after first infusion ( insulin independence 2 weeks ) while 29% were insulin dependent with detectable c - peptide , 26% had lost function , and 22% had missing data . furthermore , 70% comprising all those with one or multiple infusions achieved insulin independence at least once , of whom 71% were still insulin dependent 1 year later and 52% at 2 years translating to 50% achieving and retaining insulin independence for 1 year and 35% for 2 years for all islet - alone recipients . these results are consistent throughout the 8 years of followup included in the registry . a dramatic decrease in the prevalence of hypoglycemic episodes and substantial improvement in hba1c levels an increase from 2% preinfusion to 47%69% at year 1 post - last infusion was observed in islet - alone recipients category with hba1c less than 6.5% and absence of severe hypoglycemic episodes . factors favoring the positive primary outcomes included amongst others a higher number of infusions , greater number of total islet equivalents infused , lower pretransplant hba1c levels , related processing / infusion centers , islet viability more than 87% , larger islet size , and the use of protocols with daclizumab , etanercept or calcineurin inhibitors in the immunosuppressive regimens . in fact , while successful islet transplantation outcomes have also been reported using islets isolated from a single donor [ 17 , 18 ] , matsumoto et al . were the first to perform successful living donor islet transplantation to a recipient having brittle insulin - dependent diabetes with hypoglycemic unawareness . results obtained from recipients of autologous islet transplantation after pancreatectomy at the university of minnesotta demonstrated a remarkably limited rate of decline of insulin independence despite infusion of a lower -cell mass . of 173 islet autotransplant recipients , insulin independence was achieved in 32% within the first year . of those with insulin independence , 74% remained insulin independent at the 2-year followup , 46% at 5 years and these results are remarkably higher than the citr data and highlight the strong impact of autoimmune and alloimmune injury on graft survival in type 1 diabetics receiving an infusion of allogeneic islets [ 16 , 20 , 21 ] . the edmonton protocol can also be applied to patients undergoing either islet after kidney ( iak ) or simultaneous islet kidney ( sik ) procedures resulting in a high rate of graft function and insulin independence , although the morbidity is higher when compared to patients undergoing solitary islet transplantation for brittle t1d . in fact , an improvement in cardiovascular function for up to 3 years of follow - up was observed in type 1 diabetic patients with end - stage renal disease ( esrd ) receiving an iak transplant compared with the kidney - only group . also improvements in kidney graft function , survival rates , and stabilization of microalbuminuria among uremic patients as well as an improvement in vascular diabetic complications were observed following iak transplantations . currently , several phase i and phase ii clinical trials to study iak in t1d are ongoing ( clinicaltrials.gov identifier : nct00708604 ; nct00888628 ; nct01123187 ; nct00468117 ) . although restoration of endocrine function and glucose homeostasis can be achieved by transplantation of either whole pancreas or islets of langerhans , each procedure has distinct advantages over the other . while whole pancreas transplantation has been shown to be very effective at maintaining an euglycemic state over a sustained period of time providing the graft recipient an opportunity to benefit from the improvement of blood glucose control , it is associated with a significant risk of surgical and postoperative complications . islet transplantation on the other hand , offers substantial benefits in terms of being minimally invasive , reducing incidences of debilitating hypoglycemic episodes / hypoglycemic unawareness , lowering daily insulin requirements , improving levels of glycated hemoglobin , and affording potential insulin independence [ 2628 ] . furthermore , it allows a window of opportunity for attempting various therapeutic manipulations of islets prior to transplantation aimed at achieving superior transplant outcomes . however , despite the impressive advances in this field , a limiting supply of islets , inadequate means for preventing islet rejection and the deleterious diabetogenic and nephrotoxic side effects associated with chronic immunosuppressive therapy preclude its wide - spread applicability . also , long - term insulin independence in islet transplant recipients is frequently lost by the fifth year of followup . nonetheless , the overall positive impact of islet transplantation on metabolic control in t1d continues to spur efforts worldwide to develop new strategies directed at achieving long - lasting insulin independence following islet transplantation . current challenges to successful transplantation include amongst others ( a ) the loss of functional islet mass resulting from nonspecific inflammatory responses as well as from preexisting and/or transplant - induced , autoimmune - mediated islet destruction and alloimmune rejection and ( b ) the failure of the newly transplanted islets to revascularize successfully resulting in inefficient engraftment and primary dysfunction of islets . herein , we will review some of the various preclinical and clinical efforts to protect islet grafts from the deleterious effects of innate and adaptive allo- and autospecific immune responses as well as to promote vasculogenesis , thereby increasing longitudinal graft survival and function . immunomodulatory therapies ( e.g. , monoclonal antibody therapies , costimulatory signaling blockade , il-1 receptor antagonist therapy , cellular therapies , etc . ) act by ( a ) providing immunoregulatory cytokines such as il-4 , il-10 , or tgf- or ( b ) by inhibiting proinflammatory cytokines or ( c ) by altering the balance between th1 and th2 cells or ( d ) by protecting islets from immune destruction by encapsulation and so forth . immunosuppressive regimens on the other hand act by either ( a ) suppressing the innate and adaptive immune response through various strategies that include binding to specific cytoplasmic proteins that inhibit il-2 secretion and subsequent t - cell expansion ( cnis cyclosporine and tacrolimus ) or ( b ) by suppressing il-2r signaling thereby inactivating t cells ( sirolimus ) or ( c ) by inhibiting cell division of lymphocytes ( azathioprine ) . currently , combinatorial therapies that promote vasculogenesis / angiogenesis of islets and islet regeneration/-cell expansion and that target antigen specific / nonspecific and antibody - specific immunomodulations that readjust the underlying immunologic imbalance to stop / reverse the -cell - specific immune destruction and maintain immune tolerance together with steroid - sparing or -free immunosuppressive regimens lacking nephrotoxicity or diabetogenicity are of immense therapeutic value towards generating successful islet transplantation outcomes . one of the first inflammatory assaults encountered by the islets on contact with abo - compatible blood following transplantation via hepatic portal vein is the instant blood - mediated inflammatory reaction ( ibmir ) [ 31 , 32 ] . ibmir is a thrombotic reaction characterized by activation of both the coagulation and complement cascades that results in the disruption of islet morphology and function , posing a serious obstacle to successful islet engraftment . activation of the complement and the generation of anaphylatoxins c3a and c5a leads to recruitment of polymorphonuclear ( pmn ) leukocytes , upregulation of adhesion molecules on the endothelium and platelets , generation of ros , and induction of cytokine release [ 33 , 34 ] . islets themselves secrete both tissue factors ( tfs ) that trigger ibmir and chemokines including monocyte chemoattractant protein 1 ( mcp1 ) , chemokine c - x - c motif ligand 8(cxcl8 ) , and chemokine c - c motif ligand 2 ( ccl2 ) that might participate in recruitment of pmns and monocytes / macrophages to the site of engraftment [ 3639 ] . in fact , a significant association between tf and ccl2 released in vitro by islets and biochemical indices of coagulation was observed in patients after islet transplantation indicating that reduction of the islet proinflammatory state may be a means to reduce the early posttransplant complications and possibly improve islet engraftment . to this end , strategies attempted for improving -cell graft survival include the use of optiprep gradient media during purification of human islets . this media has been shown to significantly reduce cytokine / chemokine production including il-1 , tnf- , ifn- , il-6 , il-8 , mip-1 , mcp-1 , and rantes during the 48-hour culture after isolation thereby improving -cell survival during pretransplant culture . similarly , embedding mouse islets in reconstituted basement membrane extract ( bme ) for 24 or 48 hours partially protected islets from anoikis by decreasing caspase-3 levels and increasing 3 integrin , focal adhesion kinase ( fak ) protein level , and fak activity suggesting a beneficial role in preservation of viability and function of isolated islets . furthermore , expression of transcription factor pancreatic and duodenal homeobox 1 ( pdx-1 ) was preserved at 48 hours indicating a positive contribution of bme to cell activity . pioneering work has been done by the group of nilsson and korsgren in developing clinically applicable strategies to inhibit the activation of the coagulation and complement systems and the infiltration of leukocytes into islets that characterize the ibmir . for instance , they demonstrated the effectiveness of low mw - dextran sulphate in inhibiting ibmir in a nonhuman primate model of pancreatic islet transplantation , providing the basis for a protocol that can be used to abrogate the ibmir in pig - human clinical islet xenotransplantation . as a result of their studies , a clinical trial to assess the safety and efficacy of lmw - sulfated dextran on posttransplant islet function in people with t1d is ongoing ( clinicaltrials.gov identifier : nct00790439 ) . they also developed a biotin / avidin technique to conjugate preformed heparin complexes to the surface of islets that protected against intraportal thrombosis and inhibited the ibmir without increasing the bleeding risk or inducing acute or chronic toxicity in the islets . on a similar principle , they demonstrated that human aortic endothelial cell- ( ec- ) coated islets reduced activation of coagulation and complement , platelet / leukocyte consumption in blood as well as infiltration of cd11b+ cells [ 42 , 43 ] . furthermore , addition of mesenchymal stem cells ( mscs ) to this composite indicated enhanced ec proliferation , sprout formation , and ingrowth of ecs into the islets demonstrating the clinical potential of composite ec - msc islets in improving islet angiogenesis , revascularization , and immune regulation . optimizing the ec - islet coculture methods to enable human ec coating of pig islets would offer new strategies to improve xenogenic islet transplantation outcomes . recently , immobilization of human soluble complement receptor 1 ( scr1 ) on the islet cell surface through poly(ethylene glycol)-conjugated phospholipid ( peg - lipid ) was shown to effectively inhibit complement activation and protect islets against attack by xenoreactive antibodies and complement without loss of islet cell viability or insulin secretion ability , demonstrating an efficient means to control early islet loss in clinical islet transplantation . coating islet surfaces with peg - lipid followed by further modification with a fibrinolytic enzyme urokinase further increased the efficacy of this procedure [ 46 , 47 ] . these reports highlight the potential of tissue - targeted chemistry to reduce donor - cell - mediated procoagulant and proinflammatory responses . the anaphylatoxin c5a elicits a broad range of proinflammatory effects and most likely plays a crucial role in ibmir . in a recent mouse intraportal islet transplant study , satomi 's group demonstrated that c5a inhibitory peptide ( c5aip ) significantly suppressed the thrombin - antithrombin complex formation , improving both curative rate and glucose tolerance as well as significantly suppressing the expression of tf on granulocytes in recipient livers . suppression of tf expression attenuated cross - talk between the complement and coagulation cascades leading to improvement in islet engraftment indicating that c5aip therapy in combination with conventional anticoagulants could represent a beneficial intervention in clinical islet transplantation . similar reduction in the expression of tf and mcp-1 in human islets was demonstrated by the addition of nicotinamide to the culture medium in an in vitro loop model . surface reengineering of pancreatic islets with recombinant azido - thrombomodulin also resulted in a significant reduction in islet - mediated thrombogenicity . nilsson 's group had also demonstrated that administration of a specific thrombin inhibitor melagatran significantly reduced ibmir in vitro in a dose - dependent manner as did scr1/heparin therapy [ 52 , 53 ] or tp10/immune - suppressive drug treatment following intraportal transplantation of porcine islets to cynomlogus monkeys . other anticoagulant or complement inhibitors to block ibmir in preclinical studies include n - acetyl l - cysteine that efficiently inhibited the procoagulant activity of human recombinant tf in human islet cell preparations at clinically relevant concentrations without cellular toxicity . fetal and neonatal porcine islets express a cell surface antigen containing the epitope gal13gal14glcnac - r ( -gal ) to which humans have complement fixing antibodies that cause immediate rejection of transplanted islets [ 5659 ] . this response may be prevented by ( a ) inhibition of the functional -gal activity using knockout or knockin procedures involving human alpha ( 1,2)-fucosyltransferase or n - acetyl - glucosaminyl transferase iii gene expression or ( b ) by depletion of anti - pig antibodies or complement from serum or ( c ) the use of gt knockout ( 1,3-galactosyltransferase gene knockout [ gtko ] ) pigs as the sources of islets . expression of thrombomodulin , tf pathway inhibitor , cd39 the endothelial ecto - nucleoside triphosphate diphosphohydrolase , and haem oxygenase also demonstrate potential value for coagulation control in pig - to - human xenotransplantation . prevention of coagulation dysfunction or expression of human complement - regulatory proteins such as cd46 ( membrane cofactor protein ) [ 67 , 68 ] , human decay accelerating factor ( daf , cd55 ) [ 64 , 69 ] , cd59 [ 7072 ] or all three ; or expression of anticoagulant and antiplatelet molecules within the graft may also afford some protection . for instance , adenovirus - mediated expression of the human complement regulatory proteins daf ( cd55 ) or cd59 protected adult porcine islets from complement - mediated cell lysis by human serum as did a low - molecular mass factor viia ( fviia ) inhibitor that indirectly blocked both membrane bound and alternatively spliced forms of tf in vitro . recombinant antithrombin iii may also ameliorate both early graft damage and the development of systemic coagulation disorders in pig - to - human xenotransplantation . these strategies in parallel with physical methods such as encapsulation may contribute significantly in reducing the thrombogenicity of pig islet xenografts . several intravascular and extravascular devices involving encapsulation technology to overcome destruction of the graft by immune cells and large antibodies have been described . while intravascular macrocapsules are anastomosed to the vascular system as av shunt , extravascular macrocapsules are mostly diffusion chambers transplanted at different sites and extravascular microcapsules are transplanted in the peritoneal cavity . the major advantage of the intravascular device is that direct contact with the blood stream ensures ample oxygen and nutrient supply enhancing graft survival and function . use of vascularized bioartificial devices was among the few cases that obtained long - term allo- and xenogeneic islet survival in totally pancreatectomized dogs [ 77 , 78 ] . however , thrombosis is a major challenge , requiring intense anticoagulation therapy . the extravascular devices on the other hand offer minimal surgical risk , transplantation at extrahepatic sites , lack of thrombosis , and ease of retrieval from recipient in case of pericapsular fibrotic overgrowth . both these devices however remain vulnerable to small molecules such as cytokines / chemokines and nitric oxide as well as hypoxic stress . coencapsulation of islets with various agents such as erythrocytes and sertoli cells that release immunosuppressive factors or with factors that enhance revascularization such as vascular endothelial growth factor ( vegf ) , photosynthetic oxygen generator algae and so forth or with factors like bioengineered insulin - like growth factor - ii ( igf - ii ) that promote pancreatic -cell survival have been shown to improve insulin secretion and glycemic control posttransplantation . naturally obtained alginate hydrogels are most widely used in islet transplantation with barium alginate demonstrating long - term immunoprotection in both allo- and xenotransplantation settings . for instance , a significant decrease in the secretion of mcp1 and improvement in the islet activity was observed when sd rat islets were microencapsulated with novel sulfated glucomannan barium alginate ( sga ) and allotransplanted intraperitoneally into diabetic lewis rats . similar immunoprotection was observed when islets co - encapsulated with angiogenic protein in permselective multilayer alginate - poly - l - ornithine - alginate microcapsules were transplanted into an omentum pouch . device , consisting of islets immobilized in a thin alginate - impregnated , hydrophilized teflon membrane and simultaneously supplied with oxygen by daily refueling with oxygen - co2 mixture provided immunoprotection and sustained islet viability and function following allogeneic subcutaneous transplantation into healthy minipigs . transplantation of porcine islets microencapsulated in highly purified barium alginate into streptozotocin- ( stz- ) induced diabetic wistar rats resulted in long - term normoglycemia without immunosuppression . thus , by providing a solution to the hurdles of immunological rejection and the risk of infection with porcine pathogens in clinical xeno - islet transplantation , the use of encapsulation devices may help to circumvent the shortage of allogeneic human donor organs . alginate macroencapsulation of pig islets was also shown to correct stz - induced diabetes in primates up to 6 months without immunosuppression . in a pilot clinical trial wherein microencapsulated human islets were transplanted into the central abdominal region of non - immunosuppressed patients with t1d , improvement in glycated hemoglobin and the disappearance of hypoglycemia was observed , although the patients remained on insulin therapy . in a very recent clinical study , elliot et al . demonstrated transitory insulin independence of several months duration using the microencapsulation technique . interestingly , the treatment appeared to significantly decrease severe hypoglycemic episodes and reduce / abolish hypoglycemic unawareness episodes , even in the absence of insulin independence . approximately 60% of transplanted pancreatic islet tissue undergoes apoptosis within the first several days in experimental models of syngeneic islet transplantation [ 13 , 91 , 92 ] . infiltration of graft by pmns and tissue macrophages and elevated levels of proinflammatory cytokines following islet transplantation strongly suggest the involvement of nonspecific innate inflammatory events in mediating cellular injury . other mediators involved in the early loss of pancreatic islets include no , prostaglandins ( pgs ) , and reactive oxygen intermediates ( rois ) [ 92 , 93 ] . several studies indicate that inhibition of non specific inflammation improves the function and survival of islet grafts . for instance , yasunami 's group demonstrated that v - alpha14 nkt cell - triggered ifn- production by gr-1+cd11b+ cells mediated early graft loss of syngeneic transplanted islets . adenosine administration suppressed nkt cell - mediated ifn- production of neutrophils in the livers of graft - recipient mice , leading to prevention of early loss of transplanted syngeneic and allogeneic islets . similar results were observed in a recent study wherein recipients receiving atl therapy ( atl146e or atl313 ) achieved normoglycemia more rapidly than untreated recipients following syngeneic islet transplantation indicating improved survival and functional engraftment of transplanted marginal mass of islets . histological examination of grafts suggested reduced cellular necrosis , fibrosis , and lymphocyte infiltration in agonist - treated animals . administration of adenosine a(2a ) receptor agonists also improved in vitro glucose - stimulated insulin secretion ( gsis ) by an effect on leukocytes , suggesting a potentially significant interventional strategy for reducing inflammatory islet loss in clinical transplantation . a similar beneficial effect associated with reduction in inflammatory cell infiltration and -cell death by apoptosis sequential combination of a jnk inhibitor sp600125 and nicotinamide plus simvastatin also protected porcine islets from peritransplant apoptosis and inflammation . this combination therapy increased -cell viability index and viability of porcine islets cultured overnight as well as significantly increased the islet survival rate in vivo . intraductal administration of jnk inhibitor significantly suppressed mrna expression levels of il-1 , ifn- , tnf- , il-6 , il-8 , and mcp-1 in vivo and also decreased the concentration of il-1 and il-8 in culture supernatant in vitro possibly representing an alternative target for suppression of porcine islet inflammation . very recently , administration of bilirubin too was shown to reduce the serum levels of inflammatory mediators including il-1 , tnf- , soluble intercellular adhesion molecule 1 ( icam1 ) , mcp-1 and no , inhibit the infiltration of kupffer cells into islet grafts , restore insulin - producing ability of transplanted islets and enhance glucose tolerance in diabetic mouse recipients . il-1 plays a key role in causing pancreatic islet dysfunction and apoptosis . through a cascade of intracellular events , il-1 secreted by neutrophils and macrophages was shown to down - regulate glut2 , up - regulate inducible no synthase ( inos ) and cyclooxygenase-2 , and activate nf-b which in turn mediated the transcription of a multitude of genes , including il-1 , il-6 , tnf- , icam-1 , vcam-1 , elam-1 , inos , pge2 , cox-2 , and ep3 [ 92 , 9799 ] . the inhibition of il-1 induced cox-2 and ep3 gene expression by sodium salicylate as well as of inos and cox-2 tyrosine kinase were all shown to enhance pancreatic isle -cell function . also deficiency of nox2 decreased roi / proinflammatory cytokine production and -cell apoptosis , thereby protecting against stz - induced -cell destruction and development of diabetes in mice . inhibition of toll - like receptor ( tlr ) signalinga family of 10 functional , human , innate , immune signaling receptors called tlrs plays an important role in the host 's innate defense as well as adaptive immunity . recognition of conserved pathogen - associated microbial patterns ( prrs ) stimulates specific tlrs on apcs and t lymphocytes to induce proinflammatory cytokines , chemokines , and interferons . the endogenous tlr ligands include amongst others peptidoglycan , extradomain a of fibronectin , lipopolysaccharide , heparan sulfate , hyaluronan , high - mobility group box protein 1 ( hmgb1 ) , hsp60 , exogenous products such as bacterial lipoprotein , cpg dna , flagellin , and double stranded viral rna [ 102105 ] . murine islets constitutively express tlr2 and tlr4 , and tlr activation upregulates intra - islet production of cytokines and chemokines . a recent study indicated that tlr2 and tlr4 signaling could initiate islet graft failure and that hmgb1 was an early mediator . following transplantation into stz - induced diabetic syngeneic mice , islets exposed to lps or peptidoglycan had primary graft failure caused by recipient cd8 + t cells with intra- and peri - islet mononuclear cell inflammation . nfb activation in stressed islets was prevented in the absence of both tlr2 and tlr4 . yet another study indicated that early intraportal islet graft failure in mice was associated with increased proinflammatory cytokines , hmgb1 expression , nfb activation , caspase-3 , and tunel - positive cells . deficiency of tlr4 in donor , but not in recipient , inhibited nfb activation , reduced proinflammatory cytokines and improved viability of islet grafts . blockade of hmgb1 with anti - hmgb1 monoclonal antibody ( mab , 2 g7 ) inhibited inflammatory reactions as evidenced by reduced tnf and il-1ss production and improved islet viability . thus , these results indicate that inhibition of tlr4 activation represents a novel strategy to attenuate early graft failure following intraportal islet transplantation . receptors tlr2 or receptor for advanced glycation end products ( rage ) but not tlr4 , failed to exhibit early islet graft loss . mechanistically , hmgb1 stimulated hepatic mononuclear cells ( mncs ) in vivo and in vitro , upregulated cd40 expression and enhanced il-12 production by dcs , leading to nkt cell activation and subsequent nkt cell - dependent augmented ifn- production by gr-1+cd11b+ cells . treatment with either il12- or cd40l - specific antibodies prevented early islet graft loss . also , treatment with a hmgb1-specific antibody inhibited ifn- production by nkt cells and gr-1+cd11b+ cells following intraportal islet transplantation , indicating that the hmgb1-mediated pathway was a potential interventional target for improving the efficiency of islet transplantation . a family of 10 functional , human , innate , immune signaling receptors called tlrs plays an important role in the host 's innate defense as well as adaptive immunity . recognition of conserved pathogen - associated microbial patterns ( prrs ) stimulates specific tlrs on apcs and t lymphocytes to induce proinflammatory cytokines , chemokines , and interferons . the endogenous tlr ligands include amongst others peptidoglycan , extradomain a of fibronectin , lipopolysaccharide , heparan sulfate , hyaluronan , high - mobility group box protein 1 ( hmgb1 ) , hsp60 , exogenous products such as bacterial lipoprotein , cpg dna , flagellin , and double stranded viral rna [ 102105 ] . murine islets constitutively express tlr2 and tlr4 , and tlr activation upregulates intra - islet production of cytokines and chemokines . a recent study indicated that tlr2 and tlr4 signaling could initiate islet graft failure and that hmgb1 was an early mediator . following transplantation into stz - induced diabetic syngeneic mice , islets exposed to lps or peptidoglycan had primary graft failure caused by recipient cd8 + t cells with intra- and peri - islet mononuclear cell inflammation . nfb activation in stressed islets was prevented in the absence of both tlr2 and tlr4 . yet another study indicated that early intraportal islet graft failure in mice was associated with increased proinflammatory cytokines , hmgb1 expression , nfb activation , caspase-3 , and tunel - positive cells . deficiency of tlr4 in donor , but not in recipient , inhibited nfb activation , reduced proinflammatory cytokines and improved viability of islet grafts . blockade of hmgb1 with anti - hmgb1 monoclonal antibody ( mab , 2 g7 ) inhibited inflammatory reactions as evidenced by reduced tnf and il-1ss production and improved islet viability . thus , these results indicate that inhibition of tlr4 activation represents a novel strategy to attenuate early graft failure following intraportal islet transplantation . receptors tlr2 or receptor for advanced glycation end products ( rage ) but not tlr4 , failed to exhibit early islet graft loss . mechanistically , hmgb1 stimulated hepatic mononuclear cells ( mncs ) in vivo and in vitro , upregulated cd40 expression and enhanced il-12 production by dcs , leading to nkt cell activation and subsequent nkt cell - dependent augmented ifn- production by gr-1+cd11b+ cells . treatment with either il12- or cd40l - specific antibodies prevented early islet graft loss . also , treatment with a hmgb1-specific antibody inhibited ifn- production by nkt cells and gr-1+cd11b+ cells following intraportal islet transplantation , indicating that the hmgb1-mediated pathway was a potential interventional target for improving the efficiency of islet transplantation . protection by suppressor of cytokine signaling ( socs)the protective effect of suppressor of cytokine signalling ( socs)-3 in mouse and rat islets subjected to cytokine stimulation has been characterized by rnn et al . . using mice with -cell - specific socs3 expression as well as a socs3-encoding adenovirus construct , a significant resistance to cytokine - induced apoptosis and impaired insulin release was demonstrated by the transgenic islets . rat islet cultures transduced with socs3-adenovirus too displayed reduced cytokine - induced no and apoptosis associated with inhibition of the il1-induced nfb and mitogen - activated protein kinase ( mapk ) pathways . while transplanted socs3 transgenic islets were not protected in diabetic nod mice , they did show a prolonged graft survival when transplanted into diabetic allogeneic balb / c mice indicating that socs3 may represent a target for pharmacological or genetic engineering in islet transplantation for treatment of t1d . a more recent study using chimeric adenovirus vector ( ad5f35-socs1 ) to enhance socs1 expression in isolated sprague - dawley rat islets indicated decreased levels of active caspase 3 and intranuclear apoptosis inducing factor ( aif ) after treatment with tnf- and cyclohexamide in vitro . caspase 3 is the central executioner caspase activated by upstream cascades in a caspase - dependent apoptosis pathway while aif is a key mitochondrial protein that translocates to the nucleus in a caspase - independent apoptosis pathway . transplantation of ad5f35-socs1-infected islets into stz - induced diabetic recipients resulted in significantly prolonged functional graft survival . also , decreased caspase 3 activation and aif translocation to nucleus was observed in ad5f35-socs1-infected islet grafts in the early posttransplant period indicating that socs1 mediated protection of islet grafts from apoptosis through caspase-3-dependent and aif - caspase - independent pathways . these results were supported by another study in which transgene expression of socs-1 rendered islets significantly more resistant to cytokine - induced cell death after treatment with tnf- alone and in combination with ifn- . furthermore this resistance could be correlated with significant inhibition of the transcription factor interferon regulatory factor-1 ( irf-1 ) , reflecting enhanced cell survival signals as well as impairment of ifn-induced class i mhc upregulation [ 93 , 111 ] . importantly , socs1-tg islets significantly reversed stz - induced diabetes indicating that intragraft expression of socs1 rendered islets insensitive to the deleterious effects of cytokines ; a finding of potential significance in the development of therapies against acute allograft rejection . the protective effect of suppressor of cytokine signalling ( socs)-3 in mouse and rat islets subjected to cytokine stimulation has been characterized by rnn et al . . using mice with -cell - specific socs3 expression as well as a socs3-encoding adenovirus construct , a significant resistance to cytokine - induced apoptosis and impaired insulin release was demonstrated by the transgenic islets . rat islet cultures transduced with socs3-adenovirus too displayed reduced cytokine - induced no and apoptosis associated with inhibition of the il1-induced nfb and mitogen - activated protein kinase ( mapk ) pathways . while transplanted socs3 transgenic islets were not protected in diabetic nod mice , they did show a prolonged graft survival when transplanted into diabetic allogeneic balb / c mice indicating that socs3 may represent a target for pharmacological or genetic engineering in islet transplantation for treatment of t1d . a more recent study using chimeric adenovirus vector ( ad5f35-socs1 ) to enhance socs1 expression in isolated sprague - dawley rat islets indicated decreased levels of active caspase 3 and intranuclear apoptosis inducing factor ( aif ) after treatment with tnf- and cyclohexamide in vitro . caspase 3 is the central executioner caspase activated by upstream cascades in a caspase - dependent apoptosis pathway while aif is a key mitochondrial protein that translocates to the nucleus in a caspase - independent apoptosis pathway . transplantation of ad5f35-socs1-infected islets into stz - induced diabetic recipients resulted in significantly prolonged functional graft survival . also , decreased caspase 3 activation and aif translocation to nucleus was observed in ad5f35-socs1-infected islet grafts in the early posttransplant period indicating that socs1 mediated protection of islet grafts from apoptosis through caspase-3-dependent and aif - caspase - independent pathways . these results were supported by another study in which transgene expression of socs-1 rendered islets significantly more resistant to cytokine - induced cell death after treatment with tnf- alone and in combination with ifn- . furthermore this resistance could be correlated with significant inhibition of the transcription factor interferon regulatory factor-1 ( irf-1 ) , reflecting enhanced cell survival signals as well as impairment of ifn-induced class i mhc upregulation [ 93 , 111 ] . importantly , socs1-tg islets significantly reversed stz - induced diabetes indicating that intragraft expression of socs1 rendered islets insensitive to the deleterious effects of cytokines ; a finding of potential significance in the development of therapies against acute allograft rejection . protection by x - linked inhibitor of apoptosis proteins ( xiaps)adenovirally delivered , transient overexpression of x - linked inhibitor of apoptosis proteins ( xiaps ) in a growth - regulatable -cell line ( tc - tet ) indicated that in vitro , xiap - expressing tc - tet cells were markedly resistant to apoptosis in an ischemia - reperfusion injury model system as well as following exposure to cytokines . subcutaneous transplantation of these ad - xiap transduced tc - tet cells into immunodeficient mice resulted in reversal of diabetes in 3 days as versus 21 days for ad-gal transduced control cells . these results were recapitulated in a recent study wherein xiap overexpression inhibited -cell apoptosis in syngeneic islet transplants , reducing the number of islets as well as decreasing the number of days required to restore euglycemia , thereby raising the possibility that ex vivo xiap gene transfer in islets prior to transplantation had the potential to increase donor islet mass available for transplantation by allowing more efficient use of the limited existing supply of human islets thereby enhancing graft function and long - term transplant success . in comparison with this adenoviral delivered gene transfer approach , a nonviral approach was recently described involving the transfection of ido cdna containing plasmids into rat islets using lipofectamine . indoleamine 2,3-dioxygenase ( ido ) is an enzyme that plays a critical role in suppressing t - cell responses inducing fetomaternal tolerance . allotransplantation of ido expressing islets into stz - induced diabetic lewis rats was shown to reverse diabetes and maintain glucose metabolic control . also , survival of ido - transfected islet allografts transplanted without any immunosuppression was superior to that observed in diabetic rats receiving nontransfected islets . adenovirally delivered , transient overexpression of x - linked inhibitor of apoptosis proteins ( xiaps ) in a growth - regulatable -cell line ( tc - tet ) indicated that in vitro , xiap - expressing tc - tet cells were markedly resistant to apoptosis in an ischemia - reperfusion injury model system as well as following exposure to cytokines . subcutaneous transplantation of these ad - xiap transduced tc - tet cells into immunodeficient mice resulted in reversal of diabetes in 3 days as versus 21 days for ad-gal transduced control cells . these results were recapitulated in a recent study wherein xiap overexpression inhibited -cell apoptosis in syngeneic islet transplants , reducing the number of islets as well as decreasing the number of days required to restore euglycemia , thereby raising the possibility that ex vivo xiap gene transfer in islets prior to transplantation had the potential to increase donor islet mass available for transplantation by allowing more efficient use of the limited existing supply of human islets thereby enhancing graft function and long - term transplant success . in comparison with this adenoviral delivered gene transfer approach , a nonviral approach was recently described involving the transfection of ido cdna containing plasmids into rat islets using lipofectamine . indoleamine 2,3-dioxygenase ( ido ) is an enzyme that plays a critical role in suppressing t - cell responses inducing fetomaternal tolerance . allotransplantation of ido expressing islets into stz - induced diabetic lewis rats was shown to reverse diabetes and maintain glucose metabolic control . also , survival of ido - transfected islet allografts transplanted without any immunosuppression was superior to that observed in diabetic rats receiving nontransfected islets . protective effect of haem oxygenase-1 ( ho-1 , hmox1)overexpression of the cytoprotective protein haem oxygenase-1 ( ho-1 ) has been shown to reduce the deleterious effects of cytokine - induced apoptosis and oxidative stress in transplantable islets [ 115118 ] . for instance , ho-1 upregulation by protoporphyrins ( cobalt - protoporphyrin copp , ferrous - protoporphyrin fepp ) , powerful inducers of the ho-1 , in a murine insulinomae -tc3 cell line and in freshly isolated murine islets was shown to exert a protective effect from apoptosis induced in vitro with proinflammatory cytokines [ 115 , 116 ] or fas engagement , while in vivo ho-1 upregulation resulted in improved islet function in a syngeneic model of marginal mass islet transplantation in rodents . these results were supported by a similar study in which copp treatment of donor mice for the induction of ho-1 beginning one - day prior to islet isolation plus a 9-day posttransplantation course resulted in enhanced engraftment of syngeneic islets and improved blood glucose levels and glycemic control . short - course peritransplant administration of copp also led to long - term dba/2 islet allograft survival in a sizable proportion of c57bl/6 mice recipients . furthermore , preconditioning of islets with fepp alone led to improved graft survival in untreated recipients and further increased the proportion of long - term surviving grafts in copp - treated recipients . peritransplant administration of protoporphyrins to allograft recipients also resulted in transient powerful immunosuppression with reduced lymphocyte proliferative responses , increased proportion of regulatory t cells ( cd4+cd25 + ) and decreased mononuclear cell infiltrating the graft , paralleled by a systemic upregulation of ho-1 expression , probably contributing to the induction of donor - specific hyporesponsiveness in a proportion of the protoporphyrin - treated animals . the transgenic expression of haem oxygenase-1 in pancreatic -cells of nod mice prolonged graft survival and afforded protection from autoimmune damage . reduced levels of proinflammatory cytokines / chemokines , proapoptotic gene expression , and amounts of ros / rns from islets were observed , with islets more resistant to tnf- and ifn-induced apoptosis , providing valuable insight into the development of better strategies for clinical islet transplantation in patients with t1d . overexpression of the cytoprotective protein haem oxygenase-1 ( ho-1 ) has been shown to reduce the deleterious effects of cytokine - induced apoptosis and oxidative stress in transplantable islets [ 115118 ] . for instance , ho-1 upregulation by protoporphyrins ( cobalt - protoporphyrin copp , ferrous - protoporphyrin fepp ) , powerful inducers of the ho-1 , in a murine insulinomae -tc3 cell line and in freshly isolated murine islets was shown to exert a protective effect from apoptosis induced in vitro with proinflammatory cytokines [ 115 , 116 ] or fas engagement , while in vivo ho-1 upregulation resulted in improved islet function in a syngeneic model of marginal mass islet transplantation in rodents . these results were supported by a similar study in which copp treatment of donor mice for the induction of ho-1 beginning one - day prior to islet isolation plus a 9-day posttransplantation course resulted in enhanced engraftment of syngeneic islets and improved blood glucose levels and glycemic control . short - course peritransplant administration of copp also led to long - term dba/2 islet allograft survival in a sizable proportion of c57bl/6 mice recipients . furthermore , preconditioning of islets with fepp alone led to improved graft survival in untreated recipients and further increased the proportion of long - term surviving grafts in copp - treated recipients . peritransplant administration of protoporphyrins to allograft recipients also resulted in transient powerful immunosuppression with reduced lymphocyte proliferative responses , increased proportion of regulatory t cells ( cd4+cd25 + ) and decreased mononuclear cell infiltrating the graft , paralleled by a systemic upregulation of ho-1 expression , probably contributing to the induction of donor - specific hyporesponsiveness in a proportion of the protoporphyrin - treated animals . the transgenic expression of haem oxygenase-1 in pancreatic -cells of nod mice prolonged graft survival and afforded protection from autoimmune damage . reduced levels of proinflammatory cytokines / chemokines , proapoptotic gene expression , and amounts of ros / rns from islets were observed , with islets more resistant to tnf- and ifn-induced apoptosis , providing valuable insight into the development of better strategies for clinical islet transplantation in patients with t1d . protective regulatory role of interferon regulatory factor 1 ( irf1)a key role of irf1 in immune - mediated -cell destruction has been indicated . deletion of irf1 in islets was associated with a higher prevalence of primary nonfunction , reduced insulin secretion and shorter functioning graft survival . cytokine - exposed irf1 ( / ) islets and ins1e cells transfected with irf1 sirna showed increased expression of mcp1 ( ccl2 ) , ip10 ( cxcl10 ) , mip3 ( ccl20 ) , and inos ( nos2 ) mrna and elevated production of mcp-1 and nitrite compared with controls . in vivo , irf1 ( / ) islets displayed a higher potential to attract immune cells , reflected by more aggressive immune infiltration in the grafted islets . il-1 receptor antagonist partly restored the cytokine - induced secretory defect in vitro and completely prevented primary non function in vivo . these data indicate a key regulatory role for irf1 in insulin and chemokine secretion by pancreatic islets under inflammatory attack . a key role of irf1 in immune - mediated -cell destruction has been indicated . deletion of irf1 in islets was associated with a higher prevalence of primary nonfunction , reduced insulin secretion and shorter functioning graft survival . cytokine - exposed irf1 ( / ) islets and ins1e cells transfected with irf1 sirna showed increased expression of mcp1 ( ccl2 ) , ip10 ( cxcl10 ) , mip3 ( ccl20 ) , and inos ( nos2 ) mrna and elevated production of mcp-1 and nitrite compared with controls . in vivo , irf1 ( / ) islets displayed a higher potential to attract immune cells , reflected by more aggressive immune infiltration in the grafted islets . il-1 receptor antagonist partly restored the cytokine - induced secretory defect in vitro and completely prevented primary non function in vivo . these data indicate a key regulatory role for irf1 in insulin and chemokine secretion by pancreatic islets under inflammatory attack . beneficial effect of redox modulation strategies -cells are especially vulnerable to free radical and inflammatory damage due to reduced antioxidant defenses . a recent study was done to determine the efficacy and benefit of a redox modulation strategy using the catalytic antioxidant ( ca ) fbc-007 in improving islet preservation . the results indicated that incubation of human islets with fbc-007 before syngeneic , suboptimal syngeneic , or xenogeneic transplant protected islets from stz - induced damage and significantly increased their function . diabetic murine recipients of catalytic antioxidant - treated allogeneic islets exhibited improved glycemic control posttransplant and demonstrated a delay in allograft rejection . systemic administration of catalytic antioxidant to recipients further delayed allograft rejection suggesting that addition of a redox modulation strategy would be a beneficial clinical approach for islet preservation in syngeneic , allogeneic , and xenogeneic transplantation . -cells are especially vulnerable to free radical and inflammatory damage due to reduced antioxidant defenses . a recent study was done to determine the efficacy and benefit of a redox modulation strategy using the catalytic antioxidant ( ca ) fbc-007 in improving islet preservation . the results indicated that incubation of human islets with fbc-007 before syngeneic , suboptimal syngeneic , or xenogeneic transplant protected islets from stz - induced damage and significantly increased their function . diabetic murine recipients of catalytic antioxidant - treated allogeneic islets exhibited improved glycemic control posttransplant and demonstrated a delay in allograft rejection . systemic administration of catalytic antioxidant to recipients further delayed allograft rejection suggesting that addition of a redox modulation strategy would be a beneficial clinical approach for islet preservation in syngeneic , allogeneic , and xenogeneic transplantation . presentation of alloantigens in combination with costimulatory molecules by apcs activates signal transduction pathways , including the calcium - calcineurin pathway , triggering the t - cell response . subsequent release of il-2 amongst other molecules , activates