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gastroenterologists and surgeons who specialize in the treatment of crohn disease ( cd ) and chronic ulcerative colitis ( cuc ) have become increasingly aware of the implications of different forms of medical therapy on surgical outcomes . this is especially important in the era of top - down and multi - agent therapy for inflammatory bowel disease ( ibd ) . although relatively little literature exists regarding the relationship between pre - operative corticosteroid use and post - operative complications , several authors have found that increasingly higher doses of such immunosuppressive medications may result in an increased rate of post - operative septic complications . similarly , there is increasing concern that combination immunomodulatory therapy with multiple agents may also be associated with an increase in post - operative infectious complications . given reports of the association of monoclonal anti - tumor necrosis factor - alpha ( anti - tnf- ) antibody therapy with pulmonary infections , this concern has specifically been focused on infliximab ( ifx ) , which until recently has been the only fda - approved biologic agent for ibd [ 7 - 11 ] . a recent meta - analysis of the association between immunomodulatory therapy and post - operative complications after surgery for ibd , which included studies of azathioprine , cyclosporine a , and three studies of ifx , found that the available evidence did not support this association . since then , several studies have been published suggesting that ifx is indeed associated with an increased risk of post - operative complications . thus , the potential association between ifx and surgical complications is controversial ; studies both support and refute this potential relationship [ 5,13 - 19 ] . in a study of 270 patients by colombel et al . , 52 patients experienced intra - abdominal septic complications ( iascs ) after bowel resection for cd . analysis did not reveal any increased risk in the ifx - treated subgroup , the moderate - to - high - dose corticosteroid subgroup , or the immunomodulator subgroup . likewise , a matched case - control study from belgium of 31 cd patients also concluded that pre - operative ifx use did not result in a significantly increased rate of post - operative complications , or increased hospital length of stay , as compared to a control group of ifx - nave patients . that study did note , however , a trend towards increased early post - operative infections in the ifx group . more recently , a study of 413 ibd patients ( 156 with cd ) , of which 101 had received ifx within 12 weeks of surgery , also did not find any relationship between pre - operative ifx therapy and post - operative complications . that study , similar to others , was relatively limited by the absolute number of complications . in contrast , a more recent study from appau et al . found an association between pre - operative ifx therapy for cd and post - operative complications . they concluded that ifx therapy within 3 months of surgery was associated with an increased rate of iascs , and of hospital re - admissions . they also found that patients who received a diverting stoma had a lower risk of iascs , thus prompting the observation that a diverting stoma may be prudent when surgeons are faced with cd patients who have recently received ifx . in light of this study , the risks associated with stoma reversal surgery must be weighted against the potential increase in complications associated with ifx for cd . in a study of 151 patients with cuc , 17 ( 10% ) of whom failed ifx therapy and went to surgery and 134 of whom were ifx - nave , no association between ifx and complications was observed . however , the authors observed that ifx patients who had concurrent cyclosporine a treatment were at increased risk for overall and infectious complications . in a larger study by selvasekar et al . in which 47 cuc patients received pre - operative [ ileal pouch - anal anastomosis ( ipaa ) ] ifx therapy and 254 did not , those who received ifx were more likely to have anastomotic leak , and after multivariate adjustment for both disease severity and other medication use , ifx remained independently associated with an increased risk of ileal pouch - related and infectious complications . finally , a recent study of 85 patients with cuc who received ifx pre - operatively also found that patients who receive pre - operative ifx were at increased risk of post - operative septic complications as well as late complications . importantly , the authors also noted that patients who received ifx were more likely to have undergone a 3-stage ipaa , likely due to surgeon reluctance to perform an anastomosis in the setting of pre - operative ifx administration . disease severity determined retrospectively is often inaccurate and surrogate covariates may be inadequate substitutions for validated methods of assessing disease activity . however , it is interesting to note that in the studies of cuc , those that adjusted for disease activity showed a relationship between ifx and complications , while the converse was true for those studies that did not . another major limitation of several of these studies is the relatively long pre - operative ifx window , often 12 weeks or more ( table 1 ) . recent studies of the pharmacokinetics of ifx in ibd suggest that the elimination half - life is between 7 and 18.5 days . by 12 weeks ( 84 days , or 4.5 half - lives ) a window of 12 weeks used by several of the studies published to date may theoretically produce negative results regarding the occurrence of post - operative outcomes if a significant proportion of patients had an interval this long between ifx administration and surgery . ideally , the duration between last infusion and surgery should be included as a continuous variable , but outpatient infusion often makes these data unavailable retrospectively . referral practice patterns may too account in part for the heterogenous findings of these studies , as these may differ substantially in various regions of the world , as demonstrated by at least a 10% variation in the proportion of surgical ibd patients who received ifx . thus , as is usually true of most retrospective studies , results from a single institution may not be broadly generalizable ; the institutions themselves may be considered as a potential confounding factor when comparing results of different studies to each other . in