the mtor pathway initiating cell proliferation and hugely expanding the number of effector t cells . costimulatory signaling blockadeseveral studies indicate that blockade of costimulatory signal pathways in experimental transplantation models by cytotoxic t lymphocyte antigen-4 immunoglobulin ( ctla4ig ) or cd40lig enhances islet graft survival [ 123 , 124 ] . for instance , human islets infected with adctla4ig - ires2-cd40lig that simultaneously expressed ctla4ig and cd40lig proteins significantly prolonged graft survival of murine islet xenografts , downregulated expressions of th1-cells - related cytokines , and inhibited inflammatory cell infiltration . caspase inhibitor therapy ( ep1013 ) in combination with costimulation blockade ( ctla4-ig ) too prevented engraftment phase islet loss and markedly reduced islet mass required to reverse diabetes . furthermore , ep1013/clta4-ig cotherapy significantly increased graft survival by reducing the frequency of alloreactive ifn- secreting t cells and increasing the frequency of intragraft foxp3 + treg cells . the results of these studies indicate that by minimizing immune stimulation and reducing the requirement for long - term immunosuppressive therapy , these combination therapies have tremendous potential in improving clinical transplantation . simultaneous blockade of cd40/cd154 and icam / lymphocyte function - associated antigen ( lfa)-1 also prolonged allograft survival . larsen 's group targeted adhesion molecule lfa-1 that is preferentially expressed on donor - specific memory t ( tm ) cells and has been implicated in costimulation blockade - resistant transplant rejection . short - term induction therapy with the lfa-1-specific antibody ts-1/22 in combination with either basiliximab ( an il-2r-specific mab ) and sirolimus or belatacept ( a high - affinity variant of the cd28 costimulation - blocker ctla4ig ) prolonged islet allograft survival in nonhuman primates by masking lfa-1 on tm cells . inhibition of the generation of alloproliferative and cytokine - producing effector t cells expressing high levels of lfa-1 in vitro was also observed . neutralization of costimulation blockade - resistant populations of t cells with lfa-1-specific induction therapy has tremendous potential in transplantation . the efficacy of efalizumab , a blocking monoclonal antibody directed at lfa1 , as part of maintenance therapy regimen with sirolimus was being tested ( clinicaltrial.gov identifier nct00672204 ) . an anti - coinhibitory receptor b and t lymphocyte attenuator monoclonal antibody ( anti btla , pj196 ) has been reported to prolong fully mhc - mismatched cardiac allograft survival . a study testing the synergistic effect of anti - btla monoclonal antibody pj196 and ctla4ig costimulatory blockade in islet allotransplantation showed that downregulation of btla on the surface of lymphocytes along with accumulation of cells with regulatory phenotype at the graft site promoted islet allograft acceptance indicating that this combination may prove to be an effective adjunctive strategy for inducing long - term allograft survival . similarly , co - stimulatory signal blocker lea29y ( belatacept ) has shown promising data in transplantation studies in primates . clinical trials using lea29y in islet transplantation are currently underway , for example clinical islet transplantation consortium 's clinical trial cit-04 is designed to study islet transplantation in type 1 diabetes with lea29y ( belatacept ) maintenance therapy ( university of alberta , emory university ) and another ongoing phase ii interventional study ( clinicaltrials.gov identifier : nct00501709 ) is using belatacept for the prevention of autoimmune destruction and rejection of human pancreatic islets following transplantation for t1d . apart from costimulation blockade of the cd28/cd80/cd86 and cd40/cd154 pathways as a means of inducing peripheral tolerance , several new costimulatory molecules have been identified in recent years including ox40 that belongs to the tnf receptor family and is expressed on activated t cells . mechanistic studies indicate that anti - ox40l , treatment preserves treg numbers and ox40 blockade offers better xenoislet graft survival than ctla4ig in the spontaneous autoimmune nod model , offering a novel therapeutic target for xenoislet graft protection in type 1 diabetic patients . recently , a combination therapy consisting of anti - cd40l , anti - ox40l , and anti - cd122 mabs prolonged islet allograft survival in alloantigen - primed mice . this combination of mabs also inhibited accelerated rejection mediated by donor reactive memory t cells , known to accelerate allograft rejection , in xenoantigen - primed mice cells by inhibiting cellular and humoral immune responses . several studies indicate that blockade of costimulatory signal pathways in experimental transplantation models by cytotoxic t lymphocyte antigen-4 immunoglobulin ( ctla4ig ) or cd40lig enhances islet graft survival [ 123 , 124 ] . for instance , human islets infected with adctla4ig - ires2-cd40lig that simultaneously expressed ctla4ig and cd40lig proteins significantly prolonged graft survival of murine islet xenografts , downregulated expressions of th1-cells - related cytokines , and inhibited inflammatory cell infiltration . caspase inhibitor therapy ( ep1013 ) in combination with costimulation blockade ( ctla4-ig ) too prevented engraftment phase islet loss and markedly reduced islet mass required to reverse diabetes . furthermore , ep1013/clta4-ig cotherapy significantly increased graft survival by reducing the frequency of alloreactive ifn- secreting t cells and increasing the frequency of intragraft foxp3 + treg cells . the results of these studies indicate that by minimizing immune stimulation and reducing the requirement for long - term immunosuppressive therapy , these combination therapies have tremendous potential in improving clinical transplantation . simultaneous blockade of cd40/cd154 and icam / lymphocyte function - associated antigen ( lfa)-1 also prolonged allograft survival . larsen 's group targeted adhesion molecule lfa-1 that is preferentially expressed on donor - specific memory t ( tm ) cells and has been implicated in costimulation blockade - resistant transplant rejection . short - term induction therapy with the lfa-1-specific antibody ts-1/22 in combination with either basiliximab ( an il-2r-specific mab ) and sirolimus or belatacept ( a high - affinity variant of the cd28 costimulation - blocker ctla4ig ) prolonged islet allograft survival in nonhuman primates by masking lfa-1 on tm cells . inhibition of the generation of alloproliferative and cytokine - producing effector t cells expressing high levels of lfa-1 in vitro was also observed . neutralization of costimulation blockade - resistant populations of t cells with lfa-1-specific induction therapy has tremendous potential in transplantation . the efficacy of efalizumab , a blocking monoclonal antibody directed at lfa1 , as part of maintenance therapy regimen with sirolimus was being tested ( clinicaltrial.gov identifier nct00672204 ) . an anti - coinhibitory receptor b and t lymphocyte attenuator monoclonal antibody ( anti btla , pj196 ) has been reported to prolong fully mhc - mismatched cardiac allograft survival . a study testing the synergistic effect of anti - btla monoclonal antibody pj196 and ctla4ig costimulatory blockade in islet allotransplantation showed that downregulation of btla on the surface of lymphocytes along with accumulation of cells with regulatory phenotype at the graft site promoted islet allograft acceptance indicating that this combination may prove to be an effective adjunctive strategy for inducing long - term allograft survival . similarly , co - stimulatory signal blocker lea29y ( belatacept ) has shown promising data in transplantation studies in primates . clinical trials using lea29y in islet transplantation are currently underway , for example clinical islet transplantation consortium 's clinical trial cit-04 is designed to study islet transplantation in type 1 diabetes with lea29y ( belatacept ) maintenance therapy ( university of alberta , emory university ) and another ongoing phase ii interventional study ( clinicaltrials.gov identifier : nct00501709 ) is using belatacept for the prevention of autoimmune destruction and rejection of human pancreatic islets following transplantation for t1d . apart from costimulation blockade of the cd28/cd80/cd86 and cd40/cd154 pathways as a means of inducing peripheral tolerance , several new costimulatory molecules have been identified in recent years including ox40 that belongs to the tnf receptor family and is expressed on activated t cells . mechanistic studies indicate that anti - ox40l , treatment preserves treg numbers and ox40 blockade offers better xenoislet graft survival than ctla4ig in the spontaneous autoimmune nod model , offering a novel therapeutic target for xenoislet graft protection in type 1 diabetic patients . recently , a combination therapy consisting of anti - cd40l , anti - ox40l , and anti - cd122 mabs prolonged islet allograft survival in alloantigen - primed mice . this combination of mabs also inhibited accelerated rejection mediated by donor reactive memory t cells , known to accelerate allograft rejection , in xenoantigen - primed mice cells by inhibiting cellular and humoral immune responses . monoclonal antibodiesanti - cd3 antibody has been shown to induce tolerance in allograft transplantation , reverse autoimmunity in nod mice and slow the progression to permanent diabetes in humans with recent - onset diabetes . in female nod mice , oral anti - cd3 mab was effective in reversing diabetes , allowing pregnancies and extending longevity . treatment of diabetic transgenic mice ( nod background ) expressing the human chain of the cd3 complex with otelixizumab ( an anti - human cd3 antibody ) resulted in durable disease remission dependent on transferable t - cell - mediated tolerance . in single - donor islet transplantation studies , anti - cd3 mab [ hokt3c1(ala ala ) ] contributed to promising results . a phase ii clinical trial studying the efficacy of anti - cd3 mab in the treatment of recent onset t1d ( clinicaltrial.gov identifier nct00378508 ) is currently underway . another trial studying the efficacy of hokt31 ( ala - ala ) combined with sirolimus and delayed tacrolimus in promoting islet allograft survival has just been completed . in an adoptive transfer model , the use of anti - cd134l mab was shown to effectively prevent activation of cd4 + memory t cells and significantly prolong islet survival , similar to the manner anti - cd122 mab prevents activation of cd8 + memory t cells . short - term administrations of a combination of anti - lfa-1 and anti - cd154 monoclonal antibodies too induced tolerance to neonatal porcine islet xenografts in mice . the use of anti - cd134l and anti - cd122 mabs in addition to co - stimulatory blockade with anti - cd154 and anti - lfa-1 prolonged secondary allograft survival in an alloantigen - primed model and significantly reduced the proportion of memory t cells . it also increased the proportion of tregs in the spleen , inhibited lymphocyte infiltration in the graft , and suppressed alloresponse of recipient splenic t cells suggesting that the combination therapy of four mabs could significantly suppress the function of memory t cells and prolong allograft survival in alloantigen primed animals . similar results were obtained by the same group using a combination of ctla4ig , anticd40l , anti - lfa-1 , and anti - ox40l while another study used a combination of anti - cd40l , anti - ox40l , and anti - cd122 mabs antibodies to inhibit accelerated rejection mediated by memory t cells in xenoantigen - primed mice . anti - cd3 antibody has been shown to induce tolerance in allograft transplantation , reverse autoimmunity in nod mice and slow the progression to permanent diabetes in humans with recent - onset diabetes . in female nod mice , oral anti - cd3 mab was effective in reversing diabetes , allowing pregnancies and extending longevity . treatment of diabetic transgenic mice ( nod background ) expressing the human chain of the cd3 complex with otelixizumab ( an anti - human cd3 antibody ) resulted in durable disease remission dependent on transferable t - cell - mediated tolerance . in single - donor islet transplantation studies , anti - cd3 mab [ hokt3c1(ala ala ) ] contributed to promising results . a phase ii clinical trial studying the efficacy of anti - cd3 mab in the treatment of recent onset t1d ( clinicaltrial.gov identifier nct00378508 ) is currently underway . another trial studying the efficacy of hokt31 ( ala - ala ) combined with sirolimus and delayed tacrolimus in promoting islet allograft survival has just been completed . in an adoptive transfer model , the use of anti - cd134l mab was shown to effectively prevent activation of cd4 + memory t cells and significantly prolong islet survival , similar to the manner anti - cd122 mab prevents activation of cd8 + memory t cells . short - term administrations of a combination of anti - lfa-1 and anti - cd154 monoclonal antibodies too induced tolerance to neonatal porcine islet xenografts in mice . the use of anti - cd134l and anti - cd122 mabs in addition to co - stimulatory blockade with anti - cd154 and anti - lfa-1 prolonged secondary allograft survival in an alloantigen - primed model and significantly reduced the proportion of memory t cells . it also increased the proportion of tregs in the spleen , inhibited lymphocyte infiltration in the graft , and suppressed alloresponse of recipient splenic t cells suggesting that the combination therapy of four mabs could significantly suppress the function of memory t cells and prolong allograft survival in alloantigen primed animals . similar results were obtained by the same group using a combination of ctla4ig , anticd40l , anti - lfa-1 , and anti - ox40l while another study used a combination of anti - cd40l , anti - ox40l , and anti - cd122 mabs antibodies to inhibit accelerated rejection mediated by memory t cells in xenoantigen - primed mice . il-21-targeted therapythe efficacy of an il-21-targeted therapy ( il-21r / fc , an il-21-neutralizing chimeric protein ) on prevention of diabetes in nod mice , in combination with syngeneic islet transplantation has recently been investigated . results showed that il-21-responsiveness by cd8 + t cells was sufficient to mediate islet allograft rejection and that combining neutralization of il-21 with islet transplantation restored glucose homeostasis and resulted in recovery from autoimmune diabetes . since the absence of il-21 signaling prevented islet allograft rejection indicating a robust influence of il-21 on a graft - mounted immune response , these findings imply that therapeutic manipulation of il-21 may serve as a suitable treatment for patients with t1d . the efficacy of an il-21-targeted therapy ( il-21r / fc , an il-21-neutralizing chimeric protein ) on prevention of diabetes in nod mice , in combination with syngeneic islet transplantation has recently been investigated . results showed that il-21-responsiveness by cd8 + t cells was sufficient to mediate islet allograft rejection and that combining neutralization of il-21 with islet transplantation restored glucose homeostasis and resulted in recovery from autoimmune diabetes . since the absence of il-21 signaling prevented islet allograft rejection indicating a robust influence of il-21 on a graft - mounted immune response , these findings imply that therapeutic manipulation of il-21 may serve as a suitable treatment for patients with t1d . dna vaccination strategya therapeutic dna vaccination strategy for autoimmunity and transplantation has also recently been described wherein intradermal injection of plasmid dna encoding glutamic acid decarboxylase ( gad ) polypeptide , which is synthesized in both pancreatic islet and skin tissue , ameliorated new - onset t1d in nod mice and increased skin allograft survival in a balb / c - c57bl/6 model system in a donor - specific manner . furthermore , only cpg - methylation of plasmid dna coding for gad was required in order to significantly increase skin allograft survival after immunization of recipient ; codelivery of a cdna coding for the proapoptotic bax protein , which was shown previously to be essential for successful therapy of autoimmune diabetes in nod mice by induction of foxp3 + regulatory t cells , was not necessary . this study therefore revealed the promising potential for autoimmunity - targeting dna vaccination to be applied to clinical transplantation . a therapeutic dna vaccination strategy for autoimmunity and transplantation has also recently been described wherein intradermal injection of plasmid dna encoding glutamic acid decarboxylase ( gad ) polypeptide , which is synthesized in both pancreatic islet and skin tissue , ameliorated new - onset t1d in nod mice and increased skin allograft survival in a balb / c - c57bl/6 model system in a donor - specific manner . furthermore , only cpg - methylation of plasmid dna coding for gad was required in order to significantly increase skin allograft survival after immunization of recipient ; codelivery of a cdna coding for the proapoptotic bax protein , which was shown previously to be essential for successful therapy of autoimmune diabetes in nod mice by induction of foxp3 + regulatory t cells , was not necessary . this study therefore revealed the promising potential for autoimmunity - targeting dna vaccination to be applied to clinical transplantation . localized immunosuppression using glucocorticoid microspheresthe ability of poly(d , l - lactic ) acid ( pla ) and poly(d , l - lactic glycolic acid ( plga ) microspheres of the soft corticosteroid loteprednol etabonate ( le ) loaded within a biohybrid device to provide localized immunosuppression and reduce systemic side effects over an extended period has been evaluated in a set of early exploratory experiments with diabetic rats receiving islet transplantation . while sustained release of microspheres at low concentration showed no cytotoxicity on the viability of the min-6 insuloma cell line in vitro , animals treated using a biohybrid device loaded with microspheres showed improved results compared to those treated by delivery in solution form with an osmotic minipump . sustained local delivery formulation for glucocorticoids dexamethasone phosphate and le using pla microspheres showed promise in their ability in vivo to prolong allograft survival in rats after tapering of systemic immunosuppression , compared to control groups . also , doses delivered locally were approximately hundredfold smaller than those typically used in systemic treatments . the ability of poly(d , l - lactic ) acid ( pla ) and poly(d , l - lactic glycolic acid ( plga ) microspheres of the soft corticosteroid loteprednol etabonate ( le ) loaded within a biohybrid device to provide localized immunosuppression and reduce systemic side effects over an extended period has been evaluated in a set of early exploratory experiments with diabetic rats receiving islet transplantation . while sustained release of microspheres at low concentration showed no cytotoxicity on the viability of the min-6 insuloma cell line in vitro , animals treated using a biohybrid device loaded with microspheres showed improved results compared to those treated by delivery in solution form with an osmotic minipump . sustained local delivery formulation for glucocorticoids dexamethasone phosphate and le using pla microspheres showed promise in their ability in vivo to prolong allograft survival in rats after tapering of systemic immunosuppression , compared to control groups . also , doses delivered locally were approximately hundredfold smaller than those typically used in systemic treatments . induction of tolerance by regulatory t cells ( tregs)donor alloantigen - specific cd4+cd25 + tregs play an important role in inducing and maintaining tolerance to donor alloantigens in vivo . by actively modulating or suppressing the deleterious cd4 + and cd8 + t - cells - mediated immune response to donor alloantigens , tregs may prevent rejection and mediate linked unresponsiveness . in vivo , these cells are dependent on interleukin il-10 and ctla4 for functional activity . as part of clinical tolerance strategies , the immunosuppressive ability of peripheral t - cell - depleting agents such as anti - cd52 mab alemtuzumab ( campath-1h ) and antithymocyte globulin ( atg ) have been tested in humans with autoimmunity and in transplantation scenarios [ 139 , 149 ] . were the first to report that atg but not alemtuzumab or the il-2r antagonists caused rapid and sustained expansion of cd4+cd25 + t cells in cocultures with human peripheral blood lymphocytes and displayed enhanced expression of the regulatory markers glucocorticoid - induced tnf receptor , ctla4 and foxp3 as well as efficient suppression of a direct alloimmune response of the original responder lymphocytes . the induction of tregs depended on the production of th2 cytokines in the generating cultures . this study demonstrated the therapeutic potential of atg in promoting the generation of tregs for cellular therapy in autoimmunity and clinical transplantation . recently , a clinically relevant immunoregulatory strategy based on treatment of nod mice with murine thymoglobulin ( matg ) and ctla4-ig to prevent allo- and autoimmune activation in a stringent model of islet transplantation and diabetes reversal was investigated . the results revealed that transplant recipients experienced a complete abrogation of autoimmune responses and significant downregulation of alloimmunity in response to treatment . furthermore , this striking effect was confirmed by 100% diabetes reversal in newly hyperglycemic nod mice and 100% indefinite graft survival of syngeneic islet transplantation ( nodscid into nod mice ) . an induction immunosuppressive therapy regimen consisting of rabbit atg and the monoclonal antibody to cd20 rituximab ( rituxan ) promoted long - term islet allograft survival in cynomolgus macaques maintained on rapamycin monotherapy . several clinical trials aimed at studying the safety and effectiveness of islet transplantation combined with various immunosuppressive regimens that include atg for treatment of type 1 diabetic individuals experiencing hypoglycemia unawareness and severe hypoglycemic episodes are either completed or underway . these include amongst others an islet after kidney transplantation trial with atg and etanercept ( clinicaltrials.gov identifier : nct00468117 ) and islet transplantation trials with atg , everolimus and cyclosporine ( clinicaltrials.gov identifier : nct00286624 ) ; with rituxan ( rituximab ) , thymoglobulin ( atg ) , zenapax ( daclizumab ) and rapamune ( sirolimus ) ( clinicaltrials.gov identifier : nct00468442 ) ; with lisofylline , atg , basiliximab , sirolimus and tacrolimus , ( clinicaltrials.gov identifier : nct00464555 ) ; and with raptiva , atg , and sirolimus ( clinicaltrials.gov identifier : nct00672204 ) amongst others . in a recent study , patients receiving intraportal allogeneic islet transplants were maintained on immunosuppression consisting of atg induction and maintenance with sirolimus or mycophenolate mofetil ( mmf ) and costimulation blocker belatacept or the anti - lfa-1 antibody efalizumab which permit long - term islet allograft survival . the results indicated that both regimens were effective and well tolerated , with the calcineurin inhibitor / steroid - sparing islet protocols resulting in long - term insulin independence and belatacept proving to be an effective alternative in improving graft function and longevity while minimizing renal and -cell toxicity . because tregs strongly suppress the immune response in syngeneic islet transplantation and improve graft survival and function , several approaches are now emerging to induce / increase host tregs activity in the transplant setting , including amongst others , systemic tgf-1 therapy . a recent study demonstrated that islet - specific treg induced from the bdc-6.9 tcr transgenic mouse by activation of t cells in the presence of tgf- could suppress both spontaneous diabetes as well as transfer of diabetes into nodscid mice by diabetic nod spleen cells or activated bdc-2.5 tcr transgenic th1 effector t cells . in the latter transfer model , the authors demonstrated that infiltration of tregs into pancreas caused a reduction in the number of effector th1 t cells and macrophages , and also inhibited effector t - cell cytokine and chemokine production . transfection of effector t cells with a dominant - negative tgf- receptor demonstrated that in vivo suppression of diabetes by tgf--induced treg is tgf- dependent . donor alloantigen - specific cd4+cd25 + tregs play an important role in inducing and maintaining tolerance to donor alloantigens in vivo . by actively modulating or suppressing the deleterious cd4 + and cd8 + t - cells - mediated immune response to donor alloantigens , tregs may prevent rejection and mediate linked unresponsiveness . in vivo , these cells are dependent on interleukin il-10 and ctla4 for functional activity . as part of clinical tolerance strategies , the immunosuppressive ability of peripheral t - cell - depleting agents such as anti - cd52 mab alemtuzumab ( campath-1h ) and antithymocyte globulin ( atg ) have been tested in humans with autoimmunity and in transplantation scenarios [ 139 , 149 ] . were the first to report that atg but not alemtuzumab or the il-2r antagonists caused rapid and sustained expansion of cd4+cd25 + t cells in cocultures with human peripheral blood lymphocytes and displayed enhanced expression of the regulatory markers glucocorticoid - induced tnf receptor , ctla4 and foxp3 as well as efficient suppression of a direct alloimmune response of the original responder lymphocytes . the induction of tregs depended on the production of th2 cytokines in the generating cultures . this study demonstrated the therapeutic potential of atg in promoting the generation of tregs for cellular therapy in autoimmunity and clinical transplantation . recently , a clinically relevant immunoregulatory strategy based on treatment of nod mice with murine thymoglobulin ( matg ) and ctla4-ig to prevent allo- and autoimmune activation in a stringent model of islet transplantation and diabetes reversal was investigated . the results revealed that transplant recipients experienced a complete abrogation of autoimmune responses and significant downregulation of alloimmunity in response to treatment . furthermore , this striking effect was confirmed by 100% diabetes reversal in newly hyperglycemic nod mice and 100% indefinite graft survival of syngeneic islet transplantation ( nodscid into nod mice ) . an induction immunosuppressive therapy regimen consisting of rabbit atg and the monoclonal antibody to cd20 rituximab ( rituxan ) promoted long - term islet allograft survival in cynomolgus macaques maintained on rapamycin monotherapy . several clinical trials aimed at studying the safety and effectiveness of islet transplantation combined with various immunosuppressive regimens that include atg for treatment of type 1 diabetic individuals experiencing hypoglycemia unawareness and severe hypoglycemic episodes are either completed or underway . these include amongst others an islet after kidney transplantation trial with atg and etanercept ( clinicaltrials.gov identifier : nct00468117 ) and islet transplantation trials with atg , everolimus and cyclosporine ( clinicaltrials.gov identifier : nct00286624 ) ; with rituxan ( rituximab ) , thymoglobulin ( atg ) , zenapax ( daclizumab ) and rapamune ( sirolimus ) ( clinicaltrials.gov identifier : nct00468442 ) ; with lisofylline , atg , basiliximab , sirolimus and tacrolimus , ( clinicaltrials.gov identifier : nct00464555 ) ; and with raptiva , atg , and sirolimus ( clinicaltrials.gov identifier : nct00672204 ) amongst others . in a recent study , patients receiving intraportal allogeneic islet transplants were maintained on immunosuppression consisting of atg induction and maintenance with sirolimus or mycophenolate mofetil ( mmf ) and costimulation blocker belatacept or the anti - lfa-1 antibody efalizumab which permit long - term islet allograft survival . the results indicated that both regimens were effective and well tolerated , with the calcineurin inhibitor / steroid - sparing islet protocols resulting in long - term insulin independence and belatacept proving to be an effective alternative in improving graft function and longevity while minimizing renal and -cell toxicity . because tregs strongly suppress the immune response in syngeneic islet transplantation and improve graft survival and function , several approaches are now emerging to induce / increase host tregs activity in the transplant setting , including amongst others , systemic tgf-1 therapy . a recent study demonstrated that islet - specific treg induced from the bdc-6.9 tcr transgenic mouse by activation of t cells in the presence of tgf- could suppress both spontaneous diabetes as well as transfer of diabetes into nodscid mice by diabetic nod spleen cells or activated bdc-2.5 tcr transgenic th1 effector t cells . in the latter transfer model , the authors demonstrated that infiltration of tregs into pancreas caused a reduction in the number of effector th1 t cells and macrophages , and also inhibited effector t - cell cytokine and chemokine production . transfection of effector t cells with a dominant - negative tgf- receptor demonstrated that in vivo suppression of diabetes by tgf--induced treg is tgf- dependent . the impact of rapamycin , on human islet engraftment and function was assessed in 10 patients with t1d before islet transplantation . the results indicated that pretreatment with rapamycin was associated with a reduction in chemokines ccl2 and ccl3 pretransplantation and a dampened chemokine response post - transplantation , potentially improving clinical islet engraftment by an anti - inflammatory mechanism . in rat - to - mouse islet xenotransplantation , administration of anti - cd154 mab and rapamycin rapamycin has been shown to allow expansion , proliferation , and regulatory function of both murine and human naturally occurring cd4+cd25+foxp3 + tregs ( ntregs ) , which are pivotal for the induction and maintenance of peripheral tolerance . pothoven et al . demonstrated that rapamycin - conditioned balb / c donor bone - marrow - derived dcs ( bmdcs ) had significantly enhanced ability to induce cd4+cd25+foxp3 + itregs of recipient origin ( c57bl/6 ( b6 ) ) in vitro under treg driving conditions compared to unmodified bmdcs . these in vitro induced cd4+cd25+foxp3 + itregs exerted donor - specific suppression in vitro and prolonged allogeneic islet graft survival in vivo in rag(/ ) hosts upon coadoptive transfer with t - effector cells . the cd4+cd25+foxp3 + itregs also expanded and preferentially maintained foxp3 expression in the graft draining lymph nodes and were able to induce endogenous nave t cells to convert to cd4+cd25+foxp3 + t cells . thus , rapamycin - conditioned donor bmdcs can be exploited for in vitro differentiation into donor antigen - specific cd4+cd25+foxp3 + itregs capable of effectively controlling allogeneic islet graft rejection . strom 's group demonstrated a triple therapy approach that combined administration of rapamycin and agonist il-2- and antagonist il-15-related cytolytic fusion proteins . this treatment promoted very long - term engraftment / tolerance of allogeneic islets in both spontaneously diabetic nod mice and il-2-deficient recipients by limiting the early expansion of activated t cells , preserving and even exaggerating their subsequent apoptotic clearance , and further amplifying the depletion of these activated t cells by antibody - dependent mechanisms , while preserving cd4+cd25 + t - cell - dependent immunoregulatory networks . in t1d , treg activity has been demonstrated in the pancreatic lymph node , but the in vivo activity of tregs during suppression in pancreas remains poorly characterized . a clinical study wherein ntreg numbers and function were examined in a unique set of patients with t1d who underwent rapamycin monotherapy before islet transplantation indicated that while rapamycin monotherapy did not alter the frequency and functional features , namely , proliferation , and cytokine production of circulating ntregs , it increased their capacity to suppress proliferation of cd4+cd25 effector t - cells . these findings demonstrate that rapamycin directly affects human ntreg function in vivo , by refitting their suppressive activity , whereas it does not directly change effector t - cell function . the impact of rapamycin , on human islet engraftment and function was assessed in 10 patients with t1d before islet transplantation . the results indicated that pretreatment with rapamycin was associated with a reduction in chemokines ccl2 and ccl3 pretransplantation and a dampened chemokine response post - transplantation , potentially improving clinical islet engraftment by an anti - inflammatory mechanism . in rat - to - mouse islet xenotransplantation , administration of anti - cd154 mab and rapamycin rapamycin has been shown to allow expansion , proliferation , and regulatory function of both murine and human naturally occurring cd4+cd25+foxp3 + tregs ( ntregs ) , which are pivotal for the induction and maintenance of peripheral tolerance . pothoven et al . demonstrated that rapamycin - conditioned balb / c donor bone - marrow - derived dcs ( bmdcs ) had significantly enhanced ability to induce cd4+cd25+foxp3 + itregs of recipient origin ( c57bl/6 ( b6 ) ) in vitro under treg driving conditions compared to unmodified bmdcs . these in vitro induced cd4+cd25+foxp3 + itregs exerted donor - specific suppression in vitro and prolonged allogeneic islet graft survival in vivo in rag(/ ) hosts upon coadoptive transfer with t - effector cells . the cd4+cd25+foxp3 + itregs also expanded and preferentially maintained foxp3 expression in the graft draining lymph nodes and were able to induce endogenous nave t cells to convert to cd4+cd25+foxp3 + t cells . thus , rapamycin - conditioned donor bmdcs can be exploited for in vitro differentiation into donor antigen - specific cd4+cd25+foxp3 + itregs capable of effectively controlling allogeneic islet graft rejection . strom 's group demonstrated a triple therapy approach that combined administration of rapamycin and agonist il-2- and antagonist il-15-related cytolytic fusion proteins . this treatment promoted very long - term engraftment / tolerance of allogeneic islets in both spontaneously diabetic nod mice and il-2-deficient recipients by limiting the early expansion of activated t cells , preserving and even exaggerating their subsequent apoptotic clearance , and further amplifying the depletion of these activated t cells by antibody - dependent mechanisms , while preserving cd4+cd25 + t - cell - dependent immunoregulatory networks . in t1d , treg activity has been demonstrated in the pancreatic lymph node , but the in vivo activity of tregs during suppression in pancreas remains poorly characterized . a clinical study wherein ntreg numbers and function were examined in a unique set of patients with t1d who underwent rapamycin monotherapy before islet transplantation indicated that while rapamycin monotherapy did not alter the frequency and functional features , namely , proliferation , and cytokine production of circulating ntregs , it increased their capacity to suppress proliferation of cd4+cd25 effector t - cells . these findings demonstrate that rapamycin directly affects human ntreg function in vivo , by refitting their suppressive activity , whereas it does not directly change effector t - cell function . induction of tolerance through suppression of tlrstlr2-induced il-6 secretion from apcs has been shown to reverse the suppressive function of tregs [ 104 , 161 ] and in combination with tgf-induced th17 cells resulted in enhanced inflammation [ 103 , 104 , 162 , 163 ] and prevention of transplantation tolerance . in a very recent study to understand the mechanism by which different inflammatory signals affect transplantation tolerance and immunity , it was determined that tlr2 ligand peptidoglycan inhibited foxp3 expression in both natural tregs ( ntregs ) and tgf-driven adaptive tregs ( atregs ) independent of paracrine th1 , th2 , and th17 cytokines . while tlr2-induced inhibition of foxp3 was independent of stat1 , stat3 , stat4 , and stat6 , it was dependent on myd88 and irf . binding of induced irf1 to irf1 response elements ( irf - e ) in the foxp3 promoter and intronic enhancers negatively regulated foxp3 expression to suppress treg function . furthermore by inducing divergent chromatin changes at the foxp3 locus , inflammatory il-6 and tlr2 signals regulated treg suppressor function and reduced graft survival in an islet transplantation model . schroppel 's group demonstrated that deficiency of tlr4 in islet graft recipients prolonged graft survival . low dose rapamycin - treatment of tlr4(/ ) recipients induced permanent and prolonged engraftment of 45% of the islet graft that was dependent on the presence of cd4+cd25+foxp3 + tregs . nave cd4+cd25 t cells cultured with the tlr4 ligand lipopolysaccharide showed enhanced il-4 , il-6 , il-17 , and ifn- secretion and inhibited tgf-induced foxp3+treg generation . these results indicated that inhibition of recipient tlr4 activation at the time of transplantation decreased proinflammatory signals and allowed for treg generation . approaches such as generating tlr agonists / antagonists , creating monoclonal antibody to tlrs , blocking key molecules in the signaling pathways and downmodulating tlr signaling may be of immense benefit in the treatment of t1d and islet transplantation . tlr2-induced il-6 secretion from apcs has been shown to reverse the suppressive function of tregs [ 104 , 161 ] and in combination with tgf-induced th17 cells resulted in enhanced inflammation [ 103 , 104 , 162 , 163 ] and prevention of transplantation tolerance . in a very recent study to understand the mechanism by which different inflammatory signals affect transplantation tolerance and immunity , it was determined that tlr2 ligand peptidoglycan inhibited foxp3 expression in both natural tregs ( ntregs ) and tgf-driven adaptive tregs ( atregs ) independent of paracrine th1 , th2 , and th17 cytokines . while tlr2-induced inhibition of foxp3 was independent of stat1 , stat3 , stat4 , and stat6 , it was dependent on myd88 and irf . binding of induced irf1 to irf1 response elements ( irf - e ) in the foxp3 promoter and intronic enhancers negatively regulated foxp3 expression to suppress treg function . furthermore by inducing divergent chromatin changes at the foxp3 locus , inflammatory il-6 and tlr2 signals regulated treg suppressor function and reduced graft survival in an islet transplantation model . schroppel 's group demonstrated that deficiency of tlr4 in islet graft recipients prolonged graft survival . low dose rapamycin - treatment of tlr4(/ ) recipients induced permanent and prolonged engraftment of 45% of the islet graft that was dependent on the presence of cd4+cd25+foxp3 + tregs . nave cd4+cd25 t cells cultured with the tlr4 ligand lipopolysaccharide showed enhanced il-4 , il-6 , il-17 , and ifn- secretion and inhibited tgf-induced foxp3+treg generation . these results indicated that inhibition of recipient tlr4 activation at the time of transplantation decreased proinflammatory signals and allowed for treg generation . approaches such as generating tlr agonists / antagonists , creating monoclonal antibody to tlrs , blocking key molecules in the signaling pathways and downmodulating tlr signaling may be of immense benefit in the treatment of t1d and islet transplantation . other tolerance inducing treatmentsinterestingly , treatment of islet graft recipients with bilirubin prolonged islet allograft survival via a treg - dependent manner in which cd4+cd25 + treg cells were necessary for tolerance induction and graft acceptance . bilirubin treatment also promoted de novo generation of tregs possibly accounting for the observed protective effects . a combination treatment of hmox1 induction , carbon monoxide , and bilirubin administration led to long - term survival and tolerance toward islet allografts by promoting foxp3 + tregs and inducing and maintaining tolerance in the recipient . vag539 is a water soluble derivate of vaf347 , a low - molecular - weight compound that activates the aryl hydrocarbon receptor ( ahr ) . oral administration of vag539 promotes long - term graft acceptance and active tolerance in balb / c mice that receive mhc - mismatched pancreatic islet allograft , resulting in increased frequency of splenic cd4+cd25+foxp3 + t cells in vivo and improved cd4+cd25+foxp3 + t - cell survival in vitro . interestingly , transfer of cd11c+ dcs but not of cd4 + t or cd19 + b cells , from vag539-treated long - term tolerant hosts into mice that recently underwent transplantation resulted in donor ( c57bl/6)-specific graft acceptance and in a significantly higher frequency of splenic cd4+cd25+foxp3 + tregs . furthermore , the transfer of these cd4+cd25 + tregs into recently transplanted mice promoted islet graft acceptance . also , cell therapy with in vitro vaf347-treated bone - marrow - derived mature dcs prevented islet graft rejection , and reduced ova - specific t - cell responses in ova - immunized mice . taken together these data indicate that activation of ahr induces islet allograft - specific tolerance through direct as well as dc - mediated effects on treg survival and function . transient depletion of dividing t cells by administration of ganciclovir for 14 days , induced at the time of allogeneic islets transplantation into diabetic transgenic mice that express a thymidine kinase ( tk ) conditional suicide gene in t cells also resulted in allograft tolerance in 63% of treated mice accompanied by a 2- to 3-fold persistent increase in the proportion of cd4+cd25+foxp3 + treg within 3 weeks only in allograft - bearing mice . similar results were obtained after cytostatic hydroxyurea treatment in normal mice suggesting that the transient depletion of dividing t cells represented a novel means of immunointervention based on disturbance of t - cell homeostasis and subsequent increase in treg proportion . interestingly , treatment of islet graft recipients with bilirubin prolonged islet allograft survival via a treg - dependent manner in which cd4+cd25 + treg cells were necessary for tolerance induction and graft acceptance . bilirubin treatment also promoted de novo generation of tregs possibly accounting for the observed protective effects . a combination treatment of hmox1 induction , carbon monoxide , and bilirubin administration led to long - term survival and tolerance toward islet allografts by promoting foxp3 + tregs and inducing and maintaining tolerance in the recipient . vag539 is a water soluble derivate of vaf347 , a low - molecular - weight compound that activates the aryl hydrocarbon receptor ( ahr ) . oral administration of vag539 promotes long - term graft acceptance and active tolerance in balb / c mice that receive mhc - mismatched pancreatic islet allograft , resulting in increased frequency of splenic cd4+cd25+foxp3 + t cells in vivo and improved cd4+cd25+foxp3 + t - cell survival in vitro . interestingly , transfer of cd11c+ dcs but not of cd4 + t or cd19 + b cells , from vag539-treated long - term tolerant hosts into mice that recently underwent transplantation resulted in donor ( c57bl/6)-specific graft acceptance and in a significantly higher frequency of splenic cd4+cd25+foxp3 + tregs . furthermore , the transfer of these cd4+cd25 + tregs into recently transplanted mice promoted islet graft acceptance . also , cell therapy with in vitro vaf347-treated bone - marrow - derived mature dcs prevented islet graft rejection , and reduced ova - specific t - cell responses in ova - immunized mice . taken together these data indicate that activation of ahr induces islet allograft - specific tolerance through direct as well as dc - mediated effects on treg survival and function . transient depletion of dividing t cells by administration of ganciclovir for 14 days , induced at the time of allogeneic islets transplantation into diabetic transgenic mice that express a thymidine kinase ( tk ) conditional suicide gene in t cells also resulted in allograft tolerance in 63% of treated mice accompanied by a 2- to 3-fold persistent increase in the proportion of cd4+cd25+foxp3 + treg within 3 weeks only in allograft - bearing mice . similar results were obtained after cytostatic hydroxyurea treatment in normal mice suggesting that the transient depletion of dividing t cells represented a novel means of immunointervention based on disturbance of t - cell homeostasis and subsequent increase in treg proportion . in vivo and in vitro expansion of tregsnagahama et al . established a protocol for in vivo and in vitro alloantigen - specific expansion of naturally arising cd4+cd25 + regulatory t - cells ( treg ) to establish antigen - specific dominant tolerance to allogeneic transplants . they showed that in vivo exposure of cd4+cd25 + t cells from normal naive mice to alloantigen in a t - cell - deficient environment elicited the spontaneous expansion of alloantigen - specific cd4+cd25 + ntregs capable of suppressing allograft rejection mediated by subsequently transferred naive t cells , leading to long - term graft tolerance . similarly they demonstrated that antigen - specific expansion of ntregs can be achieved in vitro by stimulating cd4+cd25 + t cells from normal animals with alloantigen in the presence of high doses of il-2 . the expanded tregs were even capable of suppressing secondary mixed leukocyte reaction in vitro and , following adoptive transfer , were able to establish antigen - specific long - term graft tolerance . francis et al . have recently demonstrated that graft - protective treg arise in vivo both from naturally occurring foxp3+cd4 + tregs and from non - regulatory foxp3cd4 + cells . interestingly , the induction of tolerance also inhibited cd4 + effector cell priming with t cells from tolerant mice demonstrating impaired effector function in vitro . thus , by converting potential effector cells into graft - protective tregs and by expanding alloreactive naturally occurring tregs , adaptive tolerance was induced . adoptive cell therapy using patient - specific cd4+cd25 + tregs as individualised medicine to promote clinical transplantation tolerance is very promising . if this principle is to be applied to clinical tolerance induction , strategies targeting potential effector cells will have to be investigated for successful generation of alloreactive tregs that may be critical for long - term allograft survival without chronic immunosuppression . established a protocol for in vivo and in vitro alloantigen - specific expansion of naturally arising cd4+cd25 + regulatory t - cells ( treg ) to establish antigen - specific dominant tolerance to allogeneic transplants . they showed that in vivo exposure of cd4+cd25 + t cells from normal naive mice to alloantigen in a t - cell - deficient environment elicited the spontaneous expansion of alloantigen - specific cd4+cd25 + ntregs capable of suppressing allograft rejection mediated by subsequently transferred naive t cells , leading to long - term graft tolerance . similarly they demonstrated that antigen - specific expansion of ntregs can be achieved in vitro by stimulating cd4+cd25 + t cells from normal animals with alloantigen in the presence of high doses of il-2 . the expanded tregs were even capable of suppressing secondary mixed leukocyte reaction in vitro and , following adoptive transfer , were able to establish antigen - specific long - term graft tolerance . francis et al . have recently demonstrated that graft - protective treg arise in vivo both from naturally occurring foxp3+cd4 + tregs and from non - regulatory foxp3cd4 + cells . interestingly , the induction of tolerance also inhibited cd4 + effector cell priming with t cells from tolerant mice demonstrating impaired effector function in vitro . thus , by converting potential effector cells into graft - protective tregs and by expanding alloreactive naturally occurring tregs , adaptive tolerance was induced . adoptive cell therapy using patient - specific cd4+cd25 + tregs as individualised medicine to promote clinical transplantation tolerance is very promising . if this principle is to be applied to clinical tolerance induction , strategies targeting potential effector cells will have to be investigated for successful generation of alloreactive tregs that may be critical for long - term allograft survival without chronic immunosuppression . the role of tolerance - inducing dendritic cell therapies in treg and th17 cells interconversionactivated cd4 + t cells develop into th1 , th2 , or th17 subsets based on the cytokines they produce and distinct effector functions . th17 cells produce il-17a , il-17f , il-10 , il-22 , and il-21 and play a role in host defense against infections , and in inducing tissue inflammation in autoimmune disease . the broad distribution profile of il-17 and il-22 receptors guarantees induction of a massive tissue reaction . the involvement of differentiation factors ( tgf- plus il-6 or il-21 ) , the growth and stabilization factor ( il-23 ) , and the transcription factors ( stat3 , ror , and ror ) in the development and stabilization of th17 cells has recently been identified . th17-derived il-21 plays an important role in the amplification of th17 cells . based on the evidence of immunosuppressive tgf- participation in the differentiation of th17 cells , the th17 lineage appears to have a close relationship with cd4+cd25+foxp3 + tregs . th17 cells were shown to be involved in islet transplant rejection , and blockade of il-23r was shown to positively correlate with reduction in il-17 expression in a dose - dependent manner . while the combination of anti - cd154 mab and il-23r antibody was shown to prevent the acute rejection to some extent , no significant difference was observed when compared with the anti - cd154mab alone . relb(lo ) dcs generated in the presence of an nf-b inhibitor induce tregs and suppress inflammation . a very important recent study showed that while tolerizing relb(lo ) dcs were able to significantly inhibit diabetes progression when administered to 4-week - old nod mice , il-1 produced in response to islet autoantigen presentation reduced the immunosuppressive capacity of treg cells and promoted their conversion to th17 cells . this study highlights the importance to entertain caution while using tolerizing dc therapies that regulate islet autoantigen priming and prevent diabetes and that progression past the il-1/il-17 checkpoint signals the need for adapting other tolerizing strategies . activated cd4 + t cells develop into th1 , th2 , or th17 subsets based on the cytokines they produce and distinct effector functions . th17 cells produce il-17a , il-17f , il-10 , il-22 , and il-21 and play a role in host defense against infections , and in inducing tissue inflammation in autoimmune disease . the broad distribution profile of il-17 and il-22 receptors guarantees induction of a massive tissue reaction . the involvement of differentiation factors ( tgf- plus il-6 or il-21 ) , the growth and stabilization factor ( il-23 ) , and the transcription factors ( stat3 , ror , and ror ) in the development and stabilization of th17 cells has recently been identified . th17-derived il-21 plays an important role in the amplification of th17 cells . based on the evidence of immunosuppressive tgf- participation in the differentiation of th17 cells , the th17 lineage appears to have a close relationship with cd4+cd25+foxp3 + tregs . th17 cells were shown to be involved in islet transplant rejection , and blockade of il-23r was shown to positively correlate with reduction in il-17 expression in a dose - dependent manner . while the combination of anti - cd154 mab and il-23r antibody was shown to prevent the acute rejection to some extent , no significant difference was observed when compared with the anti - cd154mab alone . relb(lo ) dcs generated in the presence of an nf-b inhibitor induce tregs and suppress inflammation . a very important recent study showed that while tolerizing relb(lo ) dcs were able to significantly inhibit diabetes progression when administered to 4-week - old nod mice , il-1 produced in response to islet autoantigen presentation reduced the immunosuppressive capacity of treg cells and promoted their conversion to th17 cells . this study highlights the importance to entertain caution while using tolerizing dc therapies that regulate islet autoantigen priming and prevent diabetes and that progression past the il-1/il-17 checkpoint signals the need for adapting other tolerizing strategies . current combinatorial therapeutic strategies in clinical islet transplantationcurrently , islet transplantation followed by atg / alemtuzumab ( campath-1h , monoclonal ) . anti - cd52 ab / hokt3/anti - cd25 ( daclizumab ) induction therapy along with a sirolimus - based , prednisone - free maintenance regimen in combination with mmf and low tacrolimus as well as drugs that demonstrate powerful immunosuppressive / anti - inflammatory potency in the absence of nephrotoxicity and diabetogenicity are under investigation [ 153 , 176179 ] . other drugs currently in phases ii or iii of development include otelixizumab ( anti - cd3 ) , teplizumab ( anti - cd3 ) , rituximab ( anti - cd20 ) , abatacept ( ctla4ig ) , diappep 277 ( heat shock protein ) , and gad and oral insulin amongst others . glp-1r agonists like exendin-4 stimulate -cell proliferation and neogenesis and inhibit -cell apoptosis while dppiv inhibitors increase cell insulin content and are therefore of immense benefit in the above mentioned combination therapies for preserving and expanding -cell mass following transplantation . the activity of potent proinflammatory tnf can be inhibited by etanercept , a recombinant tnf receptor protein . a high success rate of insulin independence was achieved using a protocol in which etanercept was administered as induction therapy following single donor islet transplantation , in combination with prednisone , daclizumab , and rabbit atg [ 180182 ] . combined treatment with etanercept and exenatide in addition to the edmonton immunosuppressive protocol was shown to reduce the number of islets needed to achieve insulin independence and improve glucose control and graft survival in patients who needed a second transplantation because of progressive graft dysfunction [ 183 , 184 ] . these combinatorial strategies could also include encapsulation of islets with nanofiber scaffolds or biomatrices or permselective alginate microcapsules synthesized to release immunosuppressive drugs or drugs that stimulate vasculogenesis / angiogenesis to improve trasnplantation outcomes at extrahepatic sites . currently , islet transplantation followed by atg / alemtuzumab ( campath-1h , monoclonal ) . anti - cd52 ab / hokt3/anti - cd25 ( daclizumab ) induction therapy along with a sirolimus - based , prednisone - free maintenance regimen in combination with mmf and low tacrolimus as well as drugs that demonstrate powerful immunosuppressive / anti - inflammatory potency in the absence of nephrotoxicity and diabetogenicity are under investigation [ 153 , 176179 ] . other drugs currently in phases ii or iii of development include otelixizumab ( anti - cd3 ) , teplizumab ( anti - cd3 ) , rituximab ( anti - cd20 ) , abatacept ( ctla4ig ) , diappep 277 ( heat shock protein ) , and gad and oral insulin amongst others . glp-1r agonists like exendin-4 stimulate -cell proliferation and neogenesis and inhibit -cell apoptosis while dppiv inhibitors increase cell insulin content and are therefore of immense benefit in the above mentioned combination therapies for preserving and expanding -cell mass following transplantation . the activity of potent proinflammatory tnf can be inhibited by etanercept , a recombinant tnf receptor protein . a high success rate of insulin independence was achieved using a protocol in which etanercept was administered as induction therapy following single donor islet transplantation , in combination with prednisone , daclizumab , and rabbit atg [ 180182 ] . combined treatment with etanercept and exenatide in addition to the edmonton immunosuppressive protocol was shown to reduce the number of islets needed to achieve insulin independence and improve glucose control and graft survival in patients who needed a second transplantation because of progressive graft dysfunction [ 183 , 184 ] . these combinatorial strategies could also include encapsulation of islets with nanofiber scaffolds or biomatrices or permselective alginate microcapsules synthesized to release immunosuppressive drugs or drugs that stimulate vasculogenesis / angiogenesis to improve trasnplantation outcomes at extrahepatic sites . several studies indicate that nonimmunogenic multipotent mscs apart from playing an important role in -cell replacement therapies owing to their versatile differentiation and ex vivo expansion potential , are also cytoprotective immune modulators , exerting therapeutic effects by promoting graft protection , tissue revascularization , and -cell survival in islet transplantation . bone - marrow - derived mscs ( bm - mscs ) can enhance repair and regeneration , not only by repopulating damaged tissue , but also by reducing inflammation . also , they allow transplantation across mhc barriers since they do not possess cell surface human leukocyte antigen ( hla ) or mhc class ii molecules . kim et al . evaluated the therapeutic potential of autologous mscs in preventing graft rejection following allogeneic rat islet transplantation and demonstrated that when combined with cyclosporine a therapy , graft survival attained more than 100 days in 33% of autologous mscs - plus - csa - treated recipients . splenocytes from autologous msc - plus - csa - treated rats exhibited a reduced mlr proliferative response to donor stimulators and increased il-10 release . interestingly , il-10 induced by cd11b+ cells and il-10 activated tregs played a role in msc - mediated immune modulation in the rat islet allograft . the authors demonstrated that autologous mscs - plus - csa downregulated immune responses and induced donor - specific t - cell hyporesponsiveness by reducing the production of proinflammatory cytokines and inducing anti - inflammatory cytokine production , especially that of il-10 , during the early post - transplantation period . thus , the combined use of autologous mscs and low - dose csa exerted a synergistic immunosuppressive effect in an islet allograft model . these results were supported by the demonstration that triple - dose administration of either syngeneic or allogeneic mscs was able to prevent acute rejection and improve glycemic control in diabetic rats receiving marginal islet mass transplantation via the portal vein . reduced levels of pro - inflammatory cytokines and glucose as well as low - grade rejections were observed up to 15 days after transplantation indicating the ability of mscs to prolong graft function by preventing acute rejection . also the efficacy of mscs was independent of the administration route and comparable to that of immunosuppressive therapy indicating that mscs may play an important role in preventing acute rejection and improving graft function in portal vein pancreatic islet transplantation . it is also noteworthy to mention that xenotransplantation of three - dimensional spheroid bodies ( sbs ) formed under special induction conditions from endometrial mesenchymal stem - like cells ( sb - emscs ) into immunocompromised mice with stz - induced diabetes restored blood insulin levels to control values and greatly prolonged the survival of graft cells . these results suggest that emscs not only played a novel role in the differentiation of pancreatic progenitors , but could also functionally enhance insulin production to restore the regulation of blood glucose levels . it will be interesting to dissect the role of these cells in immunomodulation and vasculogenesis in an in vivo transplantation model . the pancreatic endocrine potential of hesc - derived cd34 + cells has recently been demonstrated by goodrich et al . who transplanted sheep with these cells by in utero intraperitoneal injections prior to development of the immune system in the fetus so that tolerance toward foreign antigens was acquired during gestation and persisted in the adult . in animals transplanted with differentiated cell populations and followed up to 55 months after transplantation , they detected human dna and insulin messenger rna in sheep pancreases as well as human c - peptide in serum . as few as 23,500 cells were able to achieve long - term sustainable -cell - like activity . these results along with the absence of teratomas , combined with the hematopoietic potential of these cells suggest that not only do hesc - derived cd34 + cells have potential for long - term in vivo endocrine cellular activity but could also be used for the induction of immunological tolerance and bone marrow chimerism prior to cellular therapy for diabetes . a phase ii islet transplantation clinical trial at the university of miami is currently ongoing using monoclonal antibody campath-1h for induction of immunosuppression combined with the simultaneous infusion of islets with donor cd34 + enriched bone marrow cells ( clinicaltrials.gov identifier : nct00315614 ) to reverse hyperglycemia , induce a state of donor - specific tolerance and eliminate the need for continuous immunosuppressive therapy . a study to investigate the immunomodulatory role of mobilized autologous hematopoietic stem cells ( hscs ) via antagonism of the cxcr4-cxl12 axis in promoting islet engraftment following allotransplantation demonstrated mobilization of hscs and prolongation of islet graft survival that was further enhanced by the addition of rapamycin to anti - cxcr4 therapy , inducing a robust and transferable host hyporesponsiveness . mobilized hscs expressed high levels of the negative costimulatory molecule programmed death ligand 1 ( pdl1 ) and suppressed the in vitro alloimmune response . thus , targeting the cxcr4-cxcl12 axis mobilized autologous hscs and promoted long - term survival of islet allografts via a pd - l1-mediated mechanism . halting the cxcr4 antagonist - mediated hsc release by administration of an ack2 ( anti - cd117 ) mab restored allograft rejection . the innate anti - inflammatory and immunosuppressive potential of human amniotic epithelial cells ( aecs ) to create localised immuneprivilege in an in vitro islet cell culture system as an alternative to immunosuppressive drug therapy was investigated . islets transduced with bioengineered cellular constructs composed of human islets and aec ( islet : aec ) demonstrated sustained , physiologically appropriate insulin secretion and reduced mitogen - induced pbl proliferation suggesting that transplanted islets may benefit from the immune - privilege status conferred on them as a consequence of their close proximity to human aec and that this approach may reduce the need for chronic systemic immunosuppression . in experimental islet transplantations , both blood perfusion as well as the tissue oxygen tension of the grafted islets are chronically decreased in the transplanted islets , indicating that reestablishment of an appropriate microvascular supply is an essential prerequisite for successful islet engraftment . the islet grafts depend upon endothelial cells and microvessels in the implantation organ for derivation of a new vascular system [ 193 , 194 ] . improved islet graft survival and function have been observed on exposure to growth factors such as basic fibroblast growth factor ( bfgf ) , endothelial cell growth factor and particularly vegf , which has been known to contribute significantly to the vascularization of transplanted islets . in humans , the vegf family of homodimeric glycoproteins consists of vegf - a , -b , -c , -d , and placental growth factor . interestingly , although pancreatic islets continuously express vegf - a , upon subjection to hypoxia following transplantation devascularized grafted islets significantly increase their expression of vegf , initiating revascularization and maintaining the vascular permeability [ 196199 ] . vegf - a stimulates ec permeability and chemotaxis through cognate vegf receptors and is a prerequisite for islet endothelial fenestration [ 194200 ] . korsgren and magnusson 's group showed that immobilizing heparin on the islet surface was useful in achieving complete coverage of islets with vegf - a as a means of attracting ecs to induce angiogenesis and revascularization , ultimately improving islet revascularization and engraftment in pancreatic islet transplantation [ 35 , 44 ] . a very recent study indicates that concomitant transplantation of isolated islets with ecs can prolong islet graft survival in diabetic rats . cartilage oligomeric matrix protein - angiopoientin-1 ( comp - ang1 ) is a specific growth factor that induces vascularization via the tie2 or tie1 receptor . using an in vitro angiogenesis assay based on a three - dimensional collagen - based culture system , park et al . recently demonstrated that the transduction of comp - ang1 into islets significantly increased angiogenesis . comp - ang1 transduced islets also attenuated hyperglycemia in syngeneic stz - induced diabetic c57bl/6 mice and enhanced glucose tolerance . in another study , following subcutaneous transplantation of balb / c islets in vegf and hepatocyte - growth - factor ( hgf ) supplemented matrigel basement membrane matrix into diabetic scid mice , histopathologic analysis of the functioning grafts harvested at 15 days revealed significantly increased blood vessel formation and increased number of islets . enhanced intercellular adhesion molecule ( icam ) and vascular cell adhesion molecule ( vcam ) within the islets was also observed suggesting stable blood vessel formation . transcription factors focal adhesion kinase phosphorylation and extracellular signal - regulated kinase1/2 phosphorylation were also increased ( 8-fold and 4.6-fold , respectively ) . these results suggest synergistic enhancement of angiogenesis by vegf and hgf following islet transplantation resulted in stable engraftment . in order to drive delivery of growth factors such as vegf and fgf into the dense islet interior , chow et al . developed heparin - binding peptide amphiphile ( hbpa)/heparin nanofiber gels that can activate heparin - binding , angiogenic growth factors . infiltration of bioactive nanofibers in the interior of islets acted as an artificial extracellular matrix ( ecm ) improving cell viability and function and enhancing their vascularization in the presence of growth factors such as fgf2 and vegf . the intraislet nanofibers helped retain fgf2 within the islet for 48 h and increased cell viability significantly for at least 7 days in culture . furthermore , enhanced insulin secretion was observed with the nanofibers for 3 days in culture . delivery of fgf2 and vegf in conjunction with the hbpa / heparin nanofibers also induced a significant amount of islet ec sprouting from the islets into a peptide amphiphile 3d matrix . mahato coexpressed human vegf ( hvegf ) and human il-1 receptor antagonist ( hil-1ra ) as well as human hgf ( hhgf ) and hil-1ra in human islets using adv - hvegf - hil-1ra adv - hhgf - hil-1ra constructs to study their effect on -cell proliferation and revascularization of islets . a dose- and time - dependent expression of hvegf and hil-1ra or hhgf and hil-1ra by islets was observed that led to a decrease in caspase-3 activity and apoptosis induced by a cocktail of tnf- , il-1 , and ifn-. also , transduction of islets with these bipartite adv vectors prior to transplantation in diabetic nodscid mice reduced blood glucose levels and increased serum insulin and c - peptide levels . immunohistochemical staining of the graft revealed positivity for human insulin , hvegf or hhgf , and von willebrand factor . transduction with adv - caspase-3-shrna also prevented islets from cytokine - induced apoptosis and improved islet transplantation . the therapeutic potential of hyperbaric oxygen therapy ( hbo ) in reducing hypoxia , enhancing vessel maturation , and improving engraftment of intraportal islet transplants by promoting angiogenesis in the critical period following transplantation has also been demonstrated . in this study , hyperbaric oxygenation combined with implantation of a foam dressing , vacuum - assisted wound closure ( foam+vac ) to create a prevascularized site was used to achieve better results in microencapsulated xenogeneic cell transplantation [ 206 , 207 ] . interestingly , following intraportal islet transplantation of porcine islets to diabetic nmri nu / nu mice , the combination of cytokines with hypoxia resulted in a strong induction of cell death that could be blocked dose - dependently by a selective ikk- inhibitor that caused systemic nf-b inhibition , significantly prolonging islet graft survival . under hypoxia , nf-b activity impaired expression of antiapoptotic genes bcl - xl , c - flip and survivin . nf-b thus appeared to have an antiapoptotic role under normoxia , while low oxygen conditions decreased its activity and transformed it to a proapoptotic transcription factor in pancreatic islets suggesting that nf-b inhibition represented a potential strategy to improve islet transplantation efficiency . significant islet loss related to reduced survival of large islets compromised by hypoxia has been observed under standard culture conditions . in order to improve the islet graft quality prior to transplantation , rat islets have been cultured for 48 h in a liquid - liquid interface culture system ( lics ) using perfluorodecalin , a method of culture that avoids exposure of islets to relative hypoxia . results indicated that this protocol optimised culture conditions , which preserved both islet viability and significantly increased their ability to engraft successfully after intraportal transplantation and could be used for islet transportation . evaluating neovascularization and correlating angiogenesis with metabolic and functional islet graft condition in diabetic mice is a crucial aspect in assessing graft survival . to this end , very recent studies demonstrated the successful use of dynamic contrast - enhanced magnetic resonance imaging ( dce - mri ) after intravenous injection of gadolinium [ 210 , 211 ] . mri has also been used to effectively image isolated mouse islets labeled with novel mri contrast agent , chitosan - coated superparamagnetic iron oxide ( cspio ) nanoparticles as long as 18 weeks after transplantation as well as islets labeled with feridex - polyethyleneimine complex [ 212 , 213 ] . a recent study using a dual - purpose therapy / imaging probe consisting of therapeutic ( sirna targeting apoptosis - related gene human caspase-3 ) and imaging ( magnetic iron oxide nanoparticles , mn ) moieties showed that treatment with the probe resulted in significantly better survival of transplanted islets that could be monitored by in vivo magnetic resonance imaging ( mri ) . bone - marrow - derived stem cells ( bmscs ) have been shown to promote islet graft function and survival by initiating angiogenesis [ 215 , 216 ] . a recent study involving cotransplantation of bone marrow cells with islets was associated with enhanced islet graft vascularization and function . a significant increase in new peri - islet vessels as well as staining for vegf was observed . the presence of pancreatic duodenal homeobox-1 ( pdx-1 ) was detected in bmscs with an increase in staining over time . 7 months indicated upregulated levels of angiogenesis factors vegf - a , pdgf , kgf , timp-1 , and angiogenin as well as lower protein levels of angiopoietin-2 . bm - induced vascularization showed significant ec distribution and islet vascularization was linked to islet growth . furthermore a 28.66-fold increase in insulin and 24.4-fold glucagon gene expression was also observed . transplantation of genetically marked whole bone marrow from tie2-cre / zeg mice into lethally irradiated wild - type mice evoked pronounced proliferation of recipient ecs while significantly increasing -cell mass and reducing the hyperglycemia of mice subjected to -cell damage by stz . a study indicated that bone marrow cells produced nerve growth factor ( ngf ) and promoted angiogenesis around transplanted islets . biochemical and histological analyses following syngeneic cotransplantation of islets and bone marrow in stz - induced diabetic mice indicated significantly low blood glucose levels high serum insulin levels , and increased serum ngf levels . a significant increase in the number of vessels within the graft area at day 14 after transplant was observed along with improvement in graft function . thus , an important adjuvant role of transplanted bmscs in both angiogenesis and -cell regeneration has been elucidated . cotransplantation of pancreatic islets and adipose - tissue - derived stem cells ( adscs ) too was shown to significantly prolong graft survival and insulin function of islet grafts in diabetic mice . adscs have angiogenic potential and anti - inflammatory properties and cotransplantation studies indicated significant revascularization ( larger number of von willebrand factor - positive cells ) and marked inhibition of inflammatory cell infiltration , including cd4 + and cd8 + t cells and macrophages , in islets - adscs grafts . creation of a rich subcutaneous vascular network with implanted adipose - tissue - derived stromal cells and adipose tissue enhanced subcutaneous grafting of islets in diabetic mice . cografting of neural crest stem cells with pancreatic islets in alloxan - induced diabetic mice too improved insulin release and enhanced -cell proliferation , resulting in increased -cell mass . multipotent human mscs possess powerful ex vivo expansion and ec differentiation potential , placing them at the forefront in the field of vasculature - directed cell - based therapy and transplantation . a recent study revealed that islets co - cultured with mscs demonstrated lower adp / atp ratios , higher gsis indexes and viability . furthermore , co - cultured islets revealed higher levels of antiapoptotic signal molecules ( xiap , bcl - xl , bcl-2 , and heat shock protein-32 ) , increased vegf receptor 2 and tie-2 mrna expression and elevated levels of phosphorylated tie-2 and focal adhesion kinase protein . islets cultured in msc - conditioned medium ( msc - cm ) for 48 hr significantly lowered blood glucose levels and demonstrated enhanced blood vessel formation upon transplantation into stz induced diabetic mice . significant levels of il-6 , il-8 , vegf - a , hgf , and tgf- were detected in msc - cm suggesting that the trophic factors secreted by human mscs enhanced islet survival and function after transplantation . similar improvement of islet graft morphology and function attributable in part to the promotion of graft revascularization was observed when lewis rat islets cocultured with syngeneic mscs were infused into the liver of stz - diabetic syngeneic recipients or when islets were cotransplanted under the renal capsule of nodscid mice with syngeneic mscs expanded in culture . other studies have demonstrated increased mean capillary density upon co - transplantation of mscs with pancreatic islets along with improved islet graft function indicating the beneficial effect of msc - mediated graft vascularization . in mice , islets co - transplanted with mscs maintained a morphology closely resembling that of islets in the endogenous pancreas , both in terms of size , and of endocrine and ec distribution . superior vascular engraftment as shown by increased ec numbers within the endocrine tissue as well as improved graft function demonstrated by normoglycemia achieved in 92% of mice indicated that mscs profoundly influenced the remodeling process , by improving islet revascularization and maintaining islet organisation [ 224 , 225 ] . in order to study the effect of mscs on islet survival and insulin secretion under hypoxia / reoxygenation- ( h / r- ) induced injury conditions that are associated with islet graft dysfunction , purified rat islets cultured with or without mscs , were exposed to hypoxia ( o(2 ) 1% ) for 8 h followed by reoxygenation for 24 and 48 h , respectively . mscs maintained a higher level of stimulation index ( si ) of gsis in islets in vitro , protected islets from h / r - induced injury by decreasing the apoptotic cell ratio and increasing hif-1 , ho-1 , and cox-2 mrna expression , and significantly increased insulin expression following islet transplantation . this study indicated that mscs could promote anti - apoptotic gene expression by enhancing their resistance to h / r - induced apoptosis and dysfunction providing an experimental basis for the therapeutic use of this strategy for enhancing islet function . apart from the beneficial therapeutic role of stem cells in immunomodulation and promoting vasculogenesis following islet transplantation , they may also play an important role in regeneration of -cells . the concept of regenerating -cells from a self renewing , expandable stock of pluripotent escs , pancreas - derived multipotent progenitor / stem cells , extrapancreatic adult stem cells ( e.g , bmscs , neural progenitor cells , ucb - scs , etc . ) or ipscs into large quantities of cells with an insulin - expressing phenotype in vitro offers an attractive alternative source for -cell replacement therapy [ 227 , 228 ] . while the immunosuppressive , anti - inflammatory , and angiogenic properties of mscs are of tremendous advantage , the ability of ipscs to generate an unlimited supply of clinically compliant , functional autologous -cells provides a definitive solution to the cited limitations of islet transplantation , namely , shortage of donor pancreases and the harmful side effects of chronic immunosuppressive therapy . based on sequential exposure of human escs to epigenetic signals that mimic in vivo pancreatic development , insulin - producing cells can be generated from escs [ 227 , 229231 ] . these differentiated cells display architectural similarity to mature primary islets , are capable of synthesizing insulin , glucagon , somatostatin , pancreatic polypeptide and ghrelin [ 229 , 230 ] , reverse hyperglycemia in diabetic mice , prolong graft survival , and respond successfully to glucose challenge in glucose - tolerant tests ( gtt ) providing definitive evidence of the ability of hescs to serve as a renewable source of insulin - secreting -cells for diabetes cell - replacement therapies . the risk of teratoma formation and tumor formation and the difficulty of purifying the differentiated progeny are major drawbacks . multipotent bm - mscs are plastic - adherent cells , expressing surface markers such as cd90 , cd73 , cd105 , cd44 , and cd29 that can be isolated and expanded with high efficiency in culture and can differentiate into cells of connective tissue lineages , including bone , fat , cartilage , and muscle . the capacity of bm - mscs to generate insulin - producing cells [ 232 , 233 ] capable of producing and releasing insulin in a glucose - dependent manner and normalizing hyperglycemia upon transplantation into a diabetic mice [ 234 , 235 ] while at the same time abrogating immune injury , altering t cell cytokine pattern toward il-10/il-13 production and preserving cd4+/cd8 + foxp3 + tregs in the periphery make bm - mscs an invaluable tool in -cells replacement therapies . in vivo differentiation of hucb cells into -cells following transplantation into stz - induced diabetic immunocompromised [ 237 , 238 ] or nod mice indicates the potential role of these cells in -cell replacement therapy . ipscs may be derived from autologous somatic cells by ectopic expression of the transcription factors oct4 , sox2 , c - myc , and klf4 or oct3/4 , sox2 , nanog , lin28 and are molecularly and functionally highly similar to escs , offering an important alternative to replenish -cell supply while simultaneously obviating immune concerns such as rejection and chronic immunosuppression . most human ipsc lines can be induced into pdx1-positive progenitor cells and further differentiated into pancreatic lineage cells using a stepwise induction strategy [ 243 , 244 ] . other alternative sources of -cells include intrahepatic biliary epithelial cells and gall bladder epithelium , human neural progenitor cells , hepatic oval cells placenta - derived multipotent stem cells , and adult pancreatic stem / progenitor cells [ 249 , 250 ] . the common embryonic origin of liver and pancreas , similarities in their glucose - sensing systems , mutually expressed transcription factors , and the high level of developmental plasticity exhibited by adult human liver cells indicate the therapeutic potential of liver stem cells / hepatocytes as a source of pancreatic progenitor tissue . several studies have demonstrated reprogramming of hepatocytes into function insulin - producing cells by expression of the pdx1 or its superactive form pdx1-vp16 fusion protein either alone or in combination with other pancreatic transcription factors using first generation , nontoxic , transiently expressed adenoviral vectors under conditions of hyperglycemia or hepatic regeneration [ 251253 ] . however , although the potential of stem cells in the future of t1d interventional therapies is immense , the accompanying risk of mutagenesis , teratoma and tumor formation needs to be stringently addressed . for now it appears that the combination of multiple therapeutic avenues is required to achieve the dream of permanently reversing / preventing t1d . areas of current research include the development of less toxic immunosuppressive regimens , the suppression of inflammatory responses immediately following transplantation , the identification of an optimal anatomical site for islet infusion , and the possibility of encapsulating transplanted islets to protect them from the alloimmune response . the generation of tregs with defined alloantigen specificity could provide dynamic control of rejection responses and offer a potential route to permanent graft survival without the need for life - long nonspecific immunosuppression . regeneration of -cells utilizing every kind cell from the pancreas , stem cells as well as cells from alternate sources , followed by transplantation using immunosuppressive regimens that would ensure maximal graft survival through protection from hypoxic and immune insults is an exciting alternative . clinical islet transplantation represents a possible definitive intervention for patients with t1d and with significant inroads in the branches of stem cell therapy , immunomodulation and gene therapy , the prospect of translating these beneficial interventions into clinical applications that promote successful long - term functional islet graft survival appears within reach .	clinical islet transplantation is a -cell replacement strategy that represents a possible definitive intervention for patients with type 1 diabetes , offering substantial benefits in terms of lowering daily insulin requirements and reducing incidences of debilitating hypoglycemic episodes and unawareness . despite impressive advances in this field , a limiting supply of islets , inadequate means for preventing islet rejection , and the deleterious diabetogenic and nephrotoxic side effects associated with chronic immunosuppressive therapy preclude its wide - spread applicability . islet transplantation however allows a window of opportunity for attempting various therapeutic manipulations of islets prior to transplantation aimed at achieving superior transplant outcomes . in this paper , we will focus on the current status of various immunosuppressive and cellular therapies that promote graft function and survival in preclinical and clinical islet transplantation with special emphasis on the tolerance - inducing capacity of regulatory t cells as well as the -cells regenerative capacity of stem cells .
a healthy 27-year - old woman was referred to our hospital for a nonresponsive , progressive corneal ulcer in the left eye . one month before visiting our clinic , she went to a local ophthalmic clinic complaining of irritation , redness , and pain in the left eye , where a corneal ulcer was diagnosed and topical gatifloxacin and tobramycin were given alternatively every hour . the medication was increased to an hourly fortified topical cefazolin at a dosage of 50 mg / ml . one week after eyedrop application , fibrin gluing with bandage contact lens was done twice , due to the impending perforation of the thinned ulcerative area . the initial culture revealed no growth . the patient was then referred to our department for further evaluation . on examination , the best - corrected visual acuity was 20 / 20 in the right eye and hand movement in the left eye . the left eye showed a large ulcer , measuring 5 3 mm , with an overlying fibrin gluing patch ( fig . severe chemosis and lid swelling were combined , but intraocular or orbital dissemination was not found on a b - scan ultrasound and an orbit computed tomography exam . the anterior chamber was maintained without chamber collapse and observed as an inflammation of 2 + cells without a hypopyon . feathery - like infiltration was observed at the margin of the lesion , even when obscured by overlying fibrin glue . corneal scraping was not performed and disease progression was observed for two days , because the lesion margin was overlaid with the gluing patch and the risk of perforation was high when the glu ing patch was handled . the ulcer lesion slowly progressed and the symptoms , including ocular pain , conjunctival injection was aggravated . we decided to remove the gluing patch and perform a corneal scraping and biopsy with a multiple amniotic membrane graft to seal the thinned or perforated cornea . necrotic , infiltrated debris had expanded , surrounding the lesion , and an iridocorneal adhesion under the ulcerative lesion was observed ( fig . corneal scraping for microbiologic evaluation was carefully done at the ulcerative base and margin and necrotic tissue was also debrided to improve drug penetration . a three - layered amniotic membrane was patched over the ulcerative lesion and the outermost layer covered the lesion over 3 mm from the margin of the lesion , including the half cornea and limbus . finally , intracameral cefuroxime ( 1 mg/0.1 ml ) and amphotericin b ( 0.5 g/0.1 ml ) were injected . on the second day after the surgery , the size of the ulcer had decreased and the conjunctival injection , lid swelling , ocular pain gradually improved . three days after the surgery , the corneal cultures grew fusarium , as well as enterococcus faecalis . topical amphotericin b ( 0.15% ) and gatifloxacin were still continuously administered hourly and the patient was also started on oral itraconazole . topical cycloplegics and antiglaucomatics were added to control the pain and lowering the intraocular pressure , respectively . topical amphotericin b and gatifloxacin were reduced to four applications per day after one week . three weeks after surgery , the outermost layer of amniotic membranes , as the temporary patch , was removed . all antifungal medications were discontinued , as the infection sign was clinically and symptomatically clear four weeks later . two months after surgery , the lesion was totally opaque and sclerotic and the thinned area was enhanced by permanent multiple amniotic grafts and the overlying epithelium was well maintained ( fig . a best - corrected visual acuity was recovered to 20 / 60 in the left eye . corneal thinning or perforations can result from a variety of ocular disease states associated with trauma , acute or chronic inflammation . regardless of the cause , perforation creates an emergent situation and prompt treatment is necessary . many surgical approaches , such as tissue adhesive , bandage contact lens , conjunctival flap , and therapeutic penetrating keratoplasty , have been introduced to treat perforated ulcers of the cornea . corneal transplant is a definitive treatment , but preserved cornea are of limited supply , and preceding temporary patching with the management of the underlying disease is emphasized . tissue glues have been used in ophthalmology to treat corneal thinning and perforations , and ocular surface disorders . several case series have reported the successful use of fibrin glues , as well as cyanoacrylate glues , in the management of corneal perforations . according to a report by sharma et al . , even though the properties of cyanoacrylate and fibrin glue seem very different , their success in sealing corneal perforations appears to be similar . in the management of 41 corneal perforations , they found that both fibrin and cyanoacrylate glues were similarly very effective in the closure of corneal perforations up to 2 mm in diameter . however , the use of tissue glue in treating infectious ulcerations has been limited in practice . the development of infectious infiltrates after prolonged cyanoacrylate glue adhesion has been documented in numerous studies . the symptoms and signs of the infectious ulcers may have been concealed by the opaqueness of the glue . furthermore , the development of resistant organisms should be considered . for this reason , the use of antibiotic prophylaxis is recommended while the cyanoacrylate glue is applied and the antibiotic may be changed periodically to minimize the growth of resistant organisms . also , the efficacy of antibiotics in treating infectious ulcerations may be limited due to tissue necrosis or overlying glue itself . in this case , a fibrin glue without a covering layer was used to manage the impending perforation in infectious keratitis . to our knowledge , this case is the initial report in which a secondary infection developed after the use of fibrin glue , although several cases of corneal ulcers by mixed microorganisms were previously reported in korea . despite the same medications , the keratitis was improved only by the removal of the overlying fibrin glue . firstly , necrotic tissue and bandage contact lenses may interrupt the antibiotics delivery , promoting microbiologic colonization . secondly , low - inflammatory properties of fibrin glue may prove disadvantageous in circumstances where vascularization is desired , such as infectious ulcers . this is similar to the principle of conjunctival flaps in the management of refractory corneal ulcers . finally , it is assumed that the application of fibrin glue in perforated infectious ulcers has the same limitations as cyanoacrylate glue . in contrast to the cyanoacrylate glues as synthetic adhesives , the benefits of fibrin glue relates to its biodegradability and flexibility . the bonding that occurs with fibrin glues simulate the final stage of the coagulation cascade , producing a biocompatible fibrin matrix similar to a natural plasma clot . although the fibrin glues have a lower tensile strength and slower polymerization , they may induce minimal inflammation , due to their being biologic and biodegradable . although there is no comparative study of the drug penetration between the cyanoacrylate and fibrin glues , it is expected that fibrin glues , which are more biocompatible than cyanoacrylate glues , may be advantageous in their management of infectious corneal perforation . in experimental studies , fibrin glue has also been used as a vehicle for delivering antibiotics and platelet gel , potentially containing natural healing factors that promote tissue regeneration [ 23 - 26 ] . however we found in this case that the application of fibrin glue to a perforated infectious ulcer aggravated the ulcer lesion and caused a secondary infection to deveop . in conclusion , fibrin glue is useful in the treatment of corneal perforations and corneal thinning , but it has also the risk of microbial colonization , and secondary infection , as with cyanoacrylate glue . the use of fibrin glue should be avoided , especially in infectious keratitis , and prophylactic antibiotics should be used even in sterile keratitis , because lesion can be aggravated and a secondary infections may develop . future studies may reveal pharmacologic properties or biochemical effects on tissue for each of the available glues , and newer compounds may be developed with better biocompatibility or drug penetration .	a healthy 27-year - old woman with a corneal ulcer underwent fibrin gluing with a bandage contact lens twice , due to an impending perforation . the ulcer lesion slowly progressed , unresponsive to topical antibiotics and amphotericin b. we removed the gluing patch and performed a corneal or scraping or biopsy with multiple amniotic membrane grafts to seal the thinned or perforated cornea . three days after the surgery , the corneal cultures grew fusarium , as well as enterococcus faecalis . three weeks after surgery , the outermost layer of amniotic membranes , serving as a temporary patch , was removed . the anterior chamber was clear without cells . the signs of infection clinically and symptomatically cleared up four weeks later . two months after surgery , the lesion became enhanced by amniotic membranes . the use of fibrin glue in infectious keratitis should be avoided , because it not only masks the underlying lesion , but it also interferes with drug penetration into the underlying lesion .
hemodialysis is one of the most prevalent treatments for these patients estimated as over 1 million worldwide . in iran , this estimate was about 11,250 patients in 2003 , 18,000 in 2007 , and 20,000 in 2012 . the patients undergoing hemodialysis usually have several complications which result from either the end stage of renal failure or the type of treatment . one of these complications is restless leg syndrome that is accompanied with sensory signs and motion disorders of limbs , especially legs . the signs of this syndrome are observed during rest or lack of activity and are reduced by moving the involved limb . although this syndrome signs are present during the day and night , they get worse at night , and it leads to patients ' sleep disorder and influences their function , especially in the evening and at night . this syndrome is more prevalent in chronic renal failure patients compared to normal population in such a way that primary estimation of its prevalence among hemodialysis patients showed it to be 2080% . the associated pathophysiology of restless leg syndrome is yet unknown , but as the syndrome is relieved by low dosage of levodopa consumption , dopaminergic system is considered to play the main role in causing such a syndrome . as medications have their own specific side effects , patients are always after non - medicational methods including complementary medicine , of which reflexology can be named . reflexology is a sort of pressure , which is often given on the feet . experts of reflexology believe that feet can be divided into several reflex points which are associated with and connected to all body organs and parts . the main mechanism of this treatment is through its effect on nervous system in such a way that the pressure to a specific part of skin stimulates nervous reflexes and their transmission to brain . on the other hand , reflexology provokes the chemical system of the nerves , balances the enzymes , and regulates endocrine function . there are other beneficial non - medicational methods to reduce the severity of restless leg syndrome . research showed that movement - based therapy methods have a positive effect on controlling some of the syndrome signs , and the patients ' daily function can be improved through physical activity strategies . movement therapy is among the effective strategies to improve motor function , prevent the complications , and lower the disabilities resulting from restless leg syndrome . among the movement therapy methods , stretching exercises are one of the oldest treatment methods , which improve muscles ' circulation and facilitate provision of nutrients to the cells . as poor circulation enhances manifestation of restless leg syndrome signs and activity modifies this condition , stretching exercises may be effective in reduction of this syndrome severity . based on the obtained results , majority of the activities of hemodialysis nurses are carried out in the absence of the physicians , such that their role in treatment of the patients has been estimated as 80% . nephrology nurses should be able to control the complications resulting from the disease , and administrate renal replacement treatment and proper non - medicational interventions for the patients . restless leg syndrome highly influences the physical and psychological aspects of hemodialysis patients and impairs their trend of life . on the other hand , the number of consumed medications is very high among these patients , and most of these medications are excreted through kidneys ; therefore , adding another medication to treatment can add up to patients ' problems , and thus , non - medicational methods seem essential for these patients . as research showed the effect of reflexology complementary medicine and stretching exercise on the level of dopamine , the researchers decided to investigate the effect of two methods of reflexology massage and stretching exercises on the severity of restless leg syndrome among patients undergoing hemodialysis . this is a three - group , two - stage ( before , after ) clinical trial ( nirct no . study population comprised all chronic end - stage renal failure patients who referred to selected hospitals in isfahan three times a week and underwent hemodialysis for 4 h in each session . inclusion criteria were patients aged 1865 years whose hemodialysis had started for at least 3 months prior to the study . those who had no idiopathic restless leg syndrome , not consuming medications to manage restless leg syndrome signs or medications worsening these signs ( three - cycle antidepressants , serotonin selective reuptake inhibitors , anti - nausea medications , antiepileptics , antipsychotics , dopamine antagonists ) , no infection , wound , and a serious complication in feet , and peripheral neuropathy or vascular problems in lower limbs were selected . whenever the subjects lost interest to remain in the study or any change occurred in the inclusion criteria at any stage , the subjects were excluded . in the first stage , after obtaining an informed written consent , all hemodialysis patients completed restless leg syndrome standard questionnaire . the clients who responded positive to all four questions of restless leg syndrome diagnostic questionnaire were considered as the clients with restless leg syndrome . next , through random numbers table , 90 subjects meeting the inclusion criteria were selected , and the goal of the study was explained to them by the researcher in their first meeting . demographic information form was completed through referring to patients ' medical files and questioning the clients . the subjects were notified that they could ask their questions and ambiguous points through telephone calls and receive the answers . in the second stage , the subjects were assigned to three groups through random allocation and use of sealed and plumed envelopes each patient randomly selected one of the envelopes and was assigned to the group mentioned in the envelope . then , through restless leg syndrome severity standard questionnaire , the score of restless leg syndrome was calculated for each patient separately and the total mean of each group was calculated . next , interventions of reflexology and stretching exercise were administrated for two groups as three sessions a week ( 12 sessions ) , each session lasting for 3040 min in the first 2 h of dialysis session in which there were no notable changes in bp respectively . the second questionnaire was restless leg syndrome severity standard measurement questionnaire in which the patients with scores less than 10 were categorized in minor ; 1120 as moderate ; 2130 as severe , and 31 or over as being in a very severe stage of the syndrome . restless leg syndrome severity questionnaires were completed by the researcher before and immediately after the interventions . validity and reliability of both questionnaires in iran were confirmed by habibzade et al . in 2011 . content validity and cronbach alpha were used to confirm the validity and reliability of the data collection tools . cronbach alphas were calculated to be 0.97 and 0.94 for restless leg syndrome diagnosis and restless leg syndrome severity investigation questionnaires , respectively . data were analyzed by paired t - test one - way analysis of variance ( anova ) and least significant difference ( lsd ) post hoc test , and the significance level was considered as p < 0.05 . findings of the present study showed that the subjects ' mean ( sd ) age was 55.45 ( 12.08 ) years , mean length of hemodialysis was 35.34 ( 29.01 ) months , and 50% of the subjects were women . statistical tests showed that frequency distribution of sex , mean age , and length of hemodialysis ( months ) were identical in the three groups and showed no significant difference . paired t - test showed a significant difference in the mean scores of restless leg syndrome severity before and immediately after intervention in reflexology and stretching exercises groups , respectively ( p < 0.001 ) , but showed no significant difference in the control group . one - way anova showed no significant difference in the mean score of restless leg syndrome severity in the three groups before intervention , but the difference was significant immediately after intervention ( p < 0.05 ) [ table 1 ] . mean score change of restless leg syndrome in three groups of reflexology , stretching exercises , and control immediately after intervention showed that the changes were significantly more in the two study groups compared to control ( p < 0.05 ) in such a way that both interventions reduced the severity of restless leg syndrome signs more , compared to the control group [ table 2 ] . comparison of restless leg syndrome mean scores in reflexology , stretching exercises , and control groups before and immediately after intervention restless leg syndrome mean score changes in reflexology , stretching exercises , and control groups immediately after intervention lsd post hoc test showed a significant difference in the mean scores of restless leg syndrome severity immediately after intervention in reflexology and control and in stretching exercises and control groups ( p < 0.05 ) [ table 3 ] , but there was no significant difference in the mean scores of restless leg syndrome severity immediately after intervention in reflexology and stretching exercises . comparison of the effects of reflexology and stretching exercises methods showed that both methods were effective , and there was no significant difference . the severity of restless leg syndrome signs was significantly reduced after reflexology therapy , compared to before intervention . stated that reflexology therapy reduced the severity of fatigue , pain , and muscular cramps of hemodialysis patients . wang et al . showed that reflexology reduced muscular cramps during the interval between two sessions of hemodialysis . ( 2011 ) reported that reflexology therapy reduced pain , controlled blood sugar , and improved nervous and temperature conductivity among diabetic nephropathy patients . lee et al . , in a systematic review study , showed the effect of reflexology therapy on fatigue , sleep disorders , and pain . the main mechanism of treatment in reflexology is its effect on the nervous system in such a way that the pressure given on a specific part of skin stimulates nervous reflexes and transmission of impulses to the brain . this mechanism seems to be effective on reduction of complications of various diseases including restless leg syndrome in hemodialysis patients . our obtained findings also showed that restless leg syndrome severity was significantly lowered after stretching exercises , compared to before intervention . giannaki et al . also showed that educating the patients with sports and administering low dosage of dopamine antagonist were effective on reduction of restless leg syndrome signs to 46% and 56% , respectively . ( 2006 ) showed that a sport program with low severity acted as an assistive treatment in improvement of physical function and efficiency of hemodialysis patients . showed that the fall risk of hemodialysis patients was reduced after a strength and balance exercises program . 's literature review study showed that sport had positive effects on reduction of cardiovascular diseases , increase in quality of life and physical function , reduction of depression and muscular disease , and reduction of mortality in patients with restless leg syndrome . smart and steele , in 15 studies , showed the effects of sports education on preservation of fat - free body mass , quadriceps muscle , prevention of knee deformation , and increase in strength of knee and pelvis joints in hemodialysis patients . reported the positive effect of aerobic sports on improvement of restless leg syndrome signs in hemodialysis patients . henrique et al . reported that regular aerobic exercises were effective on improvement of physical capacity and hypertension control of hemodialysis patients , which is consistent with the present study . although the mean changes of restless leg syndrome were higher in stretching exercises group compared to reflexology , they were not significant . therefore , stretching exercises seem to be more effective on reduction of disease complications as well as some of the problems of patients , such as restless leg syndrome . as learning these exercises is easy and almost possible for all patients and needs no special equipments and costs , the disturbing complications of this syndrome can be lowered through patients ' education of these exercises and supervising the patients ' proper practice during hemodialysis . in this way , the economic and psychological burden imposed on the patients and their families can be prevented . therefore , educating the patients and health care team concerning these two methods can be an effective action in reduction of this syndrome . the only limitation that was out of researcher 's control in the present study was subjects ' personal differences concerning social , cultural , psychological , and familial factors , which might have affected the evaluation and tolerance of the severity of signs in patients . in the end , the researchers suggest conducting a similar study with a bigger sample size and for a longer period of time , as well as investigating the severity of restless leg syndrome signs 2 and 6 months after intervention to declare the longevity of intervention and the mean score of restless leg syndrome signs ' severity . results showed that both reflexology and stretching exercises methods were effective on reduction of restless leg syndrome among the patients undergoing hemodialysis , and they are suggested in treatment of such patients .	background : restless leg syndrome prevalence is high among the patients undergoing hemodialysis . due to several side effects of medicational treatments , the patients prefer non - medicational methods . therefore , the present study aimed to investigate the effects of two methods of reflexology and stretching exercises on the severity of restless leg syndrome among patients undergoing hemodialysis.materials and methods : this study is a randomized clinical trial that was done on 90 qualified patients undergoing hemodialysis in selected hospitals of isfahan , who were diagnosed with restless leg syndrome through standard restless leg syndrome questionnaire . they were randomly assigned by random number table to three groups : reflexology , stretching exercises , and control groups through random allocation . foot reflexology and stretching exercises were conducted three times a week for 3040 min within straight 4 weeks . data analysis was performed by spss version 18 using descriptive and inferential statistical analyses [ one - way analysis of variance ( anova ) , paired t - test , and least significant difference ( lsd ) post hoc test].results : there was a significant difference in the mean scores of restless leg syndrome severity between reflexology and stretching exercises groups , compared to control ( p < 0.001 ) , but there was no significant difference between the two study groups ( p < 0.001 ) . changes in the mean score of restless leg syndrome severity were significantly higher in reflexology and stretching exercises groups compared to the control group ( p < 0.001 ) , but it showed no significant difference between reflexology massage and stretching exercises groups.conclusions:our obtained results showed that reflexology and stretching exercises can reduce the severity of restless leg syndrome . these two methods of treatment are recommended to the patients .
catecholaminergic polymorphic ventricular tachycardia ( cpvt ) is an inherited arrhythmogenic disease that can cause syncope or sudden cardiac death during emotional or physical stress in the absence of detectable structural heart disease and a prolonged qt interval on an electrocardiogram ( ecg ) . mutations in two genes , the cardiac ryanodine receptor gene ( ryr2 ) and calsequestrin 2 gene ( casq2 ) , have been identified in patients with cpvt . these mutations lead to an increase in intracellular ca concentration , resulting in life - threatening ventricular arrhythmias , possibly via delayed depolarizations . a 16-year - old male , who had suffered from recurrent episodes of syncope triggered by physical exertion was referred to our hospital after undergoing defibrillation at a local emergency department ( ed ) . he had lost consciousness at school during a volleyball test that was also emotionally stressful . an initial ecg at a local ed revealed ventricular fibrillation . although his arrhythmia was converted to sinus rhythm after defibrillation , the patient was stuporous and had repeated episodes of nonsustained ventricular tachycardia ( vt ) . after referral to our hospital , ecg monitoring in the ed and intensive care unit showed intermittent nonsustained vt and bidirectional vt ( fig . when the patient became agitated , his heart rate increased , and the ecg showed nonsustained polymorphic vt despite intravenous administration of a -blocker . he was sedated and placed on mechanical ventilation . two days later , his heart rate was regular , and no additional episodes of vt were observed . his ecg showed a sinus rhythm with a corrected qt interval of 423 msec ( fig . 2 ) . routine laboratory findings , including electrolytes and two - dimensional echocardiography , were unremarkable . a detailed history of the patient included several episodes of syncope since the age of 8 years associated with physical activities , such as sprinting , playing football , and fighting with a sibling . his father also experienced an episode of syncope associated with sprinting at the age of 9 years . the patient and his family underwent a genetic analysis , and his father and mother each had one different de novo missense mutation in ryr2 ( exon 97 c.14009t > a p.l4670h in the father and exon 37 c.5428g > c p.v1810l in the mother ) . as his episodes of syncope were associated with sympathetic activation , an exercise ecg test and epinephrine infusion test ( intravenous bolus of 0.1 g / kg followed by continuous infusion at rates of 0.1 g / kg per minute ) were performed . however , only premature ventricular complex ( pvc ) bigeminy was induced with no episodes of syncope during the exercise ecg test ( fig . 3 ) and epinephrine only increased his heart rate without an arrhythmia . with these clinical features , we made the diagnosis of cpvt . atenolol ( 37.5 mg bid ; 1.9 mg / kg / day ) was prescribed , and a follow - up exercise ecg test while on the -blocker was performed 3 days later . during the follow - up exercise ecg test the patient remains on 50 mg bid atenolol ( 2.3 mg / kg / day ) and is being followed . to date catecholaminergic polymorphic ventricular tachycardia , arrhythmogenic right ventricular cardiomyopathy , and long qt syndrome are well known causes of exercise - induced syncope that commonly manifest normal baseline ecgs.1)2 ) therefore , many provocation tests , including vt induction , an exercise ecg test , and epinephrine infusion tests , have been used to diagnose exercise - induced syncope.1)2 ) exercise - induced bidirectional vt has been considered the hallmark of cpvt for many years . however , recent studies1)2 ) have shown that ventricular bigeminy is the most common exercise - induced arrhythmia among patients with cpvt , and serious arrhythmias such as bidirectional vt and polymorphic vt are rarely induced . therefore , exercise - induced ventricular bigeminy no longer should be considered an innocent arrhythmia , at least in suspicious cases of cpvt . catecholaminergic polymorphic ventricular tachycardia may have both an autosomal dominant and an autosomal recessive pattern of inheritance . the more common type 1 autosomal dominant cpvt is caused by mutations in ryr2 on chromosome 1q42-q42 and results from defective calcium release from the sarcoplasmic reticulum , which is required for myocardial contraction.3 ) a mutation in ryr2 can increase calcium release and cause life - threatening ventricular arrhythmias . patients with ryr2 mutations become symptomatic at an early age , and men are at higher risk for cardiac events.4 ) in the autosomal recessive variant , type 2 cpvt , the causative gene is casq2 , located on chromosome 1p13 - 21.5 ) casq2 encodes calsequestrin , a calcium buffering protein in the sarcoplasmic reticulum , which binds a large amount of calcium . while the mechanism by which this mutation causes ventricular arrhythmias has not been clearly established , the mutated protein may be associated with the following : loss of polymerization of casq monomers , loss of calcium buffering capability , or indirect destabilization of the ryanodine receptor channel.6)7 ) patients with cpvt , in which many forms of exercise are associated with catecholamine release that triggers vt , should be cautioned against virtually all forms of vigorous and/or competitive physical activity.8 ) since the early reports on cpvt , -blockers have been used as primary therapy for cpvt,9)10 ) and they are indicated for both chronic treatment and acute therapy for sustained vt . other antiarrhythmic drugs such as amiodarone and class i drugs have proved to be ineffective.11 ) however , these issues were addressed in a study that evaluated both the molecular mechanisms and clinical efficacy of flecainide in mice and humans.12 ) implantable cardioverter defibrillators can be considered for primary prevention despite the potential drawbacks in young patients and concerns about provoking arrhythmic storms . left cardiac sympathetic denervation has been reported effective in patients whose symptoms were not adequately controlled by -blockers.13 ) this approach may also be considered for individuals with intractable arrhythmic storms to reduce the number of implantable cardioverter - defibrillator shocks .	a 16-year - old male with a prior history of recurrent syncope was referred to our hospital after being resuscitated from cardiac arrest developed while playing volleyball . his electrocardiogram ( ecg ) demonstrated ventricular fibrillation at a local emergency department . after referral , an ecg showed bidirectional ventricular tachycardia ( vt ) and nonsustained torsade de pointes . two days later , his heart rate became regular , and no additional episodes of vt were observed . his ecg showed sinus rhythm with a corrected qt interval of 423 msec , and two - dimensional echocardiography was unremarkable . we made the diagnosis of a catecholaminergic polymorphic vt . however , only premature ventricular complex bigeminy was induced on exercise ecg and epinephrine infusion tests , and the patient showed no episodes of syncope . his father and mother had different missense mutations in the cardiac ryanodine receptor on genetic testing . the proband had both mutations in different alleles and was symptomatic . it was recommended that the patient avoid competitive physical activities , and a -blocker was prescribed .
dna sample : a total of 658 dna samples extracted from cattle blood from cebu , iloilo , negros oriental , negros occidental , cavite and batangas in the philippines [ 33 , 34 ] were used ( fig . 1.the philippine map indicating the sampling area ( shaded ) . ) . in brief , the dna extraction was performed using a qiaamp dna blood mini kit ( qiagen , hilden , germany ) . dna concentrations were measured using a thermo scientific nano drop 2000 ( thermo fisher scientific , waltham , ma , u.s.a . ) . a dna sample prepared from blood of a japanese black cattle infected with a. marginale was used as the positive control for the subsequent pcr assays . the philippine map indicating the sampling area ( shaded ) . pcr assays : the oligonucleotide sequences of pcr primers used in the present study are presented in table 1table 1.pcr primers used in the present studyprimeroligonucleotide sequencefinal target amplicon ( bp)referencegroelgeneam265f1gactaccacatgctccatactgactg866ama424f2gtctgaagatgagattgcacaggttgam1574r1gacgtccacaactactgcattcaagam1289r2cctttgatgccgtccagagatgcamsp1a genemspa733f1tgtgcttatggcagacatttcc272983mspa2957r2aaaccttgtagccccaacttatccmspa3134r1tcacggtcaaaacctttgcttacc . briefly , for the a. marginale - specific groel nested pcr assay , 2 primer pairs , am265f1/am1574r1 and am424f2/ am1289r2 , were respectively used for the first and second round pcrs to amplify a final 866-bp amplicon . for the msp1a gene , a hemi - nested pcr was performed using two primer pairs , mspa733f1/mspa3134r1 and mspa733f1/ mspa2957r2 , for the first and second round pcrs , respectively . the presence of single or multiple infections of different genotypes were assessed based on the presence of different sizes of visualized bands . cloning and sequencing of pcr products : selected pcr amplicons were purified using either a qiaquick pcr purification kit or a qiaquick gel extraction kit ( qiagen ) . briefly , direct sequencing was initially performed using the 2nd round pcr primers . in some cases where the obtained sequence was of low quality , the pcr amplicons were cloned into a pcr 2.1-topo plasmid ( invitrogen , carlsbad , ca , u.s.a . ) . the nucleotide sequences were then determined using an abi prism 3100 genetic analyzer ( applied biosystems , foster city , ca , u.s.a . ) . sequence and phylogenetic analyses : obtained sequences were manually trimmed to include only the sequence of interest . multiple sequence alignment ( msa ) was performed using a muscle program employed in a mega5 program , as suggested by hall . phylogenetic analyses using a bayesian inference method were performed in a mrbayes 3.2 program and guided by the best model testing results of the msa in mega5 . a. marginale was detected in all the examined locations and was most and least prevalent in cavite ( 62.5% ) and cebu ( 9.6% ) , respectively . among the groel pcr - positive samples ( 19.8% ) , 93 samples ( 14.1% ) cattle infected with single ( 47 or 7.1% ) and multiple ( 46 or 7.0% ) genotypes of a. marginale were observed in the pcr assay ( table 2table 2.list of msp1a tandem - repeat forms of a. marginale detected from the philippine cattlerepeat formencoded sequencereferenceph1adsssasgvlsksdqastssqlgthis studyph2adsssagdrqqesgvssqsgqastssqlgthis studyph3tdsssasgqkqessvlsqsdqastssqlgthis studyph4adsssasgqqqdssvlsqgdqastssqlgthis studyph5tdsssasgqqqesgvlpqsgqastssqlgthis studyph6tdsssasgqqqessvlpqgdqastssqlgthis studyph7tdsssasgqqqessvlsqgdqastssqlgthis studyph8agsssasgqqqdssvlsqgdqastssqlgthis studyph9adsssagdqqqesgvssqsgqastssqlgthis studyph10tdssstgdqqqesgvssqsgqastssqlgthis studyph11adsssasgqqqessvssqlgthis studyph12adsssasdqqqesgvpsqseastssqlg ; this studyph13adsssasdqqqessvlsqsgqastssqlgthis studyph14adsssasgqqqesgvpsqseastssqlgthis studyph15adsssagdqqqessvssqsdastssqlgthis studyph16tdsssasgqrqessvlsqsdqastlsqlgthis studyph17adsssasgqqqessvlsqsdqastlsqlgthis studyph18tdsssasgqqqessvlsqsdqastlsqlgthis studyph19aysssagdqqqessvssqsgqastssqlgthis studyph20tdsssasgqkqessvlpqsgqastssqlgph21adsssagdqqqessvssqsgastssqlgthis study62tdsssagdqqqessvssqsdastssqlg61tdsssagdqqqessvssqsgastssqlgtdsssagdqqqgsgvssqsgqastssqlgrtdsssasgqqqessvssqsdastssq3adsssasgqqqessvlsqsgqastssqlg4tdsssasgqqqessvlsqsgqastssqlg13tdsssasgqqqessvlsqsdqastssqlg14tdsssasgqqqessvlsqsgastssqlg17tdsssasgqqqesgvssqsgqastssqlg21adsssagdqqqessvlsqsgqastssqlg27adsssasgqqqessvlsqsdqastssqlg46tdsssasgqqqessvlpqsgqastssqlgftdsssasgqqqessvssqsgqastssqlgmadsssasgqqqessvssqsgqastssqlgmgl10adsssasgqqqessvlsqsgastssqlgis1tdsssagdqqqesgvssqsgqastssqlgme1 ( provisional)adsssasgqqqgssvlsqsgqastssqlgaev59754 ( mexico ; unpublished ) ybanez et al . , in press ; not detected in the present study . ) . * ybanez et al . the partial groel gene fragments of a. marginale detected from the philippine cattle ( genbank acc . kc113449 - 81 ) revealed 98.6100% identities to each other and 99.2100% to already registered sequences , including those from japan ( ishigaki ; fj226455 ) , israel ( non - tailed ; af414861 ) and australia ( f12 ; af414860 ) , indicating a high conservation of the groel gene among all known a. marginale strains . on the other hand , the lengths of partial msp1a nucleotide ( genbank acc . kc181866 - 915 ) obtained in the present study were variable , ranging from 272 to 983 bp . these sequences were 10.199.9% identical to each other . meanwhile , a total of 38 kinds of tandem - repeat structures of a. marginale msp1a , including 20 novel structures that were unique to the philippine samples , were identified in the present study ( table 2 ) . these novel structures were 90.096.6% identical to those found in mexico , brazil , argentina , south africa , venezuela , japan , israel , china , u.s.a . , marginale msp1a genotypes detected from the philippine cattleareamsp1a tandem repeatnumber of repeatsbatangasph1///r///r7me1/4/m / m/4/4/47ph11/ph11/ph11/ph11/m5ph1/27/27313/13213/27 * 246/f2caviteme1/4/4/4 4ph11/ph14/33ph13/4/43ph16/ph17/mgl10 * 3ph15/622cebu13/13/14/14/13 /14/147ph4/17/ph5/ph6/ph5/ph7613/13/13/14/145ph12/m / ph12/m / m5ph21/62/62/61/61513/13/13/mgl104ph9/is1/is1/ph10413/14/14313/27/14313/27/27321/m / m346/ph20/46313/27 * 213/mgl10246/46*246/f2141171me11ph81131iloiloph4/17/ph5/ph7/ ph5/ph76ph12/m/3/3/m5ph4/17/ph5/ph5/ph75me1/4/4/4 * 4ph16/ph17/mgl10 * 3ph19/m / f313/27/13/144negros occidentalme1/4/m / m/4/46ph21/62/61/ 62/61/626me1/4/m / m/45ph2/is1/is1/is14ph18/mgl102ph3113/142negros orientalph4/17/ph5/ ph7/ph5/ph7613/27 * 2not area specific ; * ; not detected in the present study . , a total of 44 new genotypes were identified , of which 4 were not area - specific . out of 46 samples with multiple infections , 3 samples were found co - infected with 4 different genotypes and another 4 samples with 3 different genotypes . phylogenetic trees showed very low bootstrap values on monophyletic clades that contained the obtained partial sequences ( data not shown ) . * not area specific ; * * ; not detected in the present study . the present study is the first molecular - epidemiological report of a. marginale in cattle covering several geographical areas in the philippines . past studies dealt with only either water buffaloes or a few cattle in limited geographic areas [ 21 , 23 , 32 ] . the diversity of livestock vector - borne diseases is interesting to correlate with the unique geography of the philippines , which is composed of several islands . the prevalence of a. marginale in the present study ( 19.8% ) was higher than those of previous reports ( 10.316.7% ) in water buffaloes . water buffaloes living in close contact with backyard cattle is not uncommon in the philippines . therefore , cattle are in constant risk of the infection , because water buffaloes are known to serve as a reservoir for a. marginale . on the other hand , the australian centre for international agricultural research ( aciar ) and the bureau of animal industry of the philippines had a previous collaborative project ( id : as2/2000/098 ) partly dealing with the detection of bovine anaplasmosis . however , information on the national prevalence of a. marginale infection was still not made to be readily available . the project had relied on serological and peripheral blood smear examination methods , which might have sensitivity and specificity issues . the number of the positive cattle in the msp1a gene based - pcr assay was lower than that in the groel gene based - pcr assay . this might be attributed to the varying sensitivities of pcr protocols despite testing the same sample . the groel pcr assay was previously shown to be highly sensitive and specific in detecting the a. marginale in philippine cattle . furthermore , the high identities and the monophyletic clade formed by the obtained partial a. marginale groel gene fragments suggest the high conservation of the groel gene among philippine isolates regardless of the geographic locations and also provide further evidence of its usefulness in the molecular detection of the pathogen in the country . for the msp1a gene , the lower nucleotide identities and presence of many genotypes demonstrated that there is a high genetic diversity of a. marginale distributed in the philippines . in a previous study done in cebu , the registered msp1a gene sequences revealed 4 tandem - repeat structures : 2 already established structures ( 46 and m in table 3 ) and 2 novel structures containing the sequences of adsssasdqqqesgvpsqseastssqlg and tdsssasgqkqessvlpqs - gqastssqlg ( designated as ph12 and ph20 in the present study , respectively ) . although the ph20 structure was not detected in the present study , ph12 was identified together with 19 other novel structures . moreover , the 3 previously identified genotypes from cebu ( with tandem repeats ph12/m / ph12/m / m , 46/ph20/46 and 46/46 ) could not be detected in the present study . the presence of multiple infections of different genotypes indicates the superinfection of a. marginale in the philippine cattle . meanwhile , as some genotypes of a. marginale were unexclusive in each study area , there might be a common exposure or source of the infection despite geographical boundaries , or it might be due to cattle trade or movement among different islands in the philippines . on the other hand , the possible co - infection of a. marginale with other pathogens could not be discounted [ 14 , 16 ] . in a related study , co - infection of anaplasma spp . with other vector - borne disease ( vbd ) pathogens was found to be prevalent with those considered ill animals harboring concurrent infections of up to 5 vbd pathogens . therefore , studies to determine the occurrence of other vbd pathogens in the studied areas can be useful in investigating their interaction with the different genotypes of a. marginale in the susceptible host . in conclusion , a. marginale was molecularly detected from cattle populations in 6 different locations in the philippines . furthermore , the present study determined the prevalence of a. marginale and identified its different genotypes in cattle from geographically distant areas in the philippines . the information on a. marginale genotypes in the philippines is apparently the first in southeast asia . because genotypes may also vary in their pathogenicity , further studies are necessary to associate these genotypes with the clinical signs in cattle . in addition , farmers , local veterinarians , veterinary epidemiologists and the local government units in the philippines should cooperate in preventing and controlling bovine anaplasmosis , as it can cause considerable economic losses .	abstracta total of 658 cattle in 6 provinces in the philippines were screened for anaplasma marginale infection by using a diagnostic heat - shock operon ( groel ) gene - pcr assay . the screening - positive samples were further tested using the major surface antigen protein 1a ( msp1a ) gene - pcr assay . screening pcr results showed 130 cattle ( 19.8% ) were positive for the a. marginale infection . subsequent amplification using the msp1a gene only showed 93 samples ( 14.1% ) to be positive . in addition , 37 tandem - repeat structures , including 20 novel structures , and 41 distinct genotypes were identified . interestingly , multiple infections of 4 different genotypes were also observed in a. marginale - infected cattle . the present study demonstrated the prevalence and characterization of diverse genotypes of a. marginale in the philippine cattle .
increase of health expenditures is a challenge for both families and governments in most of the countries around the world . besides , raising the health services price and accessibility to purchasable coverage are important matters for healthcare providers and regulators ( 1 ) . cpi in the health sector consists of physician , dentist and hospital room visit fees , inpatient and hospital equipment expenditures , etc . studies have shown that many factors affect the inflation rate of the consumer price index for health care services ( ircpihc ) ( 2 ) . health inflation involves changes in consumer price index ( cpi ) effected by the factors , which affect supply and demand of health services . these factors include overall inflation rate , number of doctors , hospital beds , and pharmacies , existence of insurances , etc . health expenditures are the spending for health , including both the amount and price of services consumption . it contains the costs of health services ( preventive and curative ) , nutrition activities , family planning activities and emergency aid designated for health . growth in health expenditures is different from inflation rate , because this growth might occur due to the increase in the amount rather than the price of the services . this is affected by population s age , urbanization , education , level of development , etc . thus , it could be presumed that health inflation is the subset of growth in health expenditures ( 1 , 3 ) . according to the central bank of islamic republic of iran data between 1995 and 2008 , ircpihc was always higher than the general inflation . in 1995 , in addition , general inflation rate was 18% and health inflation rate was 22% in 2006 . therefore , increase in health inflation rate can decrease the patients purchasing power and enhance the catastrophic health expenditures . ultimately , inflation in the health sector increases the general inflation and decreases the individuals life conditions and human productivity ( 4 ) . the present study aimed to determine the factors , which affect the ircpihc in iran . this descriptive analytical study aimed to determine the relationship between health inflation and its determinants in iran . for estimating the model , we used the data of central bank of islamic republic of iran between 1983 and 2008 . the determinants of health inflation rate , which were used in this study , have been shown blew . the model used in this study hinf= f ( inf , doc , edu , insu , gdp , doc , bed , den , pharm ) hinf is the cpi in the health sector , inf is the total inflation rate , doc is the number of physicians per 1000 people , edu is the individuals mean years of education , insu is the percentage of the individuals covered by health insurance , gdp is per capita gross domestic product ( gdp ) at purchase price parity , bed is the number of hospital beds per 1000 people , den is the number of dentists per 1000 people , and pharm is the number of pharmacies per 1000 people . the econometrics form of this model has been presented blew : due to its cobb - dauglas form , estimating the model with general ordinary least square estimator was not possible . thus , first we had to change the form of the model and make it linear . l shows the logarithm in the model . for estimating the model , because of having long time series , we tested the unit root . on the other hand , if a variable is non - stationary , the results of estimating the model may not be true and a bias called spurious regression will occur . therefore , the validity of coefficients will be low and we can not rely on the results . hence , we used augmented dickey - fuller test and philips perron unit root test . after confirmation of having non - stationary variables , we had to find the lag order of the model . in this study , we used 3 tests to find the number of lags . after that , we used dynamic factor estimator to find out the relationship between the variables . dynamic factor estimator estimates the parameters of dynamic - factor models by using maximum likelihood . it eliminates local shocks and measurement errors because of idiosyncratic movements ( 6 ) . in our study , because of the lack of data , we could not estimate the model by cointegration approach and vector autoregressive estimator ; therefore , we used dynamic factor estimator . table 1 shows the results of augmented dickey - fuller and philips perron unit root tests . the results of unit root tests for the study variables as the table depicts , having unit roots was confirmed for lhinf , linf , and ledu and , consequently , the null hypothesis was confirmed . considering philips perron unit root test , the null hypothesis of having unit root was confirmed for lhinf , linf , ledu , and ldoc . after confirmation of having unit root , the number of lags and the r static factors entering the equation were determined . as shown in table 2 , akaike s information criterion ( aic ) , schwarz s bayesian information criterion ( sbic ) , and hannan and quinn information criterion ( hqic ) with the maximum lags of 4 were used . the results of determination of lag orders using aic , sbic , and hqic table 3 shows the results of estimating the model using factor variable estimator . according to the table , overall inflation and number of dentists per 1000 people the results were also positive for the number of pharmacies per 1000 people , but at 10% rather than 5% significance level . however , per capita gdp ( lgdp ) , number of doctors per 1000 people ( ldoc ) , and number of beds per 1000 people ( lbed ) had a negative relationship with health inflation . this relationship was negative and significant at 10% level for the percentage of insured people ( lins ) . thus , it could be indicated that these two variables were most effective in health inflation . . therefore , increase in the number of dentists will result in increase in the health inflation rate . consequently , it could be concluded that dental services were not delivered in a competitive market . because of having high demand price elasticity , insurers did not like to cover dental services . therefore , dentists could easily change the prices with poor supervision ( 7 ) . on the other hand , because of change in the consumption patterns , consumption of dental and oral care services has increased in developing countries , which could not be compensated even by increase in the number of dentists and dental care ( 8 , 9 ) . these changes have decreased in developed countries because of dental prevention programs ( 10 ) . in general , demand price elasticity of dental care is higher than that of other healthcare services ( 11 ) . hence , due to rise in demand for dental services , the price of these services will increase more than that of other services and will be more effective in health inflation rate ( 9 , 12 ) . because of having asymmetric information and high price elasticity , if the insurers cover dental services , they will be faced with high degrees of moral hazard and they will lose , so in many insurance programs there is the lack of covering oral care and there is high out of pocket payments for these services ( 11 , 12 ) . similar to dentists , the number of pharmacies per 1000 people had a positive effect on health inflation rate . the market of pharmacy is similar to a monopoly market , not a competitive market . the initial cost of a new drugstore is high and new stores can not be added to the market easily , 2 . there are some geographical regulations in drug markets that do not allow people to construct a drug store near another one ( 13 ) , and 3 . people do not know about the prices of drugs and can not evaluate their quality ( 14 ) . this asymmetric information about the prices and qualities exists in other health sectors , but it is more evident in drug market rather than dental and general physician markets . therefore , we can conclude that drug market has the behavior of monopoly markets more than other health sectors . in comparison to other markets , raising the prices is easier in a monopoly market ; thus , it has a larger effect on changing the health inflation rate ( 15 , 16 ) . the findings of the present study indicated no significant relationship between education and health inflation rate . this is due to the fact that educated people have more knowledge about personal care and have more willingness to keep themselves healthy ( 17 , 18 ) . on the other hand , educated people are more concerned about their health and , as a result , use more health outcomes . the insignificant results obtained in this study might be due to these two contradictory behaviors ( 19 ) . in the present study however , most of other studies have reported a positive relationship between gdp and healthcare expenditures ( 20 ) . when gdp increases , the level of development increases , too ; therefore , people keep themselves healthier . the findings of the current study showed a positive relationship between the number of hospital beds per 1000 people and the health inflation rate . in contrast to dental and oral care , no significant change has been observed for consumption . efficiency of hospital services and reduction in the length of hospital stay increase the supply of hospital beds ( 21 , 22 ) . additionally , when the number of hospital beds increases , the competition between inpatient suppliers will increase simultaneously . the results of this study showed a negative relationship between the number of doctors and health inflation rate . when the number of doctors increases , the competition between them increases , as well and they need to decrease their prices to attract more patients ( 5 ) . according to the literature , the relationship between the number of doctors and healthcare expenditures may be positive or negative depending on the demand elasticity of the services , which the doctors deliver ( 20 , 2326 ) . when the number of doctors increases , the number of cared people increases , too but the prices will decrease . if the services have low price elasticity , health expenditures will decrease and vice - versa ( 1 , 27 ) . in this study , a negative relationship was observed between insurance coverage and health inflation rate at 10% significance level . increase in insurance coverage empowers the insurance companies to control the prices and compel the health deliverers to stabilize the prices , eventually decreasing the health inflation rate ( 28 , 29 ) . this is due to the fact that when the prices increase , the willingness of health sector providers to increase their income for fixing their purchasing power will increase , as well ( 5 ) . we only had the data for a few years and more recent data on the inflation rate in iran were not available . further studies can investigate the relationship between health inflation rate and some other variables , such as foreign exchange rate and urbanization . growth in cpi of health , which is known as health inflation , is different from growth in health expenditures . for instance , per capita gdp increased healthcare expenditures , but decreased the health inflation rate . besides , growth in the number of hospital beds increased the health expenditures by using more beds , and increase health inflation by increasing the competition between inpatient care deliverers . 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 price inflation rate is different from increasing in health expenditures . health expenditures contain both quantity and prices but inflation rate contains prices . this study aimed to determine the factors that affect the inflation rate for health care services ( ircpihc ) in iran.methodswe used central bank of iran data . we estimated the relationship between the inflation rate and its determinants using dynamic factor variable approach . for this purpose , we used stata software.resultsthe study results revealed a positive relationship between the overall inflation as well as the number of dentists and health inflation . however , number of beds and physicians per 1000 people had a negative relationship with health inflation.conclusionwhen the number of hospital beds and doctors increased , the competition between them increased , as well , thereby decreasing the inflation rate . moreover , dentists and drug stores had the conditions of monopoly markets ; therefore , they could change the prices easier compared to other health sectors . health inflation is the subset of growth in health expenditures and the determinants of health expenditures are not similar to health inflation .
after obtaining clearance from the institutional board of studies and informed consent , horizontal strabismus patients qualifying for symmetrical recession or resection surgeries on the horizontal rectii were included . anesthesia utilized was either standard peribulbar block or general anesthesia ( propofol and nitrous oxide ) . one eye was randomized ( using an unbiased coin ) to standard paralimbal strabismus surgery ( spss ) while the other automatically qualified for miss . after separating the lids with universal eye speculum , a 5 - 0 silk traction suture ( johnson and johnson ltd . aurangabad ; nw 5079 ) was passed through the superficial sclera near the limbus at 12 and 6 oclock to expose the quadrant of interest . four points were marked on the conjunctiva : two at the upper and lower ends of the rectus muscle insertion and two at the posterior limits of the planned para - muscular conjunctival incisions [ fig . the two points on each side of the muscle insertion were then incised with a bard - parker blade ( # 11 ) after raising a fold of conjunctiva by lifting the conjunctiva between forceps , so as to obtain two incisions parallel to the upper and lower edge of the horizontal rectus muscles , with their anterior limits at the upper and lower ends of the muscle insertion [ fig . the radial length of these incisions was 1 mm less than the planned recession / resection if the latter was 5 mm , and two mm less if > 5 mm . using blunt wescott scissors , careful dissection was carried out to identify the muscle edge , under which a muscle hook was then passed [ fig . further dissection then helped delineate the muscle and the adjacent sclera up to the site of recession . a similar conjunctival incision was made at the superior border of the muscle , aided by the muscle hook already placed below the muscle . subsequently , the strap of conjunctiva , overlying the muscle tendon , between the two conjunctival incisions , was meticulously dissected free of the muscle up to the insertion to facilitate subsequent surgery in that area [ fig . this provided us access to the upper and lower edge of the muscle right up to the insertion , as also the sclera / muscle till the point where recession / resection was to be carried out . the planned site of recession on the sclera , or the site of resection on the muscle , were marked [ fig . ( a - i ) figures showing surgical steps for miss ( recession ) ( see text ) for recessions : vicryl 6 - 0 ( johnson and johnson ltd . aurangabad nw 2670 ) locking bites were taken from the muscle margins from near the insertion [ fig . if the parallel conjunctival cut edges appeared to be in good apposition , no sutures were applied ; else , a single central vicryl 8 - 0 suture approximated the incisions . good apposition was considered wherein the edges were no more than 2 mm apart at a point of maximum separation , after gently smoothening the conjunctival surface [ fig . 1i ] . in case of any complications , like excessive hemorrhage , or difficulty in suturing the muscles , were encountered , it was planned to prolong the conjunctival incisions towards the limbus and join them , effectively converting them to resemble the limbal approach ; in such an event , the miss approach would be considered to have failed . for resection : after a similar conjunctival approach as for recessions , two vicryl 6 - 0 sutures were applied at the edges of the extra - ocular muscle ( eom ) at the point of desired resection and passed through the insertion of the eom . the muscle between the suture bites was excised ; tying the sutures brought the point of resection to the insertion , effecting a resection . we recorded the duration of surgery ( in minutes ) from the start of the conjunctival incision to the completion of surgery . post - operatively graded scoring of inflammation ( o to 3 : nil , mild , moderate , severe ) was done by an observer masked to the randomization ; on day 1 , at 2 - 3 weeks , and 6 weeks : specifically redness , congestion ( these two were compared to standard photographs ) , chemosis , foreign body sensation ( fbs ) , drop intolerance were scored ; and each score combined to yield a total inflammatory score ( tis ; with a possible range from 0 to 15 ) . visible scarring ( from one meter ) and final alignment were noted at 6 weeks . we included 14 parallel designed surgeries on 10 patient ; 10 were bilateral recessions , while 4 of subjects participated a second time ( 3 months later ) undergoing planned second bilateral recestions on account of large angles not amenable to single strabismus - surgical correction . the mean duration of surgery in the spss was 29.6 ( sd 5.37 ) minutes as compared to 40.4 ( sd 7.98 ) minutes in the miss approach , with a mean difference of 10.8 minutes ( 95% ci for difference : 2.67 to 18.92 minutes . ) , which was significant ( wilcoxon test ; p = 0.013 ) . on the first post - operative day , there were significant differences ( p < 0.05 ) in the median inflammatory scores for fbs and tis ( wilcoxon test , table 2 ) . at 2 - 3 weeks , in addition , significant differences for redness and congestion . by 6 weeks , significant differences were restricted to redness and tis . at final follow - up at 6 weeks , significant scarring was observed in all the eyes , which underwent spss while it was present in 9 miss eyes ; the difference was not statistically significant ( chi - square , fisher 's exact test ; p = 0.09 ) . there was no change in visual acuity postoperatively in any of the patients during any time in follow - up . amount of strabismus , target surgery , final alignment along with status of fusion , and stereopsis at 6 weeks postoperatively are also shown in table 1 . demographic details of the patients along with post operative functional outcomes median inflammatory scores and p value during post - operative follow - up we compared post - operative comfort and cosmesis , by miss and spss , in patients undergoing symmetrical strabismus surgeries in a parallel design , randomizing one of two eyes , since that may be a superior model to compare inflammatory outcomes . on the first post - operative day , there was no significant difference between the two eyes in terms redness , congestion , chemosis , and drop intolerance ; only fbs ( p value < 0.01 ) was significantly less in the miss eyes . this could be on account of the small incisions and posterior placement of conjunctival sutures ( if any ) in the latter group . at 2 - 3 weeks follow - up , scores for redness , congestion , and foreign body sensation were significantly less in the miss eyes [ fig . 2 ] . while at 6 weeks , we did not find any significant difference , except for redness and total inflammatory score [ table 2 ] . comparative photograph at 2 - 3 weeks postoperatively , of the same patient operated through miss and spss in his introductory paper , mojon utilized the miss approach and compared them with a historical group , which underwent a limbal approach in cases undergoing horizontal rectii surgeries . although his main outcome measures ( fusion , alignment , and vision ) were different from ours , mojon did report significantly less lid swelling ( 0% vs. 21% ) in the miss group on the first post - operative day . on final analysis at 6 months , no significant difference was found . in another study on exotropes , pellanda and mojon they found significantly less conjunctival swelling and injection on the first post - operative day ( test p < 0.001 ) . they needed to convert 3/104 muscles in their miss group . in a similar analysis in esotropia , conjunctival swelling and injection were significantly less pronounced ( p value < 0.001 ) in the miss group . we believe that a comparison of grades of inflammation by the mann - whitney test would have been superior , although the results are unlikely to have been different . although we found 9 of 14 cases ( 64.3% ) of miss showing a significant scar at 6 weeks compared to all 14 cases ( 100% ) of spss , the results did not reach statistical significance ( fisher 's exact ; p value : 0.09 ) . mojon has also commented in his comparative study that minimal cicatrization was seen along the incision line , which did not hinder free movement of the conjunctiva over the sclera . on occasion ( sic ) , no scar was seen biomicroscopically . only larger studies with more statistical power may provide clear answers to this question . in our study , miss took significantly longer than spss ; but , it is likely that this would decrease as experience with miss accumulated . although comparison of alignment by its very nature can not be compared in a study like ours , we are reporting post - operative alignment outcomes [ table 1 ] , since that would be of interest to our readers . as is evident , 11 of 14 surgeries achieved correction to within 10 pd of target angle ; a success rate of 78.5% . as highlighted by kushner , the miss approach interferes less with the peri - limbal episcleral vessels , which should decrease the chances of anterior segment ischemia . although miss may appear to be superior to the paralimbal approach , it may not be much different to the parks fornix approach where too conjunctival incisions are far removed from the limbus . we are acutely aware of the small number of cases in our study and suggest that more studies with a larger sample size would yield better evidence to either support or refute our findings . since all our patients were young adults , the results should be interpreted preferable for this age group . the strength of our study is in its parallel design , a format superior in our opinion to compare inflammation scores . our small study shows that a miss approach is more comfortable and offers better cosmesis ( in terms of redness ) in the short - term ( 2 - 3 weeks ) as compared to the spss ; this difference equates by 4 - 6 weeks .	introduction : minimal access surgery is common in all fields of medicine . we compared a new minimally invasive strabismus surgery ( miss ) approach with a standard paralimbal strabismus surgery ( spss ) approach in terms of post - operative course.materials and methods : this parallel design study was done on 28 eyes of 14 patients , in which one eye was randomized to miss and the other to spss . miss was performed by giving two conjunctival incisions parallel to the horizontal rectus muscles ; performing recession or resection below the conjunctival strip so obtained . we compared post - operative redness , congestion , chemosis , foreign body sensation ( fbs ) , and drop intolerance ( di ) on a graded scale of 0 to 3 on post - operative day 1 , at 2 - 3 weeks , and 6 weeks . in addition , all scores were added to obtain a total inflammatory score ( tis).statistical analysis : inflammatory scores were analyzed using wilcoxon 's signed rank test.results:on the first post - operative day , only fbs ( p = 0.01 ) and tis ( p = 0.04 ) showed significant difference favoring miss . at 2 - 3 weeks , redness ( p = 0.04 ) , congestion ( p = 0.04 ) , fbs ( p = 0.02 ) , and tis ( p = 0.04 ) were significantly less in miss eye . at 6 weeks , only redness ( p = 0.04 ) and tis ( p = 0.05 ) were significantly less.conclusion:miss is more comfortable in the immediate post - operative period and provides better cosmesis in the intermediate period .
multiple myeloma accounts for 1% of adult malignancies and has an annual incidence of 3 to 4 per 100,000 persons in the united states , with a peak age of occurrence in the midsixties . multiple myeloma is a neoplasm of plasma cells characterized by monoclonal proliferation of plasma cells in bone marrow , infiltration of adjacent tissue and occasionally multiple organs , and production of an immunoglobulin ( usually monoclonal igg or iga ) . common presenting symptoms of multiple myalgia include bone pain ( especially back pain ) , fatigue ( typically caused by anemia ) , pathologic fracture , weight loss , paresthesia , and fever . minimally invasive surgical treatments such as vertebroplasty and kyphoplasty are effective and safe in the management of myelomatous vertebral fracture1,4,8 ) . in local recurrences , vertebroplasty can be executed before radiation therapy , thus synergizing its delayed analgesic action on pain11 ) . delayed injection of cement is safer and more effective in cases of osteolysis or fracture of the posterior vertebral wall2 ) . major spinal surgery is less effective in patients with multiple myeloma due to poor bone quality and median survival time . a 62-year - old male was admitted to the hospital with a lower back pain , pain in both lower extremities , and numbness that occurred 5 months earlier . he had slipped and fallen 2 months earlier and the back pain had since become aggravated . he had a history for hypertension , type 2 diabetes mellitus , angina pectoris , and asthma treated with oral medication . other than his erythrocyte sedimentation rate of 27mm / hr , c - reactive protein of 12mg / dl , and albumin level of 3.3 g / dl , the patient 's laboratory values on admission were normal . however , his hemoglobin was reduced from 16 to 12 g / dl 3 months later . radiographic studies including 1.5 tessler magnetic resonance imaging ( mri ) and computed tomography ( ct ) of his lumbar spine were performed on admission . mri and ct showed mild canal stenosis of l 4 - 5 , but other meaningful abnormal findings were not found ( fig . the spinal canal stenosis of l 4 - 5 existed in an mri of the lumbar spine conducted 2 years earlier , and there was no change in the degree of stenosis . we prescribed analgesics to relieve pain , and had no reason to suspect a secondary vertebral compression fracture . despite medication with analgesics for 2 months , the lower back pain was aggravated and the patient complained of gait difficulty due to back pain . an mri of the lumbar spine was performed again and showed an l1 compression fracture ( fig . 2 ) , and therefore , l1 vertebroplasty was performed ; however , patient 's pain did not improve . on an mri of the thoracolumbar spine taken one month later , t10 , l1 , and l3 compression fractures were shown , and on an mri of the whole spine 3 months later , t6 , t9 , t10 , l1 , l2 , l3 , l4 , and l5 compression fractures were shown ( fig . 3 ) . vertebroplasty of t9 , t10 , l2 , and l4 vertebral bodies ( fig . 4 ) and a bone biopsy of the t9 body were performed by vertebroplasty needle during the vertebroplasty procedure and the patient 's lower back pain was relieved . tumor markers ( cea , psa , ca 19 - 9 , ca-125 , and afp ) were demonstrated to be within normal range . on serum protein electrophoresis , an m - spike was present in the gamma globulin fraction , and the concentration of m - spike was 1.10 g / dl . serum immunoelectrophoresis and immunofixation electrophoresis revealed monoclonal gammopathy immunoglobulin a(iga ) , lambda type . on a quantitative immunoglobulin test , g / l ) , the iga level wa 16.1 g / l(normal range is 0.7 to 4 the above - mentioned results of bone biopsy , electrophoresis , immunoelectrophoresis , and immunofixation electrophoresis proved the diagnosis of multiple myeloma . the patient was referred to another hospital , where he received vad(vincristine , adriamycin , and dexamethasone ) chemotherapy 4 times and bone marrow transplantation . the mspike then decreased from 1.10 to 0.22 g / dl on serum protein electrophoresis . the patient is still alive without developing symptoms one year after diagnosis of multiple myeloma . primary osteoporosis is responsible for about 85% of vertebral compression fractures and the remaining 15% are associated with secondary osteoporosis and neoplasms13 ) . patients that suffer bone or back pain in spite of symptomatic treatment for more 2 - 4 weeks need further evaluation for multiple myeloma and other diseases , including polymyalgia rheumatica , hyperparathyroidism , vitamin d deficiency , and bone metastasis . in case of multiple myeloma , the laboratory findings usually show anemia , thrombocytopenia and neutropenia . also , the patients sometimes complain fatigue and depressive mood due to hypercalcemia . when we meet the compression fractures without hot uptake in bone scanning , more evaluations are needed . if multiple myeloma is suspected , for diagnosis , it is necessary to demonstrate m - proteins in either serum or urine , to prove the presence of 10% bone marrow clonal plasma cells , and to observe the clinical manifestations of the disease . the amount of myeloma cell mass can be predicted from the extent of bone lesions , the hemoglobin level , serum calcium level , and m - component levels in serum and urine . response to chemotherapy and patient survival are also significantly correlated with the measured myeloma cell burden3 ) . hematologic monitoring in multiple myeloma is based on determination of the serum and urine levels of paraprotein , albumin , hemoglobin , creatinine , and calcium . conventional radiological imaging is a method for staging multiple myeloma as well as for assessing complications of the skeletal system . both multidetector ct and mri are recommended for mid- and long - term monitoring5 ) . the combined use of multidetector ct and mri is also useful for initial staging of multiple myeloma10 ) . 1.5 tessler whole body stirmri can detect bone marrow infiltration in about 74% of multiple myeloma patients . moreover , whole - body mri showed a diagnostic accuracy superior to that of multislice computed tomography . in particular , the early stages of disease with less prominent osteolytic changes and diffuse infiltration patterns are more reliably visualized with the use of mri16 ) . the involvement of > 10 bone marrow focal points and diffuse infiltration on mri are more risky factors to predict vertebral fractures6 ) . both fdg - pet in combination with lowdose ct and whole - body mri are more sensitive than skeleton x - ray in screening and diagnosing multiple myeloma . however , whole - body mri is less suitable in assessing response to therapy than fdg - pet9 ) . vertebroplasty provides significant and long - lasting pain relief and reduces the risk of fracture or new collapse in contiguous vertebral bodies . vertebroplasty is an effective treatment that confers pain relief , enhanced mobility , and reduced use of medicine for all the stages of multiple myeloma associated with compression fracture12 ) . vertebroplasty was also confirmed in patients experiencing pain related to malignant spinal tumors with epidural extension , with a low complication rate15 ) . we should check for the possibility of pathologic vertebral fractures from multiple myeloma in non - osteoporotic compression fracture patients , especially when continuous fractures are present in relatively non - elderly patients , even though a history of trauma may exist . vertebroplasty is an effective method to relieve pain in cases of vertebral compression fracture due to malignancy .	multiple myeloma is a b - cell origin tumor characterized by hypercalcemia , osteolytic bony lesions , and pathologic fractures . back pain is one of the most common presenting symptoms of multiple myeloma and about 60% of patients have vertebral involvement at the time of diagnosis7 ) . minimally invasive surgeries such as vertebroplasty and kyphoplasty are effective to relieve pain and improve the quality of life when a compression fracture occurs in multiple myeloma patients . we report a case of continuous multiple compression fractures associated with multiple myeloma . we should check for the possibility of pathologic vertebral fractures resulting from multiple myeloma in non - osteoporotic compression fracture patients , and especially in cases of continuous fracture occurring in relatively non - elderly patients , even though a history of trauma may be present .
bronchocentric granulomatosis ( bcg ) is a rare , often destructive , granulomatous lesion of the bronchi and bronchioles . severe cystic lung disease secondary to pulmonary tuberculosis ( tb ) is also not commonly reported , though fibrocystic lung lesions are seen commonly . the coexistence of these two conditions together in a single patient has not been reported . here , we present this unusual association in a young female , where the diagnosis was confirmed by surgical lung biopsy . a 14-year - old , previously , healthy female presented with the complaints of fever , since 3 months followed by shortness of breath and weight loss since 1 month . there was a positive history of pulmonary tb in the mother , who was currently on treatment . a diagnosis of miliary tb was made , and she was started on anti - tubercular therapy ( att ) with 4 drugs ( isoniazid , rifampicin , pyrazinamide , and ethambutol ) . two weeks after starting att , she developed worsening of her symptoms , and a repeat x - ray chest showed bilateral pneumothoraces . bilateral intercostal chest tube insertion was done , and she was referred to our center for further management . on presentation to us , the patient was in severe distress , her pulse rate was 120/min , blood pressure was 90/60 mmhg , and respiratory rate was 36/min . on auscultation , chest revealed bilateral crackles and absent breath sounds over most of the hemithoraces . arterial blood gas analysis on 60% fio2 venturi mask showed ph - 7.45 , pao2 - 77 mmhg , paco2 - 50 mmhg , hco3 - 34.5 mmol / l , and spo2 - 92% . chest x - ray showed bilateral residual pneumothoraces with underlying collapsed lung with bilateral intercostal chest tube drainage ( icd ) tube in situ . laboratory investigations revealed hemoglobin of 8 mg / dl , total leukocyte count 12,900 cumm with neutrophils of 80% . serum urea was 18 mg / dl , and creatinine was 1.2 mg / dl . total bilirubin was 0.6 mg / dl , direct bilirubin was 0.2mg / dl , aspartate aminotransferase was 32 p - antineutrophilic cytoplasmic antibodies ( p - anca ) and c - anca were negative . high resolution computed tomography ( ct ) thorax revealed bilateral pneumothoraces with septae , multiple extensive cysts of varying sizes , and minimal ground glass haze in both the lungs [ figure 1 ] . computed tomography chest showing extensive bilateral pneumothoraces with multiple parenchymal lung cysts in an attempt to drain the pneumothoraces better , two more intercostals chest drains were inserted under ct guidance ; one on each side and attached to negative suction . however , the lungs still did not expand , and negative suction was tried but failed too . in order to establish a diagnosis and as an attempt to do some adhesiolysis to expand the lung , it was decided to do a video - assisted thoracoscopic surgery guided surgery on the left side . intraoperatively , the lung was found to be collapsed with cysts of varying sizes visible over the lung surface and multiple thick pleural strands and adhesions were seen [ figure 2 ] . intraoperatively , fiber optic bronchoscopy was performed through the endotracheal tube , and bronchial washings were taken . genexpert test was performed on the bronchial washings , which was positive for m. tuberculosis and no rifampicin resistance was detected . conventional tb culture was sent , which subsequently grew m. tuberculosis , sensitive to first - line att . histopathological examination of the surgical lung biopsy showed areas with extensive necrosis and cystic changes . silver staining was negative for fungal elements , and there was no evidence of vasculitis . there were areas in the sample which revealed extensive peribronchiolar necrotizing inflammation , with bronchial wall destruction with palisading granulomas , replete with giant cells and a few eosinophils . intraoperative video - assisted thoracoscopic surgery picture , demonstrating multiple lung cysts of varying sizes ( a - d ) histopathology panel consistent with bronchocentric granulomatosis extensive peribronchiolar necrotizing inflammation with bronchial wall destruction , pallisading granulomas replete with giant cells the patient was continued on first line att and steroids ( 4 mg of dexamethasone , twice daily ) were added . the patient was weaned of the ventilator on the 4 postoperative day , and one icd tube was removed from both the sides and the oxygenation improved marginally . on the 10 postoperative day , the patient again developed a right - sided pneumothorax , which was confirmed by a bedside ultrasound examination . however , she sustained a cardiac arrest the same day and could not be revived . bcg is a rare , destructive , granulomatous lesion of the bronchi and bronchioles that is generally believed to represent a nonspecific response to various types of airway injuries . around half of these cases are associated with asthma and allergic bronchopulmonary aspergillosis and may represent a histopathologic manifestation of fungal hypersensitivity among these patients . other reported associations include mycobacterial and fungal infections , rheumatologic diseases , granulomatosis with polyangiitis ( wegener 's ) , diabetes insipidus , red cell aplasia , pulmonary echinococcosis , bronchogenic carcinoma , and influenza infection . the presence of this lesion should generally be considered a nonspecific manifestation of lung injury and not an etiologic diagnosis . m. tuberculosis infection if implicated as a cause of lung injury , representing bcg should be treated at the earliest , to halt the lung injury . subsequent destruction of airway walls and adjacent parenchyma lead to granulomatous replacement of mucosa and submucosa by palisading , epithelioid , and multinucleated histiocytes . the radiographic presentation of bcg is varied , and pulmonary nodules , consolidation , mass lesions , etc . , have all been reported . in our patient , the picture was also confounded by the presence of multiple lung cysts of varying sizes , which led to recurrent pneumothoraces and clinical worsening in our patient . the formation of lung cysts due to tb can be attributed to number of causes . caseating necrosis of the bronchial walls leading to cystic bronchiectasis as a distal extension of fulminant tubercular bronchitis and granulomatous involvement of the bronchioles may lead to a check - valve mechanism leading to cyst formation . whether the bcg pattern is secondary to tb or a de novo entity is a matter of debate . tb is a disease of antiquity , but its varied presentations and features never cease to surprise us .	tuberculosis is known to cause both cystic lung disease and bronchocentric granulomatosis ( bcg ) . however , both are rare manifestations of this common disease . we report a case of bcg with extensive cystic lung disease in a young female who presented with fever , weight loss , and recurrent pneumothoraces with respiratory failure . early diagnosis and treatment are imperative , as appropriate therapy may be life - saving in such cases .
focal segmental glomerulosclerosis ( fsgs ) causes scarring or sclerosis of glomeruli that act as tiny filters in the kidneys , damage to which results in diminished ability to properly filter blood , resulting in the urinary loss of plasma proteins and subsequent proteinuria . a 60-year - old , white female with a history of intermittent proteinuria was referred by her primary care physician for renal dysfunction . she also had rheumatoid arthritis ( ra ) but no active synovitis and was maintained on prednisone 5 mg / d . she also complained of worsening vision in her right eye and was diagnosed with optic neuritis ( on ) . she remained stable for about 8 months when examination indicated fsgs relapse , and she reported painful ra flares . she was treated with acthar gel ( 40 mg biweekly ) for 6 months , after which proteinuria and urine protein - to - creatinine ratio decreased to about half . her on improved , and she reported that she had fewer ra flares and pain improved by 50% . this case of confirmed fsgs showed an improved response to treatment with acthar gel for fsgs with concomitant ra and on . this referral case is relevant to primary care practitioners who treat disorders that may be responsive to corticosteroid therapy . the antiproteinuric effects and ancillary improvement in ra and on symptoms during treatment with acthar gel are not entirely explained by its steroidogenic actions . acth is a bioactive peptide that , together with -melanocyte - stimulating hormone , exhibits biologic efficacy by modulating proinflammatory cytokines and subsequent leukocyte extravasation and may have autocrine / paracrine effects in joints . while acthar gel was primarily administered in this case to treat proteinuria , it also showed ancillary benefits in patients with concomitant inflammatory disease states . focal segmental glomerulosclerosis ( fsgs ) is named for the characteristic scarring or sclerosis of glomeruli that act as tiny filters in the kidneys . focal relates to the fact that only some of the filters are damaged , whereas damage to the glomeruli results in a diminished ability to properly filter blood , resulting in the urinary loss of plasma proteins ( ie , proteinuria ) , which is a hallmark of nephrotic syndrome ( ns ) . the etiology of fsgs is often unknown , but known risk factors include infection , drug toxicity , and systemic diseases such as diabetes , hypertension , and obesity.1 fsgs is a relatively common form of kidney disease in the united states and is now one of the most common patterns of glomerular injury encountered in human kidney biopsies , as well as the most common cause of proteinuria in the african american and us hispanic populations.1 in 2012 , a clinical practice guideline from the organization kidney disease : improving global outcomes ( kdigo ) was published based on a systematic literature review conducted in january 2011 , and supplemented by additional evidence through november 2011.1 the recommended initial treatment of idiopathic fsgs associated with clinical features of the ns is immunosuppressive therapy that usually begins with prednisone given at high dose for a minimum of 4 weeks to a maximum of 16 weeks as tolerated , to achieve remission , with subsequent tapering over 6 months.1 kdigo recommends consideration of calcineurin inhibitor as first - line therapy for patients with a relative contraindication to high - dose corticosteroids ( cs).1 rheumatoid arthritis ( ra ) is classified according to the 2010 american college of rheumatology and european league against rheumatism classification criteria . the current classification focuses on features at earlier stages that are associated with persistent / erosive disease.2 historically , treatment for most patients started with cs or nonsteroidal anti - inflammatory drugs . however , emerging evidence indicates that early and aggressive targeted - treatment strategies that provide consistent suppression of ra disease activity is linked to better long - term outcomes.3,4 optic neuritis ( on ) is an inflammatory , demyelinating condition that is highly associated with multiple sclerosis ( ms ) , and it may be the presenting feature in 15% to 20% of patients with ms.5 on typically affects young adults , and women are affected more often than men . on is second only to glaucoma as the most common acquired optic nerve disorder in persons younger than age 50.6 on usually improves on its own and although intravenous cs may speed recovery , it does not appear to provide long - term benefit and undergoing no treatment for acute on is an option.6,7 a meta - analysis of randomized controlled clinical trials using cs or adrenocorticotropic hormone ( acth ) showed that either modality produced significant improvement of visual disability and is therefore effective in accelerating short - term recovery.8 the author is not aware of a previous case that highlights an improved response to treatment with acthar gel ( repository corticotropin injection ) in a patient having these multiple disorders that are usually treated with cs therapy . a 60-year - old , white , obese woman was referred by her primary care physician for renal dysfunction . the patient , who has a history of intermittent proteinuria , had normal renal biopsy findings 14 years ago , and would likely benefit from another biopsy . she also has a history of hypertension , hyperlipidemia , hypothyroidism , peptic ulcer , chronic obstructive pulmonary disease , ra , and type 2 diabetes mellitus . proteinuria was 2.5 g at presentation , serum creatinine was at a normal level , and c - reactive protein level was elevated . glomerular immunofluorescence was uniformly negative for immunoglobulins igg , iga , complement c3 , c1q , and light chains . the quality of glomerular tissue preservation was adequate , with no evident cellular or proliferative changes or entrapment of immune complexes at any capillary or mesangial foci . foot processes of the visceral epithelial cells and capillary basement membranes were intact and free of defects . the patient was treated with an angiotensin - converting enzyme ( ace ) inhibitor for fsgs and was maintained on her current dosage of prednisone for ra . she also complained of worsening vision in her right eye and of seeing water bubbles . an exam with an ophthalmologist showed that the patient had orbital inflammation and on hyperemia and she was diagnosed with on . the patient s fsgs remained stable for approximately 8 months after presentation when examination showed worsening edema and highly probable fsgs relapse . serum creatinine concentration was 1.54 mg / dl , and she had 4 + proteinuria ( 319 mg / dl ) and a urine protein - to - creatinine ratio ( upcr ) of 9.3 g / g . the patient also experienced painful ra flares during ongoing treatment with 5 mg of prednisone once daily . she began treatment with a 6-month regimen of acthar gel ( 40 u twice weekly ) in april 2013 for fsgs . at her initial follow - up visit in september 2013 , proteinuria and upcr had decreased to about one - half ( 159 mg / dl and 4.3 g / g , respectively ) and serum albumin was 3.4 g / dl . at a follow - up visit later in september , spot urine upcr was 1.78 g / g and serum albumin was 3.7 g / dl . in addition , ophthalmic follow - up showed a decrease in orbital inflammation and reduction in swelling and thickness of the optic nerve in both eyes . although approved by the food and drug administration ( fda ) for treatment of on , there are limited data demonstrating the efficacy of acthar gel ( a highly purified preparation of acth ) for this condition . the patient reported that after starting treatment with acthar gel , she experienced fewer ra flares and pain had decreased by about 50% . she was also instructed to call in the case of worsening symptoms or adverse effects to medications . the patient did experience an elevation of her blood pressure and vitreous seeding was observed . this patient had a confirmed diagnosis of fsgs of unknown etiology but had a history of risk factors associated with fsgs . although less responsive than minimal - change ns , fsgs appears to respond to cs and treatment with cs is specifically recommended in kdigo guidelines.1 although not specifically studied in primary or secondary fsgs , ace inhibitors or angiotensin - receptor blockers ( arbs ) also reduce proteinuria and slow progression in proteinuric kidney disease , and patients with fsgs should receive renin - angiotensin system blockade and instructions to restrict dietary sodium.9 this patient was initially treated with the ace inhibitor lisinopril 20 mg and later switched to the arb telmisartan 80 mg . the empirical results shown in the patient in this case appear to support further consideration of acthar gel for treatment of fsgs . the role of acthar gel has not been adequately studied in ra , and it is fda - approved in ra only for short - term use . however , acth stimulates the adrenal cortex to produce cortisol in response to stress and also is an important physiologic agonist of the melanocortin ( mc ) system.10 the rationale for its use in ra is related to a study showing increased levels of -melanocyte - stimulating hormone ( -msh ) in the synovial fluid of patients with ra or juvenile ra , and it also has been shown to reduce joint pathology in rats with adjuvant - induced arthritis.11 acthar gel is a highly purified preparation of acth ( acth139 ) . acth139 is a bioactive peptide derived from a large precursor protein named pro - opiomelanocortin . acth is biochemically cleaved in vivo into another bioactive peptide , -msh , that corresponds to the first 13 amino acids of acth139 12 and exhibits biologic efficacy by modulating proinflammatory cytokines and subsequent leukocyte extravasation.13 investigations into the biologic distribution and function of mc receptors show that acth139 has anti - inflammatory or immunomodulatory functions related to the activation of mc1r , mc3r , and mc5r and is the only known mc peptide that will activate mc2r in the adrenal cortex.12 mechanisms of action now being considered for acthar gel hypothesize effects beyond steroidogenesis . the precise role of mcs in ra is uncertain , but evidence suggesting that mcs exert anti - inflammatory properties in ra is drawn from clinical studies that showed acth139 decreased joint pain , swelling , and rheumatic symptoms.13 mc peptides modulate inflammation through mc receptors and the activation of mcs initially reduce inflammatory cytokines and chemokines , later followed by the induction of anti - inflammatory mediators that facilitate the resolution of inflammatory response.13 in ra , the inflamed joint is characterized by infiltrating leukocytes as well as activated bone / cartilage cells , and studies suggest that mc peptides could act locally in an autocrine / paracrine manner.13 evidence for this is supported by an experimental murine model that mimics the flare of ra , in which the administration of an mc-3 agonist attenuated disease in incidence and severity in wild - type mice but not in mc-3 deficient mice , with sustained nf-b signaling in the osteoclasts of mc-3 deficient mice.14 this experimental model suggests that endogenous mc-3 controls the arthritic response in a model of passive serum - transfer arthritis.14 the mechanism of action of acthar gel comprises multiple components , including five mc receptors , each having varying biological activities.10 receptor - binding studies have revealed that all five mc receptors show a strong affinity for acth , which is one of four mc peptides , thereby establishing the potential for this hormone to activate these receptors.10 acth has shown efficacy as primary and secondary therapy for ns . two recent studies have prospectively examined the use of acthar gel in patients with fsgs.15,16 one was a prospective open - label trial of 15 subjects with resistant glomerular disease treated with acthar gel ( 80 u , subcutaneously , twice weekly).15 five subjects had resistant fsgs or minimal - change disease ( mcd ) and preserved renal function at baseline ( serum creatinine 0.61.2 mg / dl ) and had failed a traditional course of cs.15 all subjects with fsgs / mcd failed to achieve sustained remission with cs therapy and at least one other immunosuppressive therapy.15 among the five subjects , two subjects ( one with fsgs , one with mcd ) showed sustained improvement in upcr from baseline to 24 weeks ( 3.160.78 , mcd ; 1.940.43 , fsgs).15 of the remaining three subjects , two showed an increase in upcr from baseline to week 24 , and one showed no appreciable change.15 these results should be interpreted in the context of a population of treatment - resistant patients in whom the failure of first - line treatment increases the likelihood for the failure of second - line therapy , and so forth.15 given the small sample size , the study provides evidence to warrant further investigation in controlled trials . another study was a retrospective case series that evaluated the use of acthar gel in 21 adult patients with idiopathic ns , including one patient with fsgs.17 three patients received acthar gel as primary therapy , 18 patients had failed prior immunosuppressive therapy , nine of whom had failed at least three therapies . acthar gel was administered subcutaneously in varying regimens ranging between 80 iu and 160 iu per week.17 the patient with fsgs was a 63-year - old hispanic female who had been previously treated with cs , mycophenolate mofetil , and a calcineurin inhibitor and had proteinuria of 10,275 mg / d.17 the patient was treated with acthar gel , 80 u biweekly , for 6 months and achieved partial remission , with a decrease in proteinuria from 10,275 mg / d before treatment to 2,970 mg / d.17 in the largest retrospective case series to date , 44 patients with ns were treated with acthar gel , including 15 patients with fsgs.18 eligible cases underwent an assessment of 24-hour proteinuria level or spot upcr prior to and following 6 months of acthar gel therapy . complete remission was defined as proteinuria 500 mg / g and partial remission as proteinuria > 500 mg / g to 3,500 mg / g and with 50% reduction in proteinuria from baseline . clinical response was defined as 25% reduction in proteinuria level from baseline and the reduction in final proteinuria did not meet the definitions for complete or partial remission . twelve of the 15 patients with fsgs showed partial remission ( n=8 ) or a clinical response ( n=4 ) of approximately 30% reduction in proteinuria . fifteen of 44 patients had treatment - related side effects , mainly steroid - like such as hypertension , weight gain , and hyperglycemia . seven patients had early termination of treatment due to increased edema , fatigue , weight gain , hypertension , and seizures . therefore , all the adverse effects known to occur with elevated cortisol may occur with acthar gel administration as well.19 according to the existing experience of clinical use of acth as well as ongoing clinical trials of other mc analogs , the side effects of mc - based therapy seem mild , tolerable , and reversible.20 acth therapy through the cortisol effect might also induce mild hypertension . as an endogenous glucocorticoid , cortisol may directly elevate blood pressure by enhancing vascular tone or , because it has a fair amount of activity on the mineralocorticoid receptor , may cause sodium and water retention and volume expansion.20 for nonsteroidogenic mcs or analogs , mildly elevated blood pressure has been reported during phase ii or iii clinical trials , possibly attributable to central stimulation of sympathetic outflow mediated by the interaction of -msh with mc4r.2022 another recent study examined 24 subjects and the data for analysis were available for 23 patients.16 six subjects had steroid - dependent fsgs , 15 had steroid - resistant fsgs , and two additional subjects were treated with acthar gel as first - line therapy between january 2009 and april 2012.16 the trial included 16 subjects in a prospective investigator - initiated pilot study ( nct01155141 ) ; four had been described previously with shorter - term results and the remaining four were evaluated retrospectively in chart review.16 the classification of steroid - dependent or steroid - resistant fsgs was based on kdigo guidelines.1 treatment regimens were not entirely uniform : 12 subjects received 40 u of acthar gel subcutaneously , weekly for 2 weeks , 80 u , subcutaneously , weekly for 2 weeks , followed by 80 u , subcutaneously , twice weekly for 16 weeks of treatment . seven subjects received 40 u of acthar gel , subcutaneously , twice weekly for 2 weeks , followed by 80 u , subcutaneously , twice weekly for 6 months of treatment . the remaining five subjects received acthar gel with heterogeneous dosing as determined by the treating physician . the duration of therapy ranged from 12 to 56 weeks , with a mean follow - up time of 48 weeks.16 at the end of therapy , seven of 24 ( 29% ) patients had experienced remission ( two complete and five partial).16 in subjects achieving remission of proteinuria , median time to reduced proteinuria was 5 weeks and median time to remission of proteinuria was 16 weeks.16 although only 29% of the subjects experienced remission in this series , improved outcomes may encourage physicians to consider acthar gel in idiopathic fsgs cases that are refractory to previous cs therapy . , we recommend that acth be discontinued for patients with fsgs who have not demonstrated any significant decline in proteinuria by 1216 weeks . the pathological basis of fsgs and mcd appears to essentially be podocyte - related , as evidenced by podocyte foot processes effacement , microvillus transformation , and podocytopenia.23 knockout and transgenic models have provided proof of concept that mutations in specific podocyte proteins mediate genetic forms of fsgs.24 a study was conducted in rats with subtotal nephrectomy to examine the capability of acthar gel for providing podocyte protection.25 compared with the control group , acthar gel preserved kidney function as measured by increased renal plasma flow and glomerular filtration rate and lower serum creatinine levels . these data suggest that acthar gel reduces proteinuria and renal injury in a preclinical model of progressive glomerulosclerosis . lindskog investigated the expression of mc in human kidneys to test the hypothesis that synthetic acth directly exerts its antiproteinuric effects via mc receptors.26 gene expression of the mc receptor mc1r ( but no other mc receptor ) was identified in podocytes , glomerular endothelial cells , mesangial cells , and tubular epithelial cells , and analysis showed that podocytes expressed most of the mc1r protein.26 a specific mc1r agonist ( ms05 ) demonstrated that mc1r agonism reduced proteinuria , improved podocyte morphology , and reduced oxidative stress in rats with passive heymann nephritis ( phn).26 the nonspecific mc receptor agonist -msh ( n=8 ) also significantly reduced proteinuria by 52% compared with untreated phn ( n=8 ; p<0.05 ) . within the -msh group , this corresponds to a 55% decrease in proteinuria after 4 weeks of treatment compared with proteinuria at the start of treatment . acthar gel ( acth ) has antiproteinuric and immunomodulatory effects that are not entirely explained by its steroidogenic activity . acth is the parent molecule of -msh , and exhibits biologic efficacy by modulating proinflammatory cytokines and subsequent leukocyte extravasation , and evidence suggests that mcs also may have autocrine / paracrine effects in joints . acthar gel was primarily administered in this case to induce the remission of proteinuria in a patient with a history of intermittent proteinuria and a confirmed diagnosis of fsgs treated with ace inhibitor therapy , but who experienced worsening edema and serum creatinine with 4 + proteinuria . this case is interesting not only because of its effect in improving proteinuria , but also because it showed clinically beneficial ancillary effects on the patient s inflammatory disease states . the role of acth in the treatment of ra or on has not been adequately studied , but improvement in this patient s flares and pain in ra as well as orbital inflammation and reduction in the swelling of the optic nerve and thickness is noteworthy and may be related to its agonist effects on multiple mc receptors .	backgroundfocal segmental glomerulosclerosis ( fsgs ) causes scarring or sclerosis of glomeruli that act as tiny filters in the kidneys , damage to which results in diminished ability to properly filter blood , resulting in the urinary loss of plasma proteins and subsequent proteinuria.case presentationa 60-year - old , white female with a history of intermittent proteinuria was referred by her primary care physician for renal dysfunction . biopsy confirmed fsgs and she was treated with an angiotensin - converting enzyme inhibitor . she also had rheumatoid arthritis ( ra ) but no active synovitis and was maintained on prednisone 5 mg / d . she also complained of worsening vision in her right eye and was diagnosed with optic neuritis ( on ) . she remained stable for about 8 months when examination indicated fsgs relapse , and she reported painful ra flares . she was treated with acthar gel ( 40 mg biweekly ) for 6 months , after which proteinuria and urine protein - to - creatinine ratio decreased to about half . her on improved , and she reported that she had fewer ra flares and pain improved by 50% . this case of confirmed fsgs showed an improved response to treatment with acthar gel for fsgs with concomitant ra and on.conclusionthis referral case is relevant to primary care practitioners who treat disorders that may be responsive to corticosteroid therapy . the antiproteinuric effects and ancillary improvement in ra and on symptoms during treatment with acthar gel are not entirely explained by its steroidogenic actions . acth is a bioactive peptide that , together with -melanocyte - stimulating hormone , exhibits biologic efficacy by modulating proinflammatory cytokines and subsequent leukocyte extravasation and may have autocrine / paracrine effects in joints . while acthar gel was primarily administered in this case to treat proteinuria , it also showed ancillary benefits in patients with concomitant inflammatory disease states .
chronic recurrent non - specific parotitis is characterized by recurrent episodes of swelling and pain of unknown etiology in the parotid gland . sialography is a hallmark in the diagnosis of salivary gland disorders ; newer imaging modalities like ct - sialography , sialoendoscopy and mri can be used . various treatment modalities have been tried , from conservative approach to surgical excision depending on the recurrence rate and severity of the condition . although symptomatic treatment with antibiotics and analgesic , injection of intraductal medicament , aggressive treatment like duct ligation or excision of gland are some of the treatment modalities , there is no established algorithm as to which treatment method should be opted in such clinical situation . a 20 years old male patient reported with pain and salty taste in the mouth that had began before a week . examination revealed an elevated right parotid papilla ; ropy , cloudy appearing saliva was oozing out on milking the gland . sialography as a treatment showed a good response with no recurrence after two years of follow - up . recurrent attacks significantly affect the quality of life and also lead to progressive gland destruction . hence , conventional sialography is useful in the diagnosis and also effective as a therapeutic aid in recurrent parotitis . acute refers to sudden onset of pain and swelling in parotid gland whereas chronic recurrent parotitis ( crp ) is characterized by intermittent , painful and swelling of the gland which may or may not be associated with food intake ( 1 ) . the term chronic recurrent non - specific parotitisis used in cases where no definite etiology is identified ( 2 ) . researchers have suggested that recurrent parotitis arises due to retrograde infections eventual to stasis of saliva , allergic , immune deficiency , genetic and hereditary factors ( 2 , 3 ) . however , none of these factors have been proven as an actual cause for the disease . there is lot of disagreement regarding the treatment of recurrent parotitis due to ambiguity about its etiology . treatment of acute phase is aimed at relieving symptoms and prevention of damage to gland parenchyma ( 4 ) . proposed treatments for crp include steroids , tetracycline and 1% methyl violet as intra - ductal medicament ( 5 , 6 ) . conventional sialography can also be used as a therapeutic aid in cases of recurrent infection as it helps in salivary gland lavage , removal of small calculi and mucous plugs within the ducts ( 7 , 8) . our primary aim in this case was to relieve the pain and reduce the frequency of recurrence . to achieve this history : a 20 years old male patient presented with pain in front of the right ear and salty taste on having food that had began before a week . pain in the right parotid gland region was sudden , intermittent , throbbing in quality and initiated while the patient was chewing food and increased in intensity especially on taking citrus food . the patient gave history of at least two episodes of recurrent swelling and pain per year on the right side of the face since two - three years . physical examination : on general physical examination , the patient was febrile and lymph nodes were palpable in the submandibular region bilaterally which was single , firm , freely mobile and tender . on extra oral examination , the right parotid gland was tender on palpation and firm in consistency whereas the left parotid gland was normal . intra - oral examination revealed an elevated right parotid papilla ( figure 2 ) . on milking the gland a thick , ropy , cloudy appearing saliva was oozing out of the duct extra oral view showing the bilaterally symmetrical face intra oral view showing elevated right parotid papilla investigation : although antibiotic sensitivity test was the norm of investigation , it was not advised as the patient was already taking antibiotic which was prescribed by a private practitioner . salivary flow rate was assessed using drooling method . before unstimulated whole saliva was collected , the patient was instructed to refrain from eating and drinking for 90 minutes to avoid any salivary stimulation . later , the patient was asked to drool the saliva in a vial at every one minute for five minutes . for stimulated method , 2% citric acid was placed on the tongue at every 30 seconds for five minutes and the patient asked to drool the saliva in a vial . salivary flow rate of unstimulated and stimulated saliva was 0.3ml / min and 1ml / min respectively suggesting normal salivary flow rate . sialography was performed with 2ml of sodium diatrizoate contrast media which was slowly injected into the gland until some resistance was felt , and the patient reported slight pain in the gland area . digital opg showed uniform normal course and caliber of stensen 's duct measuring about 23 mm in diameter from opening till the periphery of the gland . terminal ductules showed areas of blobs and dots of contrast media indicating sialectasis ( figure 3 ) . fifty percent excretion of the dye was observed in digital opg after one minute ( figure 4 ) , and complete excretion of dye was observed in five minutes suggesting normal functioning of the gland . the patient was instructed to use secretogogues ( lime juice ) for three days to clear the debris and to stimulate the salivation . sialograph shows the duct which is normal in course and caliber with dots and blobs appearance at the terminal ductules approximately 50% excretion of contrast medium after 1 diagnosis and treatment : based on patient history , clinical finding , investigation and sialographic appearance a final diagnosis of chronic recurrent non - specific parotitis was made as we were not able to find out any specific etiologic factor . sialography was performed not only for the diagnostic purpose but also for gland lavage which helped in clearing the mucus plug or cellular debris . the patient was advised to continue antibiotics and analgesic for seven days and was kept under regular follow - up once in six months for a two years ' duration . the treatment seemed to be effective as there was no recurrence during the two years ' follow - up . crp patients suffer from recurrent swelling and tenderness of the involved gland which gradually leads to the destruction of the gland . reducing the frequency of recurrence and improving the quality of life are the objective of treatment . key to the successful treatment of crp is the complete removal of cellular debris and precipitated serum proteins from the ductal lumen . this can be skillful achieved with sialography , ductal dilation with lacrimal probe and gland lavage ( 9 ) . elevated parotid papilla , reduced salivary flow and secretion are viscous and milky in appearance with clumps of material interspersed ( 10 ) . the etiology of the disease is multifactorial .there are various theories to explain the pathogenesis , one theory postulates that reduced salivary flow results in decreased mechanical cleansing , allowing bacteria to colonize and invade the duct . whereas the other proposes that repeated episodes of acute infection may lead to mucus metaplasia of ductal epithelium resulting in increased mucus content of secretions , stasis and further episodes of inflammation . secretory disorders like difference in the secretion and excretion of fluid are also considered as having an important role in the pathogenesis(10 ) . studies have reported that the ultrasonagraphy and sialography appearance for recurrent parotitis is characterized by sialectasis with strictures and dilatation of the major duct ( 9 ) . sailoendoscopy revealed white wall and lack of vascularity in the ductal layer in 75% of crp cases and multiple fibrinous debris and mucous plug in 45% of juvenile recurrent parotitis ( 11 ) . in our case although sialography is primarily used for diagnostic purpose , it can also be used as treatment for recurrent parotitis and obstructive disorders ( 12 ) . sialography improves patency of duct during cannulation by flushing action of irrigant which helps in removing any epithelial debris and mucous plug . the iodine content of the contrast medium acts as an antiseptic agent , thus reducing symptoms and preventing recurrence . this treatment should be repeated once in every two days along with sialogouges until the swelling is subsided and saliva is clear . in our case , sialography was done only once and the patient showed no recurrence during the two years ' follow - up , similar with many other reported cases ( 5,7,8,13 ) . other intracanal medicaments have been tried by many authors - bowling etal reported intraductal tetracycline instillation causing acinar atrophy in rats ( 6 ) . mandel and kaynar ( 1995 ) stated that although steroid reduces swelling and inflammation , it is not effective in preventing recurrence ( 5).nahileli et al . ( 2004 ) stated that use of sailoendoscopy facilitates direct visualization of the intraglandular structures and combination of steroid lavage with ductal dilatation will help in reducing the symptoms as well as recurrence(14 ) . however , the success of the treatment depends on intraductal lavage of the affected gland rather than the type of intraductal medicament used as various studies showed no difference in the frequency of recurrence rate . watkin and hobsely found that 56% of adult and 64% of children showed good response to conservative treatment in a five years ' follow - up study ( 15 ) . bilateral sailoendoscopy and lavage with intraductal hydrocortisone resulted in 92% recurrence free rate upto 36 months in juvenile recurrent parotitis cases ( 16 ) . few case reports showed sialography as alternative treatment for this condition as it is minimally invasive procedure with favorable outcome in juvenile recurrent parotitis ( 58 ) . however , if the symptom persists or worsens , then an aggressive treatment should be opted such as duct ligation , parotidectomy and tympanic neurectomy . in conclusion , as the cause for crp is multifactorial , patients should be educated to take higher liquid content in the diet , to do self - massaging of the gland and to maintain oral hygiene so as to avoid retrograde infection . in spite of various advanced imaging techniques , there is no established algorithm as to which imaging modality should be done in a given clinical situation . in our case we preferred sialography as it is simple to perform and cost - effective . it also has added therapeutic effect , especially in conditions where it can prevent recurrence and provide maximum benefit to the patient .	backgroundchronic recurrent non - specific parotitis is characterized by recurrent episodes of swelling and pain of unknown etiology in the parotid gland . sialography is a hallmark in the diagnosis of salivary gland disorders ; newer imaging modalities like ct - sialography , sialoendoscopy and mri can be used . various treatment modalities have been tried , from conservative approach to surgical excision depending on the recurrence rate and severity of the condition . although symptomatic treatment with antibiotics and analgesic , injection of intraductal medicament , aggressive treatment like duct ligation or excision of gland are some of the treatment modalities , there is no established algorithm as to which treatment method should be opted in such clinical situation.case detaila 20 years old male patient reported with pain and salty taste in the mouth that had began before a week . examination revealed an elevated right parotid papilla ; ropy , cloudy appearing saliva was oozing out on milking the gland . unstimulated and stimulated whole salivary flow rate was assessed using drooling method . sialography was used as a diagnostic and a therapeutic aid . in our case , sialography as a treatment showed a good response with no recurrence after two years of follow - up . we highlighted the role of sialography as a therapeutic aid.conclusionrecurrent attacks significantly affect the quality of life and also lead to progressive gland destruction . preventing or reducing the frequency of recurrence remains the goal of therapeutic procedure . hence , conventional sialography is useful in the diagnosis and also effective as a therapeutic aid in recurrent parotitis .
periodontitis is a ubiquitous chronic inflammatory disease affecting the supporting structures of the teeth and if not promptly diagnosed and correctly managed can ultimately lead to tooth loss . the importance of successful management and treatment of periodontitis has gained added importance in recent years with the recognition that periodontitis is associated with a number of important systemic diseases , include cardio / cerebrovascular diseases , diabetes , respiratory diseases , gastric ulcers , rheumatoid arthritis , obesity , cognitive impairment and even cancers etc . three basic mechanisms have been postulated to play a role in these interactions ; metastatic infections , inflammation and inflammatory injury , and adaptive immunity . periodontal diseases are inflammatory diseases in which microbial etiologic factors induce a series of host responses that mediate inflammatory events . in susceptible individuals , dysregulation of inflammatory and immune pathways leads to chronic inflammation , tissue destruction , and disease . a number of risk factors contribute to the susceptibility of individuals to periodontal diseases , and to the pathogenesis and severity of the disease . these factors include smoking , diabetes , immunosuppression , genetic factors , stress , and age . studies on how the risk factors influence disease progression have mainly been focused on the inflammatory reaction . the conclusion from these studies is that a sound inflammatory host response is needed for successful periodontal defense . factors that modify this response may either cause an overwhelming reaction or an inadequate reaction , both of which may accelerate tissue destruction . the word sleep means relaxation , rest or just a light term , but it is a complex and essential biological process that is required on a daily basis for all humans regardless of age , sex or ethnic origin . in addition to maintaining normal brain functioning , sleep has important roles in controlling the functions of many other body systems vital to health and well - being . sleep deprivation is becoming increasingly common in today 's society . compared to a few decades ago , major changes in sleep culture have been observed globally because there has been a trend toward adopting a 24/7 lifestyle , longer working hours and longer work shifts . this has led to a significant reduction in total sleeping hours in both adults and children . epidemiologic data indicate that sleep disturbance and short sleep duration adversely impact human physical health and mortality risk . underlying mechanisms include decrease in overall immunity , a state of systemic inflammation with increased inflammatory markers ensues , and up - regulation of hormones . thus , in recent years , there has been an increasing body of research investigating role of sleep deprivation in pathogenesis of various chronic inflammatory and infectious diseases ; however , to the best of our knowledge , the role of sleep in chronic periodontal disease has not been investigated . therefore , the aim of the present study was to assess if there is an association of sleep deprivation with chronic periodontal diseases . a total of 60 subjects categorized in 3 groups ( n = 20 each ) viz . clinically healthy , gingivitis and periodontitis were identified and selected from among the patients visiting the department of periodontology and oral implantology , national dental college and hospital , dera bassi ( punjab ) . all study subjects underwent detailed medical history and periodontal examination before enrollment which also included information for demographics such as age , gender , and socioeconomic status . as none of the subjects belonged to poor or rich classes , subjects were categorized into low middle class , middle class , and high middle class on the basis of collected data . subjects were excluded if they were edentulous , pregnant or lactating , smokers , suffering from known systemic diseases which could alter healing response of periodontium , who had received any periodontal treatment in 6 months before study or those who had history of medication ( antibiotics or anti - inflammatory drugs ) in 3 months before study . subjects were examined by a single examiner for the assessment of gingival index ( gi ) ( loe , 1963 ) and pocket probing depth ( ppd ) . william 's periodontal probe was used to measure the ppd from the gingival margin to the bottom of the periodontal sulcus or pocket at two proximal sites of each tooth . group i healthy : gi score : 0 , ppd 3 mmgroup ii moderate to severe generalized chronic periodontitis : generalized - ppd 3 in 30% of sites ; moderate - severe periodontitis - ppd 6 mm group i healthy : gi score : 0 , ppd 3 mm group ii gingivitis : gi score 1 , ppd 3 mm group iii moderate to severe generalized chronic periodontitis : generalized - ppd 3 in 30% of sites ; moderate - severe periodontitis - ppd 6 mm all the study subjects were administered psqi questionnaire . the psqi is an effective instrument used to measure the quality and patterns of sleep in the older adult . it is brief , reliable , valid , and standardized self - reported measure of sleep quality . it differentiates poor from good sleep by measuring seven domains : subjective sleep quality , sleep latency , sleep duration , habitual sleep efficiency , sleep disturbances , use of sleep medication , and daytime dysfunction over the last month . psqi questionnaire was modified from the original in order to include the first 9 items only these items contribute to the total score . scoring of the answers was based on a 0 to 3 scale , whereby 3 reflected the negative extreme on the likert scale . the component scores were summed to produce a global score ( range 0 to 21 ) . a global sum of 5 or greater indicated a poor sleeper . all statistical analyses were carried out using statistical package for social sciences version 15.0 ( spss inc . , chicago , il , usa ) software package . anova test was used for comparison between means of groups and to determine the significance of each parameter under study . multivariate tukey honest significant difference test was used to investigate the relationship between global psqi scores and other independent variables . anova test of between - subjects effects was used to investigate the role of confounders such as age , gender , and socioeconomic status on the relationship of psqi and periodontal disease . all the study subjects were administered psqi questionnaire . the psqi is an effective instrument used to measure the quality and patterns of sleep in the older adult . it is brief , reliable , valid , and standardized self - reported measure of sleep quality . it differentiates poor from good sleep by measuring seven domains : subjective sleep quality , sleep latency , sleep duration , habitual sleep efficiency , sleep disturbances , use of sleep medication , and daytime dysfunction over the last month . psqi questionnaire was modified from the original in order to include the first 9 items only these items contribute to the total score . scoring of the answers was based on a 0 to 3 scale , whereby 3 reflected the negative extreme on the likert scale . the component scores were summed to produce a global score ( range 0 to 21 ) . a global sum of 5 or greater indicated a poor sleeper . all statistical analyses were carried out using statistical package for social sciences version 15.0 ( spss inc . , chicago , il , usa ) software package . anova test was used for comparison between means of groups and to determine the significance of each parameter under study . multivariate tukey honest significant difference test was used to investigate the relationship between global psqi scores and other independent variables . anova test of between - subjects effects was used to investigate the role of confounders such as age , gender , and socioeconomic status on the relationship of psqi and periodontal disease . sixty systemically healthy subjects ( 34 females and 26 males ) in age group of 2550 years were assessed for association of sleep deprivation with chronic periodontal disease . present study revealed that mean gi in group i , ii and iii were 0 , 1.39 0.32 and 1.88 0.18 , respectively , whereas ppd were 2.08 0.06 , 2.34 0.17 and 3.58 0.63 , respectively [ table 1 and figure 1 ] . mean psqi score in three groups was 1.20 0.83 , 1.88 0.18 , and 7.39 1.33 , respectively . intergroup comparison of psqi scores was significantly different amongst all three groups [ table 2 ] . the results showed a positive correlation of psqi with gi and ppd in gingivitis and periodontitis groups [ table 3 ] . anova test of between - subjects effects revealed that after controlling for age , gender , and socioeconomic status , association of psqi and periodontal disease was still significant between the 3 groups , viz . , healthy , gingivitis and periodontitis [ table 4 ] . mean parameters in 3 groups mean parameters in 3 groups intergroup comparison of psqi linear co - relations of psqi with gi and ppd tests of between - subjects effects for analysis of the effect of age , gender , and socioeconomic status on psqi the current investigation was aimed at assessing association of sleep deprivation with chronic periodontal disease . results of the present investigation elucidated that mean psqi was highest in the periodontitis group followed by gingivitis subjects and lowest in healthy subjects and the difference among three groups was statistically significant . a positive correlation of psqi with gi and ppd was observed in groups i and ii suggesting that psqi scores commensurate with periodontal destruction . the association was still significant after controlling for age , gender , and socioeconomic status . varieties of methods have been utilized for assessment of sleep quantity and quality like administration of the questionnaire , clinical interviews , sleep diaries , etc . good and bad sleep using a cut - off global score of 5 . it also has internal consistency and a reliability coefficient ( cronbach 's alpha ) of 0.83 for its seven components . numerous studies using the psqi in a variety of older adult population internationally have supported high validity and reliability . the study findings could not be subjected to direct comparison with any with any earlier similar investigation because to best of our knowledge this is a first investigation conducted for exploring association of sleep deprivation with periodontal disease . sleep deprivation leads to the development of unfavorable hormonal profile and modulation of host immune and inflammatory mechanisms . deprivation of sleep has been documented to increase lymphocyte activation with overproductions of interleukin-1 ( il-1 ) , il-6 , il-17 , and tumor necrosis factor alpha ( tnf- ) . an important start in the efforts to find the source of sleep loss - associated inflammation is a work showing that one night of sleep restricted to 4 h led to increased monocyte production of il-6 and tnf - alpha messenger rna . during experimental sleep deprivation of healthy volunteers , cellular adhesion molecules have been found to increase which are pro - coagulatory and pro - inflammatory markers produced by stimulated vascular endothelium ( e.g. e - selectin and intercellular adhesion molecule-1 ) . central nervous system regulation of immune responses is primarily driven by two effector signaling pathways : activation of the hypothalamic pituitary adrenal ( hpa ) axis and the sympathetic nervous system ( sns ) . sleep loss activates sympathetic activity with less robust evidence of effects on the hpa axis . whereas activation of hpa axis inhibits both antiviral and pro - inflammatory genes , sns activation suppresses antiviral responses ( th1-type gene expression such as interferon- and il-12b ) while stimulating pro - inflammatory genes ( th2-type cytokine genes such as il-4 and il-5 ) , which together provides a plausible mechanism to connect sleep disturbance with various infectious and inflammatory diseases . as many of cytokines have a significant role in the pathogenesis of chronic periodontal disease , there might occur potentiation of periodontal destruction in a sleep deprived individual . further short sleep duration has been shown not only to increase pathogen susceptibility ; but also to decrease the immunologic protection offered by standard vaccines . this might impair an individual 's capacity to perform adequate oral hygiene practices , thus increasing the risk of periodontal disease . due to multifactorial etiology of both sleep deprivation and periodontal disease other unknown confounding factors might explain this association too . thus , the present study can be taken as hypothesis generating investigation and with its preliminary results suggestive of the association of sleep deprivation with severity of periodontal disease , definitely calls on for future studies with larger samples , studying the effects after controlling of other confounding factors and including both subjective and objective measures for assessment of sleep . within limits of the present investigation , there appears to be an association between periodontal diseases and sleep deprivation . better understanding the interplay between sleep deprivation and periodontitis with more focused investigations may help in designing effective lifestyle intervention strategies for this multifactorial disease .	background : sleep deprivation has become a global phenomenon , and epidemiologic data indicate that short sleep duration adversely impacts human physical health . underlying mechanisms involve modulation of immune - inflammatory mechanisms . these changes might contribute to potentiation of destructive periodontal disease . therefore , the present study aimed to assess if there is an association of sleep deprivation with chronic periodontal diseases.materials and methods : sixty subjects were categorized into 3 groups ( n = 20 each ) viz . clinically healthy , gingivitis and periodontitis . periodontal status of subjects was assessed by gingival index and pocket probing depth . all the study subjects were administered pittsburgh sleep quality index ( psqi ) questionnaire for the assessment of sleep deprivation.results:present investigation revealed that mean psqi was highest in the periodontitis group as compared to other two groups and the difference among three groups was statistically significant.conclusion:the present study with preliminary results suggestive of the association of sleep deprivation with severity of periodontal disease , definitely calls on for future studies with larger samples .
centrosomes serve as microtubule - organizing centers in interphase and mitotic cells and play a role in cytokinesis and cell cycle progression ( doxsey , 2001 ) . they are also the precursors of primary cilia , nonmotile sensory organelles found on most vertebrate cells . ciliary dysfunctions are associated with several human diseases ( pazour and rosenbaum , 2002 ; rosenbaum and witman , 2002 ) . primary cilia in vertebrate cells appear to arise from the mother centriole of the centrosome within a membrane sheath , which forms from cytoplasmic vesicles and ultimately fuses with the plasma membrane ( sorokin , 1968 ) . the intimate relationship between the centrosome and the primary cilium suggests that functions and components may be shared between these structures . primary cilia assembly occurs by a process called intraflagellar transport ( ift ) ( kozminski et al . , 1993 ; pazour and rosenbaum , 2002 ; rosenbaum , 2002 ; rosenbaum and witman , 2002 ; han et al . , 2003 ) . interference with ift protein function results in loss or reduction of primary cilia ( pazour et al . primary cilia possess cation channels and receptors that appear to activate signal transduction pathways that control cellular function ( pazour and rosenbaum , 2002 ; pazour et al . , 2002a ; pazour and witman , 2003 ) . polycystin-2 ( pc2 ) is a calcium - selective channel on primary cilia associated with polycystic kidney disease ( somlo and ehrlich , 2001 ) . it appears to be activated by mechanical movement of primary cilia in response to fluid flow ( nauli et al . , 2003 ) , and controls the assembly of primary cilia ( thomson et al . however , little is known about the mechanism by which ift proteins and pc2 are organized at the centrioles / basal bodies ( terms used interchangeably ) . a role for centrosome proteins in primary cilia formation mutants of a drosophila protein that shares homology with the vertebrate centrosome proteins pericentrin ( pcnt ) ( flory and davis , 2003 ; zimmerman et al . , 2004 ) and akap450 ( keryer et al . , 2003 ) disrupt formation of mechanosensory and chemosensory cilia ( martinez - campos et al . , 2004 ) . drosophila mutants that affect ift also disrupt formation of drosophila sensory cilia ( han et al . , 2003 ) . however , the molecular mechanism by which centrosomes and centrosome proteins modulate primary cilia assembly has not been determined . in this report , we show that pcnt forms a complex with ift proteins and pc2 in vertebrate cells and tissues , and that pcnt depletion by small interfering rnas ( sirnas ) disrupts centriole association of ifts and pc2 and inhibits primary cilia formation . in this work , we have studied a larger isoform of pcnt using specific sirnas and antibodies unless otherwise noted ( flory and davis , 2003 ; zimmerman et al . , 2004 ) . immunofluorescence imaging demonstrated that pcnt partially overlapped with centriolin , a protein associated with the mother centriole at centrosomes ( fig . 1 a ) ( gromley et al . , 2003 ) . in addition , pcnt associated with both centrioles at the base of primary cilia ( fig . 1 , b and c ) and motile cilia ( fig . 1 d ) . higher resolution immunogold em demonstrated that pcnt was on or near the centrioles of motile cilia ( fig . ( a ) immunofluorescence image of a centrosome in rpe1 cells costained for pcnt ( green ) and centriolin ( red ; bar , 1 m ) . ( b and c ) dic ( b ) and immunofluorescence ( c ) images of a primary cilium ( arrow ) in rpe1 cell stained for pcnt ( green ) and centrioles / primary cilium ( gt335 , red ) . bar , 5 m for b and c. ( d ) immunofluorescence image of a ciliated epithelial cell from mouse trachea showing pcnt ( green ) at the base of motile cilia ( dic ; bar , 5 m ) . ( e ) immunogold electron microscopic image of a ciliated cell ( as in d ) after incubation with antibodies to pcnt and secondary antibodies bound to 5-nm gold ( bar , 250 nm ) . to test the role of pcnt in cilia organization , we depleted protein levels by sirna . we observed a 7590% reduction in protein levels and a dramatic reduction in centrosome levels of pcnt in most cells ( fig . c ; arrow in c ) when compared with cells treated with control sirnas targeting lamins a / c ( fig . 2 , a and b ) or cells that did not respond to sirna treatment ( fig . in contrast , centrosome localization of -tubulin was only slightly affected under these conditions ( fig . primary cilia were induced in retinal pigmented epithelial cells ( rpe1 ) treated with sirnas targeting pcnt or lamin a / c . cilia were detected with antibodies to polyglutamylated tubulins ( gt335 ; gromley et al . , 2003 ) and by differential interference contrast ( dic ) microscopy . in most cells treated with sirnas targeting pcnt , primary cilia failed to assemble ( fig . 2 , e , g , and h ) , whereas control cells treated with sirnas targeting lamin or ninein assembled normal full - length primary cilia ( fig . 2 , d , f , and h ; unpublished data ) . pcnt silencing inhibits primary cilia formation . ( a ) pcnt and lamin protein levels ( western blot ) after sirna as indicated . ( b ) fluorescence intensity of individual centrosomes ( bars ) after treatment with sirnas targeting pcnt or lamin . centrosomal pcnt is reduced to levels below the lowest control levels ( lamin ) in 87% of cells . ( c ) immunofluorescence image of rpe1 cells after pcnt silencing showing reduced centrosomal pcnt in one cell ( green , arrow ) and normal level in the other . low ( d and e ) and high ( f and g ) magnification immunofluorescence images of cilia and centrioles stained with gt335 after treatment with pcnt ( e and g ) or lamin ( d and f ) sirnas . bar in e , 5 m ( for d and e ) ; bar in f , 1 m ( for f and g ) . ( h ) graph showing percentage of cells that lack cilia after treatment with indicated sirnas . ( i k ) electron micrographs showing centriole structure in cells with reduced pcnt . k ) . to address the mechanism of ciliary loss in cells with reduced pcnt , we examined centriole function , structure , and composition . consistent with previous results from our group and others ( dammermann and merdes , 2002 ; martinez - campos et al . , 2004 ; zimmerman et al . , 2004 ) , we found that microtubule organization and nucleation were not significantly disrupted ( unpublished data ) . 2 , e and g ) , but this was also observed after functional abrogation of proteins that did not affect primary cilia ( e.g. , ninein ; unpublished data ) . because vertebrate primary cilia formation and function requires ift proteins ( murcia et al . , 2000 ; pazour et al . , 2000 ) and the cation channel pc2 ( somlo and ehrlich , 2001 ; pazour et al . , 2002b ; rosenbaum and witman , 2002 ; , we reasoned that pcnt might cooperate with these proteins in primary cilia organization . to test this ift57 and ift88 localized primarily to the distal end of the mother centriole near the base of the primary cilium and to the tips and in spots along the length of primary cilia ( fig . localization of these ifts to the distal portion of the mother centriole was consistent with known sites of ift protein localization in chlamydomonas reinhardtii ( cole et al . ift20 was found on the proximal portion of mother centriole and the lateral aspect of the daughter centriole ( fig . 3 c ) , an area thought to be involved in interconnecting the two centrioles . epithelial cells , ift proteins partially localized with pcnt to sites at the base of the motile cilia where basal bodies are found ( fig localization of ift proteins and pc2 , and mislocalization of pcnt in cells with reduced ift20 . ( a d ) rpe1 cells stained for ift57 , ift88 , ift20 , and pc2 ( green ) and for basal bodies / cilia ( gt335 , red ) . bar in d , 1 m . ( e ) pcnt ( green ) partially colocalizes with ift20 ( red ) at the base of motile cilia ( seen by dic ) in mouse epithelial cells . ( f g ) untreated rpe1 cells ( f f ) or rpe1 cells stably expressing sirna targeting ift20 ( g g ) showing centrosomal levels of ift20 ( f and g ) , pcnt ( f and g ; bar , 5 m ) , or merge ( f and g ) . insets , enlargements of f and g. ( h and i ) fluorescence intensity of ift20 ( h ) and pcnt ( i ) at individual centrosomes ( bars ) in cells stably expressing ift20 sirna or mock , as indicated below graph . next , we addressed the centriolar anchoring mechanism of pcnt , ifts , and pc2 . we found that pcnt was dependent on ift proteins for localization to basal bodies using cells that stably express sirnas targeting ift20 . 3 , f f and h ) . in cells with reduced centriole - associated ift20 pcnt localized to both centrioles at the base of cilia , partially colocalized with ift proteins ( fig . 4 a , ift57 ) and totally overlapped with pc2 ( fig . 4 c ) . 4 b , b , c , and c ; top cell ) , ift57 ( fig . 4 , bb ) , ift20 , ift88 ( unpublished data ) , and pc2 ( fig . 4 , cc ; top cell ) . in contrast , adjacent nontransfected cells or cells treated with lamin sirnas had robust staining for ift57 and pc2 ( fig . these results show that pcnt and ifts are codependent in their localization to basal bodies of primary cilia . pcnt colocalizes to basal bodies with ift proteins and pc2 , and pcnt silencing mislocalizes ift proteins and pc2 from basal bodies and centrosomes . ( a c ) ift57 ( a b red ) and pc2 ( c c , red ) are mislocalized from basal bodies in rpe1 cells with reduced pcnt ( b b , arrows ; c c , green , small arrows ) , but not in rpe1 cells treated with lamin sirnas ( a a ; bar , 10 m ) or in the cell with control level of pcnt ( c and c , bottom ) . insets : higher magnification of a , b , and c as indicated by arrows . previous reports showed that ift protein complexes and pcnt complexes had similar s values on sucrose gradients ( 1718s ; dictenberg et al . to determine whether pcnt interacted with ift proteins , we isolated ift complexes by a multistep procedure ( san agustin and witman , 2001 ) and found that pcnt a and b cofractionated with ift88 in the final gel filtration column ( fig . 5 a ) . based on recent data showing that ift71 is present on centrosomes and spindle poles ( iomini et al . , 2004 ) , we analyzed centrosome preparations ( doxsey et al . , 1994 ) for the presence of ift proteins . . 5 b ) and other ift proteins ( unpublished data ) were present in pooled fractions containing centrosome proteins ( -tubulin , pcnt ) , but not in pooled fractions lacking centrosomes . moreover , our immunofluorescence imaging showed that ift20 , 57 , 88 , and pc2 were abundant at centrosomes in interphase cells and spindle poles during mitosis in rpe1 cells ( unpublished data ) . ( a ) pooled ift fractions from a sucrose gradient from mouse testes were applied to an fplc column and fractions were loaded on sds gels and probed with pcnt antibodies or ift88 antibody . ( b ) pooled peak centrosome fractions from sucrose gradients ( + ) containing -tubulin , pcnt , and ift88 as indicated and pooled noncentrosome fractions ( ) . ( c , top ) pcnt nh2- and cooh- terminal antibodies ( pcn , pcc ) independently immunoprecipitated endogenous ift88 from lysates of ciliated 488 cells . ( c , middle ) pcc / n immunoprecipitated ift88 from ciliated rpe1 cells , as did antibodies to ift88 and ift57 but not rabbit igg . ift20 fusion protein from a cell line stably overexpressing the protein , as does a glutathione column ( ift20 ) , but not nonimmune igg . blots were probed with anti - gfp antibodies , immunoprecipitation with pcnt ( right ) ; ib , immunoblot antibody . ( d ) pcn immunoprecipitated pc2 from mitotic rpe1 cells , whereas beads alone did not ( bd ) . ( e ) pc2 antibody , but not rabbit igg , immunoprecipitated ift57 from ciliated 488 cells . immunoprecipitation of pcnt using antibodies ( that recognize both small , pcnt a and large , pcnt b isoforms ) raised to two independent domains pulled down endogenous ift88 from two ciliated cell lines ( fig . 5 c , two top panels ) ; endogenous ift57 from testes and ciliated cells ( unpublished data ) , and ectopically expressed gst - gfp - ift20 ( fig . 5 c , bottom ) and endogenous pc2 from mitotic cells ( fig . 5 d ) . we observed no coimmunoprecipitation of any ift protein or pc2 when pcnt antibody was omitted ( fig . 5 , c and e ; igg ) . in reciprocal experiments we found that pc2 immunoprecipitation pulled down endogenous ift57 from ciliated cells ( fig . together , these biochemical data suggest that pcnt , pc2 , and ift proteins form a complex in the cytoplasm of vertebrate cells . the data in this manuscript show that pcnt binds ift proteins and pc2 and is required for primary cilia formation in human cells . this suggests a model in which pcnt recruits protein complexes involved in cilia assembly and calcium signaling to centrioles at the base of primary cilia ( and perhaps flagella ) . because drosophila pcnt / akap450 and ift were shown separately to function in primary cilia assembly ( han et al . , 2003 ; martinez - campos et al . , 2004 ) , it is possible that pcnt has a conserved function in ift organization during cilia formation in both drosophila and vertebrate cells . ift does not appear to play a role in assembly or function of drosophila sperm flagella ( han et al . , thus , it is unlikely that defects in flagellar motility in drosophila pcnt / akap450 mutants ( martinez - campos et al . , 2004 ) are a consequence of disruption of the pcnt ift interaction . however , both vertebrate pcnt ( dictenberg et al . , 1998 ; takahashi et al . , 2002 ; zimmerman et al . , 2004 ) and drosophila pcnt / akap450 ( kawaguchi and zheng , 2003 ) interact with complexes containing -tubulin , and -tubulin has recently been shown to be required for flagellar motility in trypanosomes ( mckean et al . , 2003 ) . thus , it is possible that disruption of the interaction between pcnt / akap450 and -tubulin complexes could account for lack of motility in drosophila flagella . another possibility is that the observed structural alterations in centrioles in spermatocytes from drosophila pcnt / akap450 mutants ( martinez - campos et al . , however , in this work we did not detect changes in centriole structure in cells depleted of pcnt . given the recent findings that pcnt and other centrosome proteins are integral components of cilia and flagella and that ift proteins and pc2 are integral components of centrosomes and spindle poles ( iomini et al . , 2004 ; unpublished data ) , it is likely that perturbation of proteins in one of these compartments affects the function of the other . because defects in centrosomes and spindle poles are well documented in cells with abrogated pcnt and pcnt orthologues , they could also contribute to defects in centrosome derivatives such as cilia and flagella . moreover , pcnt and ift proteins require molecular motors to mediate transport events , so it is possible that they utilize some of the same components to accomplish these functions ( zimmerman and doxsey , 2000 ) . it is clear from this discussion that a better understanding of the precise role of pcnt in cilia and flagella assembly / function will require additional studies . on a final note , it is interesting that centrosomes in drosophila pcnt / akap450 mutants are disorganized but appear to assemble normal mitotic spindles ( martinez - campos et al . , 2004 ) . it is possible that residual functional protein remaining in drosophila mutants is sufficient for spindle function . however , recent results from vertebrate cells indicate that there are several forms of pcnt ( flory and davis , 2003 ) , and that a smaller form of the protein is required for spindle organization and function , possibly through its role in anchoring -tubulin complexes or ift proteins at spindle poles ( zimmerman et al . , 2004 ) . a larger pcnt isoform that shares homology with drosophila pcnt / akap450 does not have a dramatic effect on spindle organization ( zimmerman et al . , 2004 ) . it is likely that the multiple pcnt isoforms contribute to a multitude of cellular functions . cells used in this work , rpe1 ( morales et al . , 1999 ) , a mouse inner medullary collecting duct ( imcd3 ) , primary cells isolated from tg737 wild - type mouse ( 488 ) ( pazour et al . , 2000 ) , and freshly isolated primary mouse trachea cells were grown as described in american type culture collection . trachea dissected from mice in pbs were opened and scraped with a wooden applicator stick . sirnas ( 21-nt ; dharmacon research , inc . ) targeting pcnt b ( genbank / embl / ddbj accession no . xm_036857 ; nt 301319 ) , pcnt a / b , or ninein ( dammermann and merdes , 2002 ) and lamin a / c ( gromley et al . , 2003 ) were delivered to cells at 200 nm ( oligofectamine ; invitrogen ) . we also used a stable rpe1 cell line expressing ift20-specific sirnas ( 5-ggaagagtgcaaagacttt-3 ; follit and pazour , in preparation ) . ift protein fractions were prepared as described previously ( san agustin and witman , 2001 ) using additional protease a inhibitors ( complete mini tablets ; roche ) in lysis buffer . primary cilia were induced after sirna treatment ( 72 h ) by culturing rpe1 cells in medium with 0.25% serum and sirnas for 48 h and were identified using gt335 antibody and dic microscopy . cells were prepared for immunofluorescence , imaged , deconvolved ( metamorph ; universal imaging corp . ) , and displayed as two - dimensional projections of three - dimensional reconstructions to visualize the entire cell volume as described in gromley et al . we used methanol as fixative , then confirmed using formaldehyde fixation as previously shown ( dictenberg et al . , 1998 ) . immunogold em was performed as described previously ( doxsey et al . , 1994 ) . rt - pcr for amplification of pcnt b ( forward primer 5-aacactctccatgattgccc-3 and reverse 5-taccctcccaatctttgctg-3 ) and -tubulin was performed as described previously ( gromley et al . , 2003 ) . cells were lysed in lysis buffer consisting of 50 mm tris - hcl ( ph 7.5 ) , 10 mm na2hpo4 ( ph 7.2 ) , 1 mm edta , 150 mm nacl , 1% igepal ca-1630 , and complete mini tablets . testes lysates were prepared as described previously ( san agustin and witman , 2001 ) . protein a / g plus - agarose ( santa cruz biotechnology , inc . ) or glutathione sepharose 4b ( amersham biosciences ) was washed in lysis buffer , added to the cell extracts , and incubated for 2 h at 4c . 5% sds - page gels were used to detect pcnt and pc2 , and 10% gels to detect ifts . no bands were seen with control iggs under any of these conditions or when control iggs were used at concentrations > 10-fold higher than experimental samples . cell extracts used in this work for the pcnt ift interactions came from cells grown in 0.25% serum for 48 h to induce cilia formation . we used affinity - purified antibodies against the nh2 and cooh termini of pcnt a / b ( pcn , pcc ; doxsey et al . , 1994 ; dictenberg et al . , 1998 ) , pcnt b ( a gift of t. davis , university of washington , seattle , wa ; flory et al . , 2000 ) , ift proteins ( pazour et al . , 2002a ) , pc2 ( scheffers et al . , 2002 ) , centriolin ( gromley et al . , 2003 ) , gt335 ( a gift of p. denoulet , universit pierre et marie curie , paris , france ; wolff et al . , 1992 ) , -tubulin ( sigma - aldrich ) , and lamin a / c ( cell signaling technology ) . cells used in this work , rpe1 ( morales et al . , 1999 ) , a mouse inner medullary collecting duct ( imcd3 ) , primary cells isolated from tg737 wild - type mouse ( 488 ) ( pazour et al . , 2000 ) , and freshly isolated primary mouse trachea cells were grown as described in american type culture collection . trachea dissected from mice in pbs were opened and scraped with a wooden applicator stick . sirnas ( 21-nt ; dharmacon research , inc . ) targeting pcnt b ( genbank / embl / ddbj accession no . xm_036857 ; nt 301319 ) , pcnt a / b , or ninein ( dammermann and merdes , 2002 ) and lamin a / c ( gromley et al . , 2003 ) were delivered to cells at 200 nm ( oligofectamine ; invitrogen ) . we also used a stable rpe1 cell line expressing ift20-specific sirnas ( 5-ggaagagtgcaaagacttt-3 ; follit and pazour , in preparation ) . ift protein fractions were prepared as described previously ( san agustin and witman , 2001 ) using additional protease a inhibitors ( complete mini tablets ; roche ) in lysis buffer . primary cilia were induced after sirna treatment ( 72 h ) by culturing rpe1 cells in medium with 0.25% serum and sirnas for 48 h and were identified using gt335 antibody and dic microscopy . cells were prepared for immunofluorescence , imaged , deconvolved ( metamorph ; universal imaging corp . ) , and displayed as two - dimensional projections of three - dimensional reconstructions to visualize the entire cell volume as described in gromley et al . we used methanol as fixative , then confirmed using formaldehyde fixation as previously shown ( dictenberg et al . , 1998 ) . immunogold em was performed as described previously ( doxsey et al . , 1994 ) . rt - pcr for amplification of pcnt b ( forward primer 5-aacactctccatgattgccc-3 and reverse 5-taccctcccaatctttgctg-3 ) and -tubulin was performed as described previously ( gromley et al . , 2003 ) . cells were lysed in lysis buffer consisting of 50 mm tris - hcl ( ph 7.5 ) , 10 mm na2hpo4 ( ph 7.2 ) , 1 mm edta , 150 mm nacl , 1% igepal ca-1630 , and complete mini tablets . testes lysates were prepared as described previously ( san agustin and witman , 2001 ) . protein a / g plus - agarose ( santa cruz biotechnology , inc . ) or glutathione sepharose 4b ( amersham biosciences ) was washed in lysis buffer , added to the cell extracts , and incubated for 2 h at 4c . 5% sds - page gels were used to detect pcnt and pc2 , and 10% gels to detect ifts . no bands were seen with control iggs under any of these conditions or when control iggs were used at concentrations > 10-fold higher than experimental samples . cell extracts used in this work for the pcnt ift interactions came from cells grown in 0.25% serum for 48 h to induce cilia formation . we used affinity - purified antibodies against the nh2 and cooh termini of pcnt a / b ( pcn , pcc ; doxsey et al . 1998 ) , pcnt b ( a gift of t. davis , university of washington , seattle , wa ; flory et al . , 2002a ) , pc2 ( scheffers et al . , 2002 ) , centriolin ( gromley et al . , 2003 ) , gt335 ( a gift of p. denoulet , universit pierre et marie curie , paris , france ; wolff et al . , 1992 ) , -tubulin ( sigma - aldrich ) , and lamin a / c ( cell signaling technology ) .	primary cilia are nonmotile microtubule structures that assemble from basal bodies by a process called intraflagellar transport ( ift ) and are associated with several human diseases . here , we show that the centrosome protein pericentrin ( pcnt ) colocalizes with ift proteins to the base of primary and motile cilia . immunogold electron microscopy demonstrates that pcnt is on or near basal bodies at the base of cilia . pcnt depletion by rna interference disrupts basal body localization of ift proteins and the cation channel polycystin-2 ( pc2 ) , and inhibits primary cilia assembly in human epithelial cells . conversely , silencing of ift20 mislocalizes pcnt from basal bodies and inhibits primary cilia assembly . pcnt is found in spermatocyte ift fractions , and ift proteins are found in isolated centrosome fractions . pcnt antibodies coimmunoprecipitate ift proteins and pc2 from several cell lines and tissues . we conclude that pcnt , ifts , and pc2 form a complex in vertebrate cells that is required for assembly of primary cilia and possibly motile cilia and flagella .
approximately 50% of human genes are predicted to be alternately spliced ( 1,2 ) , and up to 90% of the entire genome to be transcribed ( 3,4 ) . recent work has shown that many non - coding genes are of direct functional significance , that there is strong conservation in regions once characterized as simply junk dna , and that there are many putative novel exons and transcripts in relatively well - characterized protein coding regions ( 4,5 ) . many loci also contain multiple interwoven and overlapping genes , possibly translated in different reading frames , adding additional layers of complexity ( 5 ) . in particular , it is desirable both to use multiple reporters for individual genes in order to pursue splicing events and to increase coverage to explore the less well - characterized regions of the genome . as knowledge of the complexities of gene expression increases , so does the demand for increasingly feature - dense arrays . advances in microarray technology are beginning to address these issues by offering significantly increased feature densities and a concomitant rise in the number of available reporters . the affymetrix human exon 1.0st array , for example uses 6.5 million probes , comprising 1.4 million probesets , to target 1.2 million individual exons , and similar arrays exist for mouse and rat . the aim is to target every known and predicted exon in the genome ( 68 ) . these arrays offer a number of opportunities , but place significant challenges on the bioinformatics tools necessary to exploit them . in particular , sufficient annotation must be provided , for example to identify which exons , transcripts and genes are targeted by each probeset , and the levels of evidence used to define each of these features within the genome ( 7 ) . it offers detailed annotation of the intron exon structure of each gene , their mappings to known transcripts , and their location relative to affymetrix exon array target sequences . data can be explored using a fully interactive real - time scrollable browser , built using the google maps api ( figure 1 ) . other groups have also considered ajax - based approaches to this problem , most notably gbrowse - ajax ( available on sourceforge.net ) . however , these are currently at the proof of concept / prototype stage . ( a ) scrollable genome map . ( b ) toolbar . ( c ) hierarchical annotation of map features . ( d ) context - dependent annotation . an associated bioconductor ( 9 ) package , exonmap ( 7 ) , provides an interface between the database and r , a popular programming language for data analysis . x : map is unique because although some of these data are provided by , for example , affymetrix through their netaffx ( 10 ) database , comprehensive mappings to the genome , filtering for match specificity and provision of the data in a form that supports both high - throughput and gene - centric analysis , are not available in an integrated form elsewhere . x : map is built by searching , in silico , every 25-mer probe sequence represented on the microarray against the entire genome [ downloaded from ensembl ( 11 ) ] and recording the location of each exact match in a relational database . the search strategy is discussed in ref . genome searches are performed because many of the probesets on the array target less well - characterized regions [ e.g. genscan ( 13 ) predictions ] , and because the large amount of intergenic transcription observed means that it is necessary to consider each probe 's potential to hybridize outside annotated genes . an additional search is also performed against a database of known transcript sequences ( ensembl cdna data ) to identify probes that cross exon boundaries ; these would not be found at the genome level . this is all done via a series of groovy ( groovy.codehause.org ) scripts initiated from a master java process . groovy was used because it allowed a much faster turnaround during prototyping and testing , while maintaining reasonable performance ( groovy scripts are compiled to java before they are executed ) . these data are used to populate a set of new tables in a local copy of ensembl ( 11 ) , indexed for speed of querying . an additional set of stored procedures are imported to provide a well - defined api for both the website and the bioconductor package . the ensembl database does not support foreign keys , but these new tables link to the existing ones using the same unique identifiers as those used by ensembl . the overall architecture of x : map is shown in figure 2a . in total , this phase of the build process takes 3 h for ensembl v45 ( human , mouse and rat combined ) on a twin - xeon 64 bit machine with 16 gb ram running red hat enterprise linux . figure 2.the x : map build process . the entire human genome is searched against the array 's 25-mer probe sequences by the java matching engine . results are stored in a relational database as a set of additional tables accompanying a local installation of ensembl . the database is used both by an r client that supports analysis using bioconductor and a webserver that supports a web 2.0/ajax genome browser . the browser serves a set of pre - computed images generated offline by a coarse - grained parallel task ( b ) . tile generation jobs are placed in a javaspace by a master process ( main java process ) . these are consumed by a series of worker processes distributed across a variety of linux machines . the entire human genome is searched against the array 's 25-mer probe sequences by the java matching engine . results are stored in a relational database as a set of additional tables accompanying a local installation of ensembl . the database is used both by an r client that supports analysis using bioconductor and a webserver that supports a web 2.0/ajax genome browser . the browser serves a set of pre - computed images generated offline by a coarse - grained parallel task ( b ) . tile generation jobs are placed in a javaspace by a master process ( main java process ) . these are consumed by a series of worker processes distributed across a variety of linux machines . the x : map genome browser makes use of pre - drawn tiled images in order to improve run - time speed . these are generated in a second phase , managed as a coarse - grained parallel task distributed across a number of linux servers and workstations ( figure 2b ) using a producer / consumer model . in this way , spare cycles available within existing hardware are exploited . during this phase of the build , images are generated using a disparate set of 11 linux servers and workstations in 3 days . tiles are then processed to generate three alternate levels of magnification ( 5 days ) . in the end , for all three species , layers and zoom levels , this process generates 52 gb of image data , comprising 10.27 million individual image tiles . the original exon array annotation supplied by affymetrix grouped probesets into three sets based on the reliability of the annotation used to define the putative genomic regions of interest . x : map groups probesets based on ensembl annotations , and currently provides data for ensembl genes , ensembl ests ( predictions based on est data ) and genscan predictions ( 11 ) . in this way , a direct mapping is formed between the probesets and the data sources used to provide the annotations found at the location the probeset matches the genome . the arrays use a set of short 25-mer probes to target each feature of interest , together referred to as a probeset. the majority of exon array probesets contain four probes . each vertical line in the background of figure 1a represents a probe . it is coloured green if that sequence can match the genome in only one location , grey if it matches at more than one site . x : map considers a probeset to be multi - targeting if one or more of its probes matches at multiple genomic locations . x : map also categorizes probesets according to whether they hit within an exon ( exonic ) , within a gene but outside an exon ( intronic ) or between genes ( intergenic ) . the relative proportions of probesets in each of these categories are summarized for human mouse and rat arrays in table 1 . it can be seen , therefore , that a substantial number of probesets on the array target outside known ensembl genes , or within genes , but outside known exons , and that many of these match instead to est or genscan predictions . even though relatively few probesets ( < 10% ) contain one or more probes capable of hybridizing to multiple sites , their identification is important because many are highly promiscuous and each may match multiple regions of interest ( 7 ) . table 1.relative proportions of probesets in different categories for human , mouse and exon arrayshumanmouseratensembl version45_36 g45_36 f45_34ototal probeset hits143215012358731044729multitarget1294049%871817%711637%exonic30103021%25742121%22287621%intronic probesets all32900723%28418223%19494119% within est exons188511%253032%5057<1% within genscan168521%156831%158482%intergenic probesets all65019645%59147548%53773251% within est exons319132%376913%126531% within genscan492363%488424%520315%data were filtered to remove multi - target probesets and then matched against ensembl genes . intronic and intergenic probesets were then searched against ensembl ests and genscan predictions to assign matches to putative exons . relative proportions of probesets in different categories for human , mouse and exon arrays data were filtered to remove multi - target probesets and then matched against ensembl genes . intronic and intergenic probesets were then searched against ensembl ests and genscan predictions to assign matches to putative exons . one of the issues of presenting genomic data is the need to represent structures such as genes that are many thousands of residues long , while being able also to represent other features , such as a probe hybridization location that are a few ( 25 bp ) residues in length . this requires a browser that can not only offer a variety of different zoom levels but can also support swift navigation up- and downstream of the current location . other important considerations are the need to maximize the amount of screen real estate available to present data , the potential for clutter if too many features are presented at the same time and a simple but capable search interface . x : map , which is designed specifically for high - density microarrays was required to show for each gene , transcript and exon the probesets that match it , their specificity and for each probe , their locations of potential hybridization , and for each individual exon , its sequence . these requirements were addressed by using a visual map with multiple levels of zoom . in order to maximize the responsiveness of the browser this is implemented by generating a set of pre - drawn images ( for the entire genome ) at various levels of magnification . these are then stored on the server as a series of small , tiled , image files , which are managed by the client using the google maps api , which handles the task of requesting the appropriate tiles from the server , of assembling them into a larger contiguous image , as well as the scrolling and zooming of the map . the result is a fully interactive map that can be scrolled dynamically , and with a user interface that , through the use of the google maps api , will be already familiar to many users . in order to maximize the amount of data that can be displayed , the genome is drawn horizontally ( most monitors are wider than they are tall ) and extends the full width of the browser window . data are presented as a set of transparent layers that can be added or removed from the image . by default , only the ensembl gene layer is shown ; est and genscan predictions can be overlaid as required . clearly , since the ensembl annotation pipeline also makes use of ests and in silico predictions , there is a significant degree of overlap between these data , and statistical analyses using exonmap ( see below ) should take this non - independence into account . the tools menu ( figure 1b ) can be hidden or shown by clicking on its tab . when a region of the map is selected , neighbouring features are listed in a selection box ; selection from which causes information pertaining to that item to be displayed as a tree ( figure 1c ) . if the menu is hidden , the tab is animated ( it briefly moves left and right a few pixels ) providing a visual cue to draw attention to the fact that its state has changed . the hierarchy ( figure 1c ) shows the nested relationships between genes , transcripts , exons , probesets , probes and their match locations . selecting an item in the hierarchy causes contextual annotation to be displayed ( figure 1d ) along with hyperlinks to external databases . thus genes and transcripts are annotated with hugo symbols , descriptions and hyperlinks to external databases such as ensembl and genecards ( 14 ) , exons with their reading frame and the appropriate fragment of genomic sequence , and probes and probesets with probe sequences and links to netaffx ( 10 ) . icons are used to identify probesets that are annotated as non - specific , exonic , intronic and intergenic , both in the hierarchy and the annotation view . detailed descriptions of the different icons and visual cues used within the browser can be found on the help pages accompanying the web site . a bioconductor package , exonmap , can connect to the database ( via the rdbi package ) in order to extract annotation data for use within a full statistical analysis environment . the package provides a series of functions allowing mappings to be made between probeset , exon , transcript and gene identifiers , and filterings to be performed to include / exclude exon , intron and intergenic probesets and those that are non - specific to the genome . finally , a set of visualization functions can be used to map expression data onto genomic features ( figures 3 and 4 ) . figure 3.genes selected as alternatively spliced between mcf7 and mcf10a cell lines , coloured by fold change . fold change colouring ranges from 2 ( intense blue ; up in mcf7 ) to 2 ( intense red ; up in mcf10a ) . expression data are mapped onto these and coloured according to fold change between mcf7 and mcf10a . the pattern of expression across the length of the gene clearly corresponds to the known gene structure . genes selected as alternatively spliced between mcf7 and mcf10a cell lines , coloured by fold change . fold change colouring ranges from 2 ( intense blue ; up in mcf7 ) to 2 ( intense red ; up in mcf10a ) . expression data are mapped onto these and coloured according to fold change between mcf7 and mcf10a . the pattern of expression across the length of the gene clearly corresponds to the known gene structure . ( b ) line plot for the same data . a possible workflow for data analysis using x : map and exonmap is to first identify a set of differentially expressed probesets . these are then mapped , using x : map , to their target genes , and filtered for non - specificity due to multiple targeting . the result is a list of genes for which at least one probeset is differentially expressed . then , for each of these genes , it is possible to retrieve expression data for every targeting probeset . fold change ( e.g. splicing index ) , anova ( e.g. midas ) or variance - based approaches can then be used to characterize genes according to consistency in differential expression across their length ( 15,16 ) . figure 3 , for example , was generated by using limma ( 17 ) to identify all probesets with statistically significant differential expression in a triplicate comparison between two cell lines , the human breast cancer cell line mcf7 , and the non - tumorigenic breast epithelial cell line , mcf10a ( n = 90 051 ; fold change threshold > 2 ; adjusted p value = 0.05 , benjamini and hochberg correction ) . this list was then filtered to remove multi - target probesets and those that did not map cleanly to exons ( n = 49 326 ) . variance was calculated for each gene , and used to select the 200 most varying up- and downregulated genes . figure 4a shows , for example , the gene lama3 , with expression mapped to its transcript structure . a set of exons at the 3 end of the gene figure 4b shows mean intensity for mcf7 and mcf10a plotted for the same gene against sequence position . it can be seen that the expression levels for both cell lines are similar at the 5 end , but that expression levels increase in mcf10a at the 3 end ( rather than decreasing for mcf7 ) . this provides strong evidence that the changes observed are due to over - expression of the known short isoform in mcf10a , rather than under - expression of the 5 end of the gene in mcf7 . currently , x : map focuses on affymetrix exon arrays , however , many of the approaches can be generalized to other array types , and in particular , to tiling and snp arrays . we intend to add these array types to x : map , and also to extend the types of annotation represented within the database to include data types more pertinent for these arrays . an advantage of the approach taken by x : map is that , since all visualization data are pre - computed , making more annotation available via the web - interface will not result in a loss of run - time performance . we are also exploring the possibility of adding an additional client - rendered layer to allow dynamic presentation of users own expression data alongside the existing map annotation . all database data can be downloaded from the x : map website , exonmap from the bioconductor website and the mcf7 and mcf10a sample data from ref . ( 6 ) .	affymetrix exon arrays aim to target every known and predicted exon in the human , mouse or rat genomes , and have reporters that extend beyond protein coding regions to other areas of the transcribed genome . this combination of increased coverage and precision is important because a substantial proportion of protein coding genes are predicted to be alternatively spliced , and because many non - coding genes are known also to be of biological significance . in order to fully exploit these arrays , it is necessary to associate each reporter on the array with the features of the genome it is targeting , and to relate these to gene and genome structure . x : map is a genome annotation database that provides this information . data can be browsed using a novel google - maps based interface , and analysed and further visualized through an associated bioconductor package . the database can be found at http://xmap.picr.man.ac.uk .
parkinson 's disease ( pd ) is the second - most common neurodegenerative disease , and its motor characteristics include resting tremors , rigidity , bradykinesia , and postural instability it has been suggested that losses in proprioception may result from a lack of proper regulation of motor control and body reflexes . for example , proprioception of the knee joint is essential for neuromotor control of the lower limbs . therefore , assessing proprioception may play an important role in evaluating changes in postural instability , gait , and risk of falls . few studies have assessed proprioception in individuals with pd compared to healthy individuals [ 413 ] . the relationship between proprioception and the severity and laterality of motor symptoms and the type of predominant symptom ( akinetic rigid versus tremor ) has not been evaluated in previous studies . the present study aimed to evaluate knee proprioception in individuals with pd and the association between proprioception and motor symptoms , cognitive status , postural instability , and disease severity . this controlled , cross - sectional study was approved by the ethics committee of the universidade federal de cincias da sade de porto alegre , number 988/12 , and was performed according to the code of ethics of the world medical association ( declaration of helsinki ) . this study consisted of individuals with parkinson 's disease group ( pg ) and a control group ( cg ) made up of healthy age- and sex - matched individuals . the number of participants needed for this study was determined using a calculation based on the results of the study by duman et al . ( 2012 ) , which evaluated kinetic postural proprioception in individuals with knee osteoarthritis and established a target angle of 45 for knee flexion . the authors found that the standard deviation for the angular error was 1.60 for the left knee and 1.24 for the right knee . we adopted a margin of error of 0.5 for each standard deviation and assumed a significance level of 0.05 ; therefore , the total number of individuals needed for this study was 20 participants in each group . all participants who agreed to participate in the study signed an informed consent form and were scheduled for the procedure . the pg participants included individuals of both sexes with a clinical diagnosis of pd according to the london brain bank criteria and were ranked between stages 1 and 3 according to the hoehn and yahr staging scale . for both groups , the inclusion criteria were as follows : age older than 45 years , ability to walk alone without the assistance of devices , a score of 26 points or higher on the montreal cognitive assessment scale , and the cognitive ability to understand the tasks . individuals with a history of knee surgery or lower limb fractures in the past 6 months or amputation of any part of the leg , who presented with any restricting pain at the time of the assessment , who were not right - handed , or who had any other neurological disease or diabetes mellitus were excluded from both groups . pd patients were classified into the tremulous form or akinetic rigid type depending on the predominant symptoms , which were assessed using the updrs scale . all ratings were performed 1 hour after taking antiparkinsonian medication for patients in the on phase . the participants in the pg were assessed through an interview and scales to assess cognitive ability ( montreal cognitive assessment , moca ) , motor function ( unified parkinson 's disease rating scale , updrs ) , postural instability ( pull test and stabilometric analysis ) , and disease stage ( hoehn and yahr scale ) . kinetic postural proprioception of knee flexion and knee extension were assessed using an isokinetic dynamometer ( biodex multi - joint system 4 pro ) . the researcher who conducted the proprioceptive assessment of the participants was blinded to the other ratings , which were performed by a second evaluator . the cg participants responded to an anamnesis and moca scale , and they were subsequently evaluated for kinetic postural proprioception and postural instability . static balance was assessed by analyzing oscillations of the center of pressure ( cop ) using a baropodometer ( footwork ist / am3 intermetique ) . the baropodometer consists of a pressure platform that uses capacitive sensors with a thickness of 4 mm , an active area of 490 mm 490 mm , and a sampling rate of 40 hz . the participants were instructed to remain immobile for 30 seconds using self - selected lateral spacing of the feet , a quasistatic posture on the baropodometer platform , and a constant gaze at a point on the wall located 3 m in front of the person at eye level . three measurements were performed with a 1 min interval between each measurement , and an average cop value was calculated for the three assessments . kinetic postural proprioception was evaluated with an isokinetic dynamometer according to the manufacturer 's guidelines ; the same method was used in both groups . specifically , to test the position sense of the knee , the participants remained seated in a chair tilted to 70 with their hips and knees flexed , and one leg was attached at the ankle using specific support equipment . for testing , the lower limb was stabilized , and the knee remained aligned with the mechanical axis of the dynamometer through a point marked on the lateral femoral condyle in the sagittal plane ( figure 1 ) . a single examiner , who was blinded to the other tests , performed the experiments , and the assessment orientations remained the same for all the participants . the equipment was programmed for the proprioceptive assessment of the right and left knees with 45 and 75 of flexion as the testing parameters . initially , the lower limb was positioned at the reference angle ( 90 for knee flexion ) . subsequently , the target angle was reached with the help of a device at a speed of 10/s , and the leg remained in this position for 15 seconds to allow the patient to memorize it . once the patient memorized the position , the limb was passively returned to the reference angle and remained in this position for 10 seconds to rest . then , the participant was asked to place the limb at the memorized target angle and press the stop button on the equipment to record the angle reached . the first round of this process was performed for the participants to become familiar with the test . after the training period , the participants performed three additional attempts , and we calculated the difference between the original target angle and the angle reached , the angular error , and the average proprioceptive error in both tests ( 45 and 75 ) for the three attempts for each lower limb and for bilateral proprioception . only nonparametric tests were used for the data analysis , eliminating the requirement of a normal distribution . to compare the control and patient groups , we used the wilcoxon test for paired samples . to compare the variables within the group of patients , we used the mann whitney and kruskall - wallis tests , for comparisons between two or more variables , respectively . the spearman correlation was used to assess the relationship between two quantitative characteristics of the patients . the sample consisted of 40 participants ( 20 pd patients and 20 age- and gender - matched controls ) . there were no statistically significant differences between the pd and control group in terms of cognition ( p = 0.072 ) . the mean duration of disease among the individuals with pd was 6.1 years ( table 1 ) . half of the pd participants ( 10 ) presented with initial symptoms on the right side and the other half presented with initial symptoms on the left . all participants in the pg were undergoing some type of physiotherapy treatment for motor disorders arising from pd , whereas none of the individuals in the cg were undergoing physiotherapy because they were healthy subjects . the lateral predominance of motor symptoms at the time of assessment remained in the same proportion . based on the participants ' reports during the anamnesis , the first noticeable symptom of pd was rigidity in the majority of the participants ( 55% ) . with respect to the analysis of the center of pressure , oscillations of the cop were higher in patients with pd compared to controls ( p = 0.002 ) . pd patients had higher angular errors in the proprioceptive assessments than the participants in the control group ( table 2 and figure 2 , p = 0.002 ) . pd patients had impaired proprioception performance compared with the control participants , which was observed by analyzing both the side less affected by the disease ( p = 0.050 ) and the side more affected by pd ( p = 0.004 ) . the significant differences between the pg and the cg were observed even when the side with the initial symptoms of pd was analyzed ( p = 0.040 for the right side and p = 0.006 for the left side ) . the analysis of the factors correlated with angular proprioceptive errors in pd is shown in table 3 and described separately in the following . predominant motor symptoms . regarding the type of parkinsonian symptom ( tremor , rigidity , or bradykinesia ) that most interfered with proprioception , patients with tremor as the predominant symptom had larger angular proprioceptive errors ( p = 0.017 ) . we verified that there is a statistically significant correlation between proprioceptive deficits and the degree of staging of the more affected side ( r = 0.461 , p = 0.041 ) and in the bilateral analysis ( r = 0.054 , p = 0.011 ) . impaired proprioception was observed in both sides and was significantly correlated with the degree of functionality as assessed using the updrs ( p = 0.027 in the bilateral analysis ) . other variables . the duration of the disease , presence of dyskinesia , on / off phenomenon , oscillations in the cop , and pull test results did not influence the proprioceptive angular errors . in the present study , pd patients had higher angular errors in proprioception than healthy individuals . pd patients also presented with higher postural instability when evaluated using oscillations of the cop . pd patients showed greater impairment in reaching the target knee angle than did the controls . this deficit in reaching known targets has also been found in other studies [ 46 , 8 ] . these results support the existence of a proprioceptive deficit in pd patients . the proprioception impairment in pd patients was present even in the side less affected by the disease . at the time of the proprioceptive assessment , some authors have suggested that antiparkinsonian medications may negatively affect the proprioceptive system in pd [ 4 , 17 , 18 ] . however , some studies have suggested that proprioception impairment in pd patients could be related to levodopa - induced dyskinesia . we observed that participants who had tremor as the first noticeable symptom of pd had the largest proprioceptive errors . to date , no previous studies have linked the first motor symptom of pd with proprioceptive changes caused by the disease . bradykinesia and postural instability are the most common motor symptoms associated with deficits in proprioception [ 7 , 10 , 11 , 19 , 20 ] . it is important note that rigidity is always a sign and not a symptom although patients reported this as a symptom and most likely this represents a misperception of bradykinesia . determining the most common , disabling symptoms or the symptoms that may influence other motor disorders is important for planning physiotherapy treatment , which , according to the literature , should be focused on the main motor disorders reported by the patient . one possibility for the absence of a correlation between the cop results and the pull test with angular errors of proprioception could be the compensation of the visual system in the postural stability tests . it is known that the visual system aids in minimizing proprioceptive deficits and postural reactions because vision compensates for instabilities , preventing falls . therefore , the results of the postural instability test would likely have been different if the tests were conducted without the influence of vision , in which case there might have been a relationship between postural instability and the proprioceptive alteration of parkinsonian participants . postural instability in pd is not fully understood ; however , it is believed to be the result of a complex interaction between compensatory strategies and the impairment caused by the disease at different levels of the nervous system [ 21 , 22 ] . several posturographic studies investigating cop [ 23 , 24 ] in both static and dynamic conditions have shown that pd patients sway significantly more than healthy subjects because they tend to exceed their limits of stability to a much greater extent . these results are in accordance with our results , which showed that patients with pd had greater oscillations of the cop than the control subjects . the impaired motor ability of the participants in the pg ( based on the updrs - iii scale ) and more severe disease staging was significantly correlated with higher angular errors in the proprioceptive assessment . this relationship between impaired motor symptoms and higher deficits in proprioception was also observed in previous studies [ 4 , 12 , 20 ] . although the severity of the disease influenced the proprioceptive aspects in the sample , the time of diagnosis was not significantly associated with proprioception . this finding may be because sensory deficits increase with duration of disease , and the mean disease duration of the study participants was relatively low . from the findings of this study and based on previous studies , we observed that there was a proprioceptive deficit in pd and , therefore , in the joint position sense . however , the real origin of this alteration is still unknown because the function of the muscle spindles , particularly the receiver of the joint position sense , appears to be normal in pd . in addition , there is no evidence of the existence of an alteration in the ascending somatosensory pathways in pd . some authors argue that proprioceptive deficits in pd may be associated with dysfunction in the sensory integration of cues used to guide movements within the basal nucleus ( bn ) because intact bn circuitry is essential for the perception of joint position and movement , and damage to this circuit can result in kinesthetic deficits . therefore , the bn could be responsible for alterations in proprioception in individuals with pd . however , if the bn was responsible for the proprioceptive alterations in pd , studies that evaluate the effect of dopaminergic medications on proprioceptive deficits should find similar results for all patients , regardless of disease severity . therefore , some authors [ 11 , 12 , 26 ] have suggested that dopaminergic dysfunction within the bn is not primarily responsible for dysfunction of the proprioceptive system . to corroborate this hypothesis , jacobs and horak hypothesized that disturbances in proprioception in pd may be primarily due to a dysfunction in the supplementary motor area ( sma ) because this is one of the main projections of the bn and is sensitive to dopamine and significantly contributes to the planning and direction of movement . furthermore , the cells of the sma showed a loss of selectivity in the response to passive joint movement in parkinsonian monkeys , which demonstrates the importance of the sma in the proper processing of proprioceptive inputs . in this study , we did not find any significant relationship between proprioceptive errors and disease duration , the presence of dyskinesia and motor fluctuations , oscillations of the cop , and the pull test . the present study showed that the participants with pd have higher angular proprioceptive errors , which supports the hypothesis that pd affects the proprioceptive system . there were also significant differences in fluctuations of the center of pressure between individuals with pd and healthy controls , indicating that individuals with pd tend to have greater postural instability than healthy people . the analysis of the factors that have a relationship with proprioception showed that tremors were the dominant symptom and that impaired motor ability significantly influenced the presence of proprioceptive alterations in the parkinsonian individuals . in this study , the postural instability in individuals with pd and the relationship between proprioceptive deficits and motor skills and the severity of pd highlight the importance of focusing on these aspects during the rehabilitation process . based on our results , we suggest that physiotherapy for pd should include proprioceptive and balance exercises to improve motor skills and , in turn , improve the functionality and quality of life of individuals with pd .	background . changes in proprioception may contribute to postural instability in individuals with neurological disorders . objectives . evaluate proprioception in the lower limbs of patients with parkinson 's disease ( pd ) and the association between proprioception and cognitive ability , motor symptoms , postural instability , and disease severity . methods . this is a cross - sectional , controlled study that evaluated proprioception in pd patients and healthy age- and sex - matched individuals . kinetic postural proprioception of the knee was evaluated using an isokinetic dynamometer ( biodex multi - joint system 4 pro ) . participants were evaluated using the montreal cognitive assessment ( moca ) , the hoehn and yahr rating scale and postural instability ( pull test and stabilometric analysis ) , and motor function ( updrs - iii ) tests . results . a total of 40 individuals were enrolled in the study : 20 pd patients and 20 healthy controls ( cg ) . the pd patients had higher angular errors on the proprioceptive ratings than the cg participants ( p = 0.002 ) . oscillations of the center of pressure ( p = 0.002 ) were higher in individuals with pd than in the controls . proprioceptive errors in the pd patients were associated with the presence of tremors as the dominant symptom and more impaired motor performance . conclusion . these findings show that individuals with pd have proprioceptive deficits , which are related to decreased cognitive ability and impaired motor symptoms .
metastasis of gastrointestinal carcinomas to bilateral ovaries is usually seen , and the metastatic tumors are called krukenberg tumors . a few cases have been reported of breast metastatic tumors from both gastrointestinal tract and gastrointestinal tumors metastatic from breast 1 - 3 . however , literature of metastatic gastrointestinal tract carcinomas to bilateral breasts and ovaries at the same time under breastfeeding condition can not be found . here we describe an unusual case of gastrointestinal signet ring cell carcinoma ( srcc ) metastatic to the bilateral breasts and ovaries during breastfeeding period . in july 2009 , a 27-year - old woman with two children was breastfeeding her second 4-month - old baby . at the same time , two breasts had no painfulness and other uncomfortable feelings except of hardness . and then , her baby refused to suck her breasts . in september 2009 , she saw a doctor in her countryside hospital . 1 ) . however , since the doctor said that it was just mastitis , she was not worry about it . in the beginning of 2010 , she was presented with weakness , abdominal distension , slight pain and 5 kg weight loss , but there were not bloodiness , diarrhea and changes in stool - shape . after physical examination and abdominal ultrasound , doctor found a lump in bilateral ovaries , and considerable ascitic fluid . she was transferred to our hospital and re - examined , and the same signs were found under ultrasound ( fig 2 ) . because her bilateral breasts were still very hard , coarse needle biopsy was operated on march 3 , 2010 . the hematoxylin - eosin ( h&e ) staining revealed that a quantity of signet ring - like cells were diffused among fibrous tissues . er , pr , e - cadherin , and ck7 were positive in breast gland epithelium , and negative in tumor cells . ck20 , ca199 , cdx2 and villin were positive in tumor cells ( fig 3 ) , cerbb2 and ca125 negative . these results demonstrated that the tumor was a metastatic carcinoma from gastrointestinal tract , and not a breast primary one . after one course of chemotherapy and on april 13 , operations of bilateral ovaries were carried out . during operation , intraoperative frozen - section examination appeared ring cells among fibrous tissues of ovaries as observed in breast sections . moreover , it was found that there were nodules on uterine and oviduct serosas , omentum , mesentery , and rectal surface with hemorrhagic ascites . interestingly , h&e and ihc of lesions of bilateral ovaries and those nodules in abdomen were all consistent with those of the breast tumors ( fig 4 ) . on june 16 , 2010 , the patient took fourth course of chemotherapy . during this course , she appeared digestive obstruction symptom - several days ' diarrhea and then stopping exhaust . the clinic doctors doubted her intestine was blocked by a tumor . because of her poor physique , she did n't take endoscopy detection . finally , the patient died with cachexia on sep 12 , 2010 . even so , a clear conclusion could be drawn from the clinical and pathological data that tumors of bilateral breasts and ovaries were metastatic from gastrointestinal tract . an analysis of 112 krukenberg tumors in ovaries in 2006 found primary carcinomas were detected synchronously with , or subsequent to , detection of the only two thirds krukenberg tumor 4 . in present case , many tumors with the similar h&e images were found in bilateral breasts , ovaries and abdomen . where is the primary site ? a variety of immunologic markers have been applied to distinguish srccs from the breast , stomach , and gastrointestinal tract . firstly , although er is an effective mark in breast carcinoma , one study showed that approximately 20% of breast srccs can be negative for er 5 . while in the other study , up to 30% of gastrointestinal adenocarcinomas were er positive 6 . secondly , adenocarcinomas of the breast , ovary and gastrointestinal tract show different ck7 and ck20 expression patterns . in breast and ovary adenocarcinomas , ck7 is positive and ck20 is negative . in contrast , ck20 is positive and ck7 is negative in gastrointestinal ones 5,6 . in a word , the combination of ck7 , ck20 and er could play some role in differentiating srcc origination . in recent years for example , villin is mainly produced by epithelial cells that develop a brush border . cells producing villin are reported to be found in the epithelial cells of the intestinal mucosa and gall bladder . another antibody , cdx2 , is considered to be a special marker for gastrointestinal carcinomas . it is a caudal - type homeobox , intestine - specific transcription factor that is expressed early in intestinal development and may be involved in the regulation of proliferation and differentiation of intestinal epithelial cells . one study 7 has demonstrated cdx2 expressed in all cases of colorectal adenocarcinoma and almost 50% of gastric adenocarcinomas , meanwhile , only rare cases of adenocarcinomas of the genitourinary and gynecologic tracts , breast , lung , and head and neck show significant levels of cdx2 . another study 8 found that cdx2 negative in breast srcc , 89% and 90% in gastrointestinal tract and gastric srcc respectively . in comparison with villin which is sensitive and special for gastrointestinal adenocarcinoma , cdx2 is an easier marker for evaluation because it expresses in nuclei while villin is restricted to the apical surface and there may be more false - positives . ihc of the present case showed that er , pr , cerbb2 , e - cadherin and ck7 were negative , ck20 , cdx2 and villin were positive . according to the above theories and studies , its gastrointestinal tract origination is assured . why the tumor in this case advanced so rapidly and severely in the only half - year ? as known , during pregnancy and breastfeeding periods , estrogen hormone is always presented in high levels . some studies indicate that pregnancy and breastfeeding do not influence tumor advance compared with other people with similar stage and age 9 . a poorer prognosis of the tumors in pregnancy and breastfeeding women diagnosed at a later stage attributed to pregnancy . for example , the symptoms of colorectal cancer include nausea , vomiting , abdominal pain , and rectal bleeding . physicians and pregnant women often assumed these are normal pregnant reactions and therefore miss early diagnosis 10 . some studies believes that during pregnancy and breastfeeding periods , the increased vascularity of the breast , high circulating levels of hormones and immune suppressed state of pregnancy accelerate cancer development 11 . similar to malignant melanoma , pregnant women may present with more invasive or advanced disease due to hormonal or growth factor effects 12 . moreover , one study of a clinicopathologic analysis of 112 cases of krukenberg tumors of the ovary found that in the patients with a recent pregnancy , the abdominal mass developed rapidly after delivery . , we do not know when the gastrointestinal tract tumor grew and migrated to breasts and ovaries . however , we believe that pregnancy and breastfeeding accelerate the tumor development . according to our and others ' experiences , srcc type krukenberg tumors are often seen in young women 13 , 14 and breast metastasis are rare . until now , case like ours has not been reported . we speculate the tumor could not aggravate so rapidly if there is no pregnancy and breastfeeding . in conclusion , tumors in pregnant and breastfeeding woman should be thought of and not be disguised by pregnancy symptoms . we obtained informed consent from the patient for her breast photos used in the article . we obtained informed consent from the patient for her breast photos used in the article .	we report a case of a metastatic signet ring cell carcinoma ( srcc ) of gastrointestinal tract . the case is very rare because gastrointestinal tract srcc was metastatic to bilateral breasts and ovaries ( krukenberg tumor ) in the same young woman almost synchronously under breastfeeding condition . though the primary site was not detected , ihc results fully verified that the tumor originated from gastrointestinal tract . the severe situation of the patient was believed to be connected to her high estrogen hormone .
fracture of long bones would require the time for healing ranging from several weeks to months . since the introduction of antibiotics , direct bone surgeries and fixation with metals have changed to a conventional method ( 1 ) . use of internal fixators has increased the rate of bone fracture treatment and decreased the time interval for normal life . this matter is not acceptable by majority of patients and may result in mental and somatic problems . regarding the increased use of internal fixators in recent years and diversity of available products , the failure of these tools has also increase . it may be seen as different forms including major deformity , mechanical fracture , corrosion , and avulsion from the bone ( 2 ) . also different factors are contributing for failure including inappropriate material and make - up , inappropriate fixator selection and surgical technique , and lack of consideration of optimal weighting by patient ( 2 , 3 ) . the failure before treatment completion is an important complex complication leading to numerous problems and reoperation leading to costs and injuries ( 1 ) . two different types of alloys including stainless steel 316 and pure titanium are two optimal materials for making the internal fixators introduced in astm and iso standards ( 4 - 6 ) . regarding to optimal technique and fixator would result in higher tolerability and better healing ( 7 ) . since the failure rate has increased , this study was performed to determine the etiologies of implant fracture in patients with fractures of the implants of lower limbs long bones for better programming to reduce the rate of failure of fixators . in this cross - sectional study , two types of fractured implants in the body were analyzed and underwent metalogical , mechanical , and modeling and stress - bending analysis among 11 subjects among 21 total sample size attending to a referral academic hospital in 2013 and 2014 who had reoperation for femoral and tibial bones repair . background diseases such as osteoporosis , osteopenia , malignancy , diabetes mellitus , high - energy trauma , and the fixator types other than stainless steel and titanium were the exclusion criteria . at this stage was performed by spark emission spectrophotometer ( made in germany ) in environmental situations by standard of astm e406 - 81 and the results were compared with iso-5832 - 1 amounts . then optic metallography was performed by optical microscope . determination of size of particles in microstructure of metal , cycle of thermal operation , production procedure and type of superficial operation , microstructrual cracks , and type of impurity in cross - section were the objectives in this study . the used chemical to determine the border of particles was , cucl2 , hcl , and alcohol . the third matalological test was electron microscopy evaluation in which the cross - section was assessed by scanning electron microscope ( sem ) to determine the fracture cause by evaluation of borders and lines and cracks and corrosions . the goal of mechanical assessment was determination of mechanical tolerance of implant ; the hardness of samples was measured and altered to stretch tolerance by experimental data . the hardness was measured by macrohardness tester by wicker s method . for alteration of hardness to stretch tolerance , reference data were used ( 11 ) . the results were compared with stretch stress of implants in iso5832 standard . by considering the large predicted stresses in dhs plaque , it underwent stress - strain analysis . for geometric modeling the solidwork software was used and the model was completely made according to real sample . the borderline conditions were prepared with fixation of screws location and insertion of external forces to femoral head . regarding difference of inserted forces on implants according to patients activity , the reference data were used ( 12 ) including the forces on femoral head in slow walking , normal walking , rapid walking , stair coming up and down , sitting , standing , dribbling , and bending and up righting the knee measured by sensor . for determination of chemical composition , the quantometry test was performed and the weight percent of elements of alloys were determined . this test was performed by spark emission spectrometer ( made in germany ) in standard situation of astm e406 - 81 . determination of particle size in microstructure of metal , thermal function cycle , production process , type of superficial performance , microstructural cracks , and evaluation of impurity particles on cross - sections were the goals for this test . preparation of samples according to astm e 3 - 10 was in four stages including soft wearing , hard processing , final processing , and making h. the soft wear was performed by silicium carbide on special papers by three sizes including 320 , 400 , and 600 and using silicium carbide particles sizing of 33 , 23 , and 17 microns . the initial wearing was performed by moving the sample on a surface to make the scratches in an orientation . then the samples were put in rotary cycle and processed homogenously by diamond powder of 6 micron size under significant pressure . in final process stage , the fine scratches and remaining bend layers were removed . in this stage alumina powder ( gamma type ) with 0.05 micron size was used . h formation stage was performed to determine the border of particles with putting the samples in a solution of cucl2 , hcl , and alcohol . the other used standards were astm e 407 - 07 for metal microh , astm e 883 - 02 for microscope imaging , and stm e 112 - 96 for measurement of mean particles size . for samples number 10 and 11 the cross - sections were assessed by electron microscope device of scanning electro microscope ( sem ) to determine the cause of fracture by evaluation of cracks and fractures . regarding complexity of loading of dhs implants and uncertainty about the stresses by segment method the stress - strain analysis was performed . for determination of type of loading and locking on the segments the data from study by bergmann et the percent for categorical and the mean and standard deviation were reported for numeric variables . for determination of chemical composition , the quantometry test was performed and the weight percent of elements of alloys were determined . this test was performed by spark emission spectrometer ( made in germany ) in standard situation of astm e406 - 81 . determination of particle size in microstructure of metal , thermal function cycle , production process , type of superficial performance , microstructural cracks , and evaluation of impurity particles on cross - sections were the goals for this test . preparation of samples according to astm e 3 - 10 was in four stages including soft wearing , hard processing , final processing , and making h. the soft wear was performed by silicium carbide on special papers by three sizes including 320 , 400 , and 600 and using silicium carbide particles sizing of 33 , 23 , and 17 microns . the initial wearing was performed by moving the sample on a surface to make the scratches in an orientation . then the samples were put in rotary cycle and processed homogenously by diamond powder of 6 micron size under significant pressure . in final process stage , the fine scratches and remaining bend layers were removed . in this stage alumina powder ( gamma type ) with 0.05 micron size was used . h formation stage was performed to determine the border of particles with putting the samples in a solution of cucl2 , hcl , and alcohol . the other used standards were astm e 407 - 07 for metal microh , astm e 883 - 02 for microscope imaging , and stm e 112 - 96 for measurement of mean particles size . for samples number 10 and 11 the cross - sections were assessed by electron microscope device of scanning electro microscope ( sem ) to determine the cause of fracture by evaluation of cracks and fractures . regarding complexity of loading of dhs implants and uncertainty about the stresses by segment method the stress - strain analysis was performed . for determination of type of loading and locking on the segments the data from study by bergmann et the percent for categorical and the mean and standard deviation were reported for numeric variables . the forces on femoral head in three orientations characteristics in patients chemical composition of samples hardness and stretch tolerance in samples the amount of stress on implant and the ratio to allowed limit causes of implant failure the samples were 11 cases after exclusion of 20 ones . the mean age was 35.8 8.2 years ( ranging from 21 to 52 ) . the mean weight was 71.6 5.4 kg and the mean bmi was 20.6 1.22 kg / m ( tables 1 and 2 ) . the chemical composition is showed in table 3 . except samples 2 , 8 , and 10 , metalogical microstructure for all except the sample number 5 , the size of particles was fine and in standard level . in samples 2 , 8 , 10 , and 11 , there was no delta - ferrit phase but the other samples had some amounts especially samples number 5 , 7 , and 9 . the impurity particles were low in majority of samples but it was high in samples number 8 , 9 , 10 , and 11 . finally the effects of mechanical load were seen in all samples especially in sample numbers 3 , 4 , and 9 . also the stretch tolerance of samples numbers 1 and 11 was low and near to standard . samples numbers 2 , 8 , and 10 had most stretch tolerance and far from standard ( table 4 ) . the most stress was in second hole exactly in location of fracture showing 1.2 gigapasckal . it means a stretch tolerance of 640 megapasckal . as shown in table 5 , down also the percent of chromium and nickel was out of standard limits ( 17 - 19 for chromium and 13 - 15 for nickel ) . regarding the important role of these elements in corrosion and mechanical tolerance this may be contributing for implant failure ( 13 - 19 ) . in optic metallographic evaluation , the dense points of carbide particles were seen on the surface of implant that would result in crack and subsequent stress fractures . in implant hardness test , the mean wicker s number was 193 showing a stretch resistance of 640 megapasckal which is smaller than standard . the maximal stress in the piece was in location of fracture showing 1100 megapasckal which is more than 640 megapasckal for stretch tolerance . it shows the effect of bad chemical composition and low mechanical tolerance in presence of implant failure . in metallographic results , the cause of failure may be the hardness in manufacturing process ( probably due to cold method ) and subsequent corrosive failure . the severe corrosion was seen in cross - section of fracture which was accompanied with fine and gross cracks . however low sample size may result in low probability for utilization of the results about the failure of implants some conclusions may be said . majority ( nine out of eleven ) of samples had failure due to incongruence with standards . some samples were made from alloys with chemical composition not in accordance with standards resulting in lowered corrosive and mechanical tolerance and modeling . also some samples had inappropriate manufacturing method leading to large particles and disturbing phases , or cold method effects . despite partial increase in mechanical tolerance regarding the increased rate of internal fixators use , exact supervision on alloys and manufacturing processes and checking for immune status and function would prevent further problems . application condition is another cause of failure in some samples ( three out of eleven ) and even it was the main failure cause in two cases ( despite standard status of implant ) . the weight - loading conditions should be respected in patients under internal fixation for fractures . the tolerance of high stress forces on implants during some operations such as up staring is difficult and proper weight - loading and optimal physical activity should be learned to the patients . also before increasing the loading weight , the fracture repair status and callus formation process should be evaluated radiographically for higher confidence about tolerance to mechanical loads . many samples ( six out of eleven ) were locking plate . these implants are usually used with space from bone periosteum leading to higher bending stresses . for this reason the corrosion was also another important factor for failure of internal fixators and was seen in five out of 11 samples in current study . the duration of presence of implants in patient s body is an important factor for corrosion . delayed removal would result in lack of sufficient mechanical load for callus maturation and bone regeneration in fracture region . also corrosive complications would result in decreased mechanical tolerance of implants leading to higher probability of fractures . hence paying attention to proper definite time of implant removal it may be concluded that following the standard chemical composition and use of optimal making method are the most important works for prevention of failure of implants . however further studies with larger sample size is required to obtain more definite applicable results .	background : orthopedics implants are important tools for treatment of bone fractures . despite available recommendations for designing and making the implants , there are multiple cases of fracture of these implants in the body . hence , in this study the frequency of failure of implants in long bones of lower extremities was evaluated.methods and materials : in this cross - sectional study , two types of fractured implants in the body were analyzed and underwent metalogical , mechanical , and modeling and stress - bending analysis.results:the results revealed that the main cause of fractures was decreased mechanical resistance due to inappropriate chemical composition ( especially decreased percentages of nickel and molybdenum).conclusions : it may be concluded that following the standard chemical composition and use of optimal making method are the most important works for prevention of failure of implants .
yeast strains the strains used in this study are listed in table 1 . yeast strains were grown in either ypd medium ( 2% peptone , 1% yeast extract , 2% glucose ) or minimal medium ( 0.67% yeast nitrogen base , 2% glucose , appropriate supplements ) at temperatures required for individual experiments ( 17 , 24 , 30 , or 37 c ) . analysis of plasmid - borne forms of cpy * , dpy * , and opy * were performed in strains lacking any endogenous cpy ( prc1::kanmx ) . to make strains of the required genotypes , we re - engineered the sec61 - 3 allele in target strains in the following way . the sec61 - 3 mutation was introduced into pbw11 ( 15 ) by site - directed mutagenesis with primers 1 and 2 generating plasmid pcw11 table 2 . a 2.4-kb kpni - psti fragment containing the mutagenized sec61 - 3 allele was cloned into kpni - psti of yip352 , generating an integrative vector pcw14 , which was subsequently linearized with xba1 and transformed into yeast . loss of the integrative vector part was selected for on media containing 5-fluo - orotic acid , and the resulting single copy genomic copy of sec61 - 3 was confirmed by pcr and dna sequencing . table 1yeast strains used in this studystraingenotypesource mwy61 mataprc1::kanmx leu2 his3 trp1 ura3 der1::kanmx this study mwy63 mataprc1::kanmx leu2 his3 ura3 ade2 pep4 - 3 sec61 - 3 this study mwy64 mataprc1::kanmx leu2 ura3 ade2 pep4 - 3 this study mwy71 mataprc1::kanmx leu2 his3 trp1 ura3 ade2 sec65 - 1 this study mwy77 mataprc1::kanmx leu2 his3 ura3 doa10::kanmx this study mwy82 mataprc1::kanmx leu2 his3 trp1 ura3 ade2 sec62 - 1 this study w303-cd3 mata leu2 his3 trp1 ura3 ade2 der3 prc1 - 1 this study yeast strains used in this study table 2oligonucleotides used in this studynamesequence prc1 - 1_f ccgagctcgcgatgttggtcatccagtacacttcggtagc prc1 - 1_r ggaatcagcacatagttcttgaaccagctcag cpy_nde1_f ctactcaacttaaagtatacataccatatgaaagcattcaccagtttactatg cpy_nde1_r catagtaaactggtgaatgctttcatatggtatgtatactttaagttgagtag dpapb_f2 gaggacatatgagagtcggtattatcttcgttttgttgatttggggtactgttt dpapb_r2 gtgccgtaggccttctatactcttcaacaacaaaacagtacccaaaatcaacaa ost2f ttctttgagctccgaggcagcagtctctgtggagggggtacc ost2r ccggatatcggctcgtattgggcagcagaagacacgttgaaaaaacatagg primer 1 ctcaaatggccttgagcgagttggcctactacatccaacc primer 2 ggttggatgtagtaggccaactcgctcaaggccatttgag oligonucleotides used in this study plasmid constructions plasmids encoding cpy * ( pmw319 ) , dpy * ( pmw339 ) , and opy * ( pmw342 ) were generated as follows . the cpy * encoding allele of prc1 ( prc1 - 1 ) was amplified from genomic dna with primers prc1 - 1_f and prc1 - 1_r . a 2379-bp hindiii - saci dna fragment was cloned into prs315 and prs316 to generate pmw319 and pmw320 , respectively . a 645-bp bamhi - saci fragment from pmw320 was cloned into pbluescript ks+ to generate pmw321 . a ndei restriction site was introduced by site - directed mutagenesis with primers cpy_ndei_f and cpy_ndei_r to generate pmw325 . signal sequence coding sequence of dpap b was amplified with primers dpapb_f2 and dpapb_r2 and following digestion with ndei and stui cloned into pmw325 to generate pmw330 . finally , a 748-bp saci - bamhi fragment from was cloned from pmw330 into pmw319 to generate pmw339 . an ost1 sequence was pcr - amplified with primers ost2f and ost2r , digested with saci and ecorv , and cloned into saci - stui of pmw319 to generate pmw342 . to be able to make strains of the required genotypes , we decided to regenerate the sec61 - 3 allele in target strains in the following way : the sec61 - 3 mutation was introduced into pbw11(22 ) by site - directed mutagenesis with primers 1 and 2 generating plasmid pcw11 . membrane association experiments the microsomes were prepared as previously described ( 16 ) . the microsomes were treated with 100 mm na2co3 at 0 c for 30 min and spun at 100,000 g for 60 min . equal proportions of pellets and supernatants were analyzed by western blotting with appropriate antibodies . antibodies against cpy , kar2p , and sec61p have previously been described ( 1720 ) . the cells were grown in minimal medium to a600 = 0.2 and labeled with [ s]methionine for 20 min ( 5 min for experiments looking at translocation ) . chase was performed by the addition of cold methionine and cysteine to final concentrations of 2 mm each . 5 a600 units of cells were taken for each time point , and the cell extracts were prepared for immunoprecipitation as described ( 17 ) . radiolabeled proteins were visualized using a fujifilm fla3000 phosphorimaging device , and quantitation was performed on signals within the linear range of detection using the aida image analyzer v.3.44 software . properties of new erad substrates a single mutation ( g255r ) in the yeast vacuolar protease carboxypeptidase y ( cpy ) results in a misfolded protein ( cpy * ) , which is a substrate for the erad - l pathway ( 21 ) . preprocpy * is translated in the cytosol and translocated post - translationally into the er . we were interested in addressing the role of sec61p in erad using cpy * as a substrate , but all known mutant alleles of sec61 are deficient in post - translational translocation . the sec61 - 3 mutant allele is no exception but differs in that it also exhibits a cold - sensitive defect in co - translational translocation . this mutant translocates srp - dependent precursors efficiently at 30 c but is defective in post - translational translocation at this temperature and is completely deficient in all translocation at 17 c ( fig . we therefore reasoned that an srp - dependent erad substrate might translocate normally in sec61 - 3 cells at 30 c and might therefore allow us to test specifically for any effect on erad following the loss of sec61p function at 17 c . the signal sequence of a precursor determines whether it will follow either the sec62- or srp - dependent pathway , with more hydrophobic sequences tending to require the latter ( 22 ) . we therefore sought to create srp - dependent derivatives of cpy * by replacing its targeting sequence with that of either dpap - b ( dpy * ) or ost1p ( opy * ) ( fig dpap - b is a type ii integral membrane protein with an amino - terminal signal anchor domain whose targeting is srp - dependent ( 23 ) . ost1p is a type i integral membrane protein with a cleavable signal sequence ( 24 ) whose hydrophobicity led us to predict a likely dependence upon srp . figure 1.translocation properties of cpy * , dpy * , and opy. a , wild type ( sec61 ) cells and sec61 - 3 mutant cells each expressing cpy were grown at 30 c and either shifted to 17 c or incubated with tunicamycin ( t ) for 2 h prior to radiolabeling as described under experimental procedures . immunoprecipitated forms of cpy * and dpap b were analyzed by sds - page : translocated glycosylated forms ( g - pcpy * and mdpap b ) , translocated but unglycosylated forms upon treatment with tunicamycin ( pcpy * and pdpap b ) , and untranslocated precursor ( ppcpy * and pdpap b ) . all are based on cpy * and contain the g255r mutation ( black dot ) , which prevents cpy * folding . predicted signal sequence cleavage sites indicated ( arrows ) , dpy * has the dpap b signal sequence , and opy * has the signal sequence from ost1p . c , translocation of cpy * , dpy * , and opy * was analyzed in wild type and sec61 - 3 cells as described in a. d , wild type ( wt ) , sec62 - 1 cells , or sec65 - 1 cells expressing either cpy * , dpy * , or opy * were grown at 24 c and then either shifted to 37 c for 1 h or treated with tunicamycin prior to radiolabeling and immunoprecipitation as described above . translocation properties of cpy * , dpy * , and opy. a , wild type ( sec61 ) cells and sec61 - 3 mutant cells each expressing cpy were grown at 30 c and either shifted to 17 c or incubated with tunicamycin ( t ) for 2 h prior to radiolabeling as described under experimental procedures . immunoprecipitated forms of cpy * and dpap b were analyzed by sds - page : translocated glycosylated forms ( g - pcpy * and mdpap b ) , translocated but unglycosylated forms upon treatment with tunicamycin ( pcpy * and pdpap b ) , and untranslocated precursor ( ppcpy * and pdpap b ) . all are based on cpy * and contain the g255r mutation ( black dot ) , which prevents cpy * folding . predicted signal sequence cleavage sites indicated ( arrows ) , dpy * has the dpap b signal sequence , and opy * has the signal sequence from ost1p . c , translocation of cpy * , dpy * , and opy * was analyzed in wild type and sec61 - 3 cells as described in a. d , wild type ( wt ) , sec62 - 1 cells , or sec65 - 1 cells expressing either cpy * , dpy * , or opy * were grown at 24 c and then either shifted to 37 c for 1 h or treated with tunicamycin prior to radiolabeling and immunoprecipitation as described above . figure 2.membrane association of novel derivatives of cpy. microsomes from wild type cells expressing cpy , dpy * , or opy * were treated with na2co3 as described under experimental procedures . total ( t ) , pellet ( p ) , and supernatant ( s ) fractions were analyzed by western blots using antibodies against cpy , kar2p , or sec61p as indicated . membrane association of novel derivatives of cpy. microsomes from wild type cells expressing cpy , dpy * , or opy * were treated with na2co3 as described under experimental procedures . total ( t ) , pellet ( p ) , and supernatant ( s ) fractions were analyzed by western blots using antibodies against cpy , kar2p , or sec61p as indicated . er translocation of dpy * and opywhen expressed in wild type cells , both dpy and opy * were efficiently translocated and glycosylated , indicating that their respective er targeting signals are functional . our hypothesis predicted that dpy * and opy * would translocate efficiently in sec61 - 3 cells at 30 c , and this proved to be the case ( fig . interestingly , these proteins also translocate well at 17 c , suggesting that the srp - dependent targeting of a polypeptide that is competent for post - translational import can overcome the sec61 - 3 translocation defect . this finding suggested that the translocation defect in sec61 - 3 cells might be kinetic in nature , and this has been confirmed by pulse - chase analysis ( see supplemental fig . 1 ) . to confirm that dpy * and opy * were being targeted via the srp - dependent pathway , we also examined their translocation in sec65 - 1 cells , which express a temperature - sensitive form of srp ( 23 ) . as expected , preprocpy * translocation is unaffected in sec65 - 1 cells with only the er glycosylated form of procpy * being evident at either 24 or 37 c . in contrast , dpy * and opy * translocate efficiently at 24 c but accumulate as precursor forms at 37 c ( fig . these results demonstrate that dpy * and opy * require functional srp for their er targeting . a further characteristic of a genuinely srp - dependent precursor is that it does not depend on sec62p for translocation . sec62 - 1 cells have defects in post - translational translocation at their permissive temperature ( 24 c ) and are temperature - sensitive for growth at 37 c ( 25 ) . we therefore examined translocation of dpy * and opy * in sec62 - 1 cells . a profound defect in translocation of cpy * was observed at both 24 and 37 c , but dpy * and opy * translocation were unaffected ( fig . we therefore conclude that unlike cpy * , both dpy * and opy * translocate via the co - translational srp - dependent pathway . most importantly , the sec61 - 3 mutation has no detectable effect on translocation of these new precursors . membrane association of dpy * and opythe signal peptide of preprocpy is cleaved during translocation by signal peptidase . opy * was predicted to be similarly cleaved , whereas dpy * was predicted to insert into the bilayer as an integral membrane protein . to test these predictions we used carbonate extraction of microsomes to examine the membrane association of the various proteins . we found that dpy * behaved as an integral membrane protein , whereas both cpy * and opy * were readily extracted by carbonate ( fig . 2 ) . we therefore conclude that opy * is soluble , whereas dpy * is membrane associated . dpy * and opy * thus behave entirely differently from cpy * with regards to their mode of translocation and differently from one another with regards to their solubility / membrane association properties . figure 3.dpy * and opy * are substrates for erad in wild type cells . a , wild type cells expressing cpy * , dpy * , or opy * were pulse - labeled with [ s]methionine as described under experimental procedures . after the addition of cold methionine ( time 0 ) , the samples were taken at 30 min intervals , whole cell extracts were subjected to immunoprecipitation with anti - cpy antibodies , and labeled proteins were analyzed by sds - page . b , quantification of data shown in a. dpy * and opy * are substrates for erad in wild type cells . a , wild type cells expressing cpy * , dpy * , or opy * were pulse - labeled with [ s]methionine as described under experimental procedures . after the addition of cold methionine ( time 0 ) , the samples were taken at 30 min intervals , whole cell extracts were subjected to immunoprecipitation with anti - cpy antibodies , and labeled proteins were analyzed by sds - page . b , quantification of data shown in a. figure 4.erad of dpy * and opy * is der1p - dependent . a , wild type and der1 cells expressing cpy * , dpy * , or opy * were analyzed as in fig . b , quantification of data shown in a. erad of dpy * and opy * is der1p - dependent . a , wild type and der1 cells expressing cpy * , dpy * , or opy * were analyzed as in fig . b , quantification of data shown in a. figure 5.erad of dpy * and opy * is unaffected in sec61 - 3 cells at 30 c . a , pulse - chase experiment using wild type and sec61 - 3 cells at 30 c as described in fig . b , quantification of data shown in a. erad of dpy * and opy * is unaffected in sec61 - 3 cells at 30 c . a , pulse - chase experiment using wild type and sec61 - 3 cells at 30 c as described in fig . b , quantification of data shown in a. dpy * and opy * are subject to der1p - dependent erad - l next we examined whether the translocated forms of dpy * and opy * were subject to erad . wild type cells expressing cpy * , dpy * , or opy * were subjected to pulse - chase studies demonstrating that all three substrates degraded with similar kinetics ( fig . 3 ) . signal cleaved opy * behaved as a lumenal protein in our carbonate extraction studies , and so one might expect that it would be a substrate for the der1p - dependent erad - l pathway ( 7 ) . this was confirmed by pulse - chase studies in der1 cells in which the rate of opy * degradation was indistinguishable from that of cpy * ( fig . 4 ) . dpy * shares the same lumenal lesion as cpy/opy but in a membrane - tethered form . recent studies suggest that erad - m can supersede the erad - l pathway for substrates that have lesions in both a membrane anchor and a lumenal domain ( 26 , 27 ) ; thus any lesion in the dpy * membrane anchor might have led to degradation via the der1p - independent erad - m pathway . 4 ) and so conclude that this degradation occurs as a result of the lumenal lesion in this protein . finally we found that the rates of degradation of dpy * and opy * were unaffected in doa10 cells ( data not shown ) , confirming that neither was dependent on the erad - c pathway ( 28 ) . we therefore conclude that dpy * and opy * are efficiently degraded via the erad - l pathway . figure 6.erad of dpy * and opy is blocked in sec61 - 3 cells at 17 c . 5 except cells were incubated at 17 c after the addition of cold methionine , and samples were collected every 60 min for 4 h. b , quantification of data shown in a. c , wild type and sec62 - 1 cells expressing dpy * or opy * were analyzed as described in fig . d , wild type cells expressing dpy * were analyzed as described in fig . 3a with or without the addition of 10 g / ml cycloheximide ( chx ) . erad of dpy * and opy is blocked in sec61 - 3 cells at 17 c . 5 except cells were incubated at 17 c after the addition of cold methionine , and samples were collected every 60 min for 4 h. b , quantification of data shown in a. c , wild type and sec62 - 1 cells expressing dpy * or opy * were analyzed as described in fig . d , wild type cells expressing dpy * were analyzed as described in fig . 3a with or without the addition of 10 g / ml cycloheximide ( chx ) . opy * requires functional sec61p next we sought to examine any requirement for sec61p . we have earlier shown that dpy * and opy * can be efficiently translocated into the er in sec61 - 3 mutant cells at 30 c . we therefore tested whether these translocated substrates were competent for erad under these same conditions . wild type or sec61 - 3 mutant cells expressing either dpy * or opy * were labeled at 30 c , and samples taken at various time points during a chase performed at the same temperature . we found the half - life of both dpy * and opy * in sec61 - 3 cells to be indistinguishable from that in wild type cells under these conditions ( fig . these results demonstrate that dpy * and opy * are available for erad after translocation at 30 c in sec61 - 3 cells and that their degradation occurs with essentially wild type kinetics . next we tested for any effect on erad following inactivation of the sec61p - dependent translocase at 17 c . the er was preloaded with erad - competent substrate by pulse labeling at 30 c in either wild type or sec61 - 3 mutant cells . the cells were then shifted to 17 c and chased in the presence of unlabeled methionine for the times indicated ( fig . we observed a dramatic increase in the stability of both substrates in sec61 - 3 cells when compared with wild type controls . thus functional sec61p is required for the er - associated degradation of both dpy * and opy. it remains formally possible that the severe translocation phenotype associated with sec61 - 3 at 17 c might lead to an indirect effect on erad , perhaps by blocking the import of some essential factor . to rule this out we first examined erad in sec62 - 1 mutant cells in which post - translational translocation is specifically blocked but found no significant difference in the rate of degradation of either substrate when compared with wild type controls ( fig . thus erad of these novel substrates does not require ongoing post - translational translocation . of course sec61 - 3 cells are also deficient in co - translational translocation at 17 c . we therefore tested the effects on our erad substrates of a complete block in all protein translocation by treating wild type cells with cycloheximide to inhibit protein synthesis . the cells were pulse - labeled at 30 c and then chased at either 30 or 17 c in the presence or absence of cycloheximide ( fig . we found no delay in the degradation of dpy in the presence of drug . because protein synthesis is not required for erad , it naturally follows that ongoing co - translational import of factors into the er can not be required . this is consistent with numerous studies in which erad has been observed in cells treated with cycloheximide ( 29 ) . a variety of studies implicate der1p in erad , but its precise function remains unknown . it is clearly required for the degradation of a range of misfolded luminal proteins including cpy * ( 7 ) . in contrast , the majority of membrane proteins tested appear to be degraded independently of der1p ( 2 , 26 , 30 , 31 ) . one notable exception is the type i integral membrane protein , kww , whose degradation is defective in der1 mutant cells ( 2 ) . the misfolded lesion in kww is located within its luminal domain leading to the suggestion that der1p is required for the degradation of substrates with misfolded lumenal domains via a pathway now known as erad - l ( 2 ) . a number of studies have similarly implicated sec61p in erad , but the role of sec61p in the initial translocation of such substrates into the er complicates the analysis of degradation kinetics . we have sought to temporally separate the known role of sec61p in translocation from any subsequent role in erad by exploiting the properties of the sec61 - 3 mutant . we have created two new erad substrates , both of which are imported into the er in an srp - dependent manner . the first , dpy * , inserts into the er membrane as a type ii integral membrane protein , whereas the second , opy * , is subject to signal peptide cleavage and is released into the er lumen . both dpy * and opy * are substrates for der1p - dependent erad and so , like cpy * , must be substrates for the erad - l pathway . as expected , both dpy * and opy * were efficiently translocated in sec61 - 3 cells at the permissive temperature of 30 c . these translocated forms of dpy * and opy * were evidently available to the erad machinery because both were degraded with kinetics that were indistinguishable from those observed in wild type cells . this allowed us to load the er in sec61 - 3 cells with erad - competent substrate and to then inactivate sec61p by shifting cells to 17 c . our data demonstrate that the inactivation of sec61 - 3p at 17 c results in a rapid and substantial block in the erad of both dpy * and opy*. this can not be explained by some indirect effect of a block in sec61 - 3-dependent protein import because neither sec62 - 1 nor cycloheximide treatment had any similar effect on erad . our results demonstrate that functional sec61p is essential for degradation of erad - l substrates . this finding supports a model in which sec61p forms a bi - directional protein - conducting channel for the transport of polypeptide chains both into and out of the lumen of the endoplasmic reticulum . it will be interesting to determine whether or not different accessory factors might engage with sec61p to determine the directionality of transport .	misfolded proteins in the endoplasmic reticulum ( er ) are exported to the cytosol for degradation by the proteasome in a process known as er - associated degradation ( erad ) . cpy * is a well characterized erad substrate whose degradation is dependent upon the hrd1 complex . however , although the functions of some of the components of this complex are known , the nature of the protein dislocation channel remains obscure . sec61p has been suggested as an obvious candidate because of its role as a protein - conducting channel through which polypeptides are initially translocated into the er . however , it has not yet been possible to functionally dissect any role for sec61p in dislocation from its essential function in translocation . by changing the translocation properties of a series of novel erad substrates , we are able to separate these two events and find that functional sec61p is essential for the erad - l pathway .