one of the largest studies of the association of ifx and serious infections and mortality , the treat registry study of 6290 patients found that after adjustment for corticosteroid use and disease severity , ifx was not independently associated with increased risk , although both corticosteroids and disease severity were associated with those adverse outcomes . although this study did not include surgical endpoints , the implications are nonetheless important , especially given the lack of large - scale surgical data . in a study of 270 patients by colombel et al . , 52 patients experienced intra - abdominal septic complications ( iascs ) after bowel resection for cd . analysis did not reveal any increased risk in the ifx - treated subgroup , the moderate - to - high - dose corticosteroid subgroup , or the immunomodulator subgroup . likewise , a matched case - control study from belgium of 31 cd patients also concluded that pre - operative ifx use did not result in a significantly increased rate of post - operative complications , or increased hospital length of stay , as compared to a control group of ifx - nave patients . that study did note , however , a trend towards increased early post - operative infections in the ifx group . more recently , a study of 413 ibd patients ( 156 with cd ) , of which 101 had received ifx within 12 weeks of surgery , also did not find any relationship between pre - operative ifx therapy and post - operative complications . that study , similar to others , was relatively limited by the absolute number of complications . in contrast , a more recent study from appau et al . found an association between pre - operative ifx therapy for cd and post - operative complications . they concluded that ifx therapy within 3 months of surgery was associated with an increased rate of iascs , and of hospital re - admissions . they also found that patients who received a diverting stoma had a lower risk of iascs , thus prompting the observation that a diverting stoma may be prudent when surgeons are faced with cd patients who have recently received ifx . in light of this study , the risks associated with stoma reversal surgery must be weighted against the potential increase in complications associated with ifx for cd . in a study of 151 patients with cuc , 17 ( 10% ) of whom failed ifx therapy and went to surgery and 134 of whom were ifx - nave , no association between ifx and complications was observed . however , the authors observed that ifx patients who had concurrent cyclosporine a treatment were at increased risk for overall and infectious complications . in a larger study by selvasekar et al . in which 47 cuc patients received pre - operative [ ileal pouch - anal anastomosis ( ipaa ) ] ifx therapy and 254 did not , those who received ifx were more likely to have anastomotic leak , and after multivariate adjustment for both disease severity and other medication use , ifx remained independently associated with an increased risk of ileal pouch - related and infectious complications . finally , a recent study of 85 patients with cuc who received ifx pre - operatively also found that patients who receive pre - operative ifx were at increased risk of post - operative septic complications as well as late complications . importantly , the authors also noted that patients who received ifx were more likely to have undergone a 3-stage ipaa , likely due to surgeon reluctance to perform an anastomosis in the setting of pre - operative ifx administration . many of these retrospective studies suffer from common limitations . first is lack of adjustment for disease severity ( table 1 ) . disease severity determined retrospectively is often inaccurate and surrogate covariates may be inadequate substitutions for validated methods of assessing disease activity . however , it is interesting to note that in the studies of cuc , those that adjusted for disease activity showed a relationship between ifx and complications , while the converse was true for those studies that did not . another major limitation of several of these studies is the relatively long pre - operative ifx window , often 12 weeks or more ( table 1 ) . recent studies of the pharmacokinetics of ifx in ibd suggest that the elimination half - life is between 7 and 18.5 days . by 12 weeks ( 84 days , or 4.5 half - lives ) a window of 12 weeks used by several of the studies published to date may theoretically produce negative results regarding the occurrence of post - operative outcomes if a significant proportion of patients had an interval this long between ifx administration and surgery . ideally , the duration between last infusion and surgery should be included as a continuous variable , but outpatient infusion often makes these data unavailable retrospectively . referral practice patterns may too account in part for the heterogenous findings of these studies , as these may differ substantially in various regions of the world , as demonstrated by at least a 10% variation in the proportion of surgical ibd patients who received ifx . thus , as is usually true of most retrospective studies , results from a single institution may not be broadly generalizable ; the institutions themselves may be considered as a potential confounding factor when comparing results of different studies to each other . in one of the largest studies of the association of ifx and serious infections and mortality , the treat registry study of 6290 patients found that after adjustment for corticosteroid use and disease severity , ifx was not independently associated with increased risk , although both corticosteroids and disease severity were associated with those adverse outcomes . although this study did not include surgical endpoints , the implications are nonetheless important , especially given the lack of large - scale surgical data . whether pre - operative ifx use is associated with post - operative complications after surgery for ibd remains controversial ; current evidence favors this association after surgery for cuc but does not favor an increased risk after surgery for cd . the potentially practice - changing implications of this debate are occurring now ; increasingly some surgeons are waiting until anti - tnf- agents are washed out before operating or , if that is not possible , performing resections for cd with temporary diverting stomas or advising 3- rather than 2-staged ipaa . larger studies and/or formal meta - analysis of the relationship between ifx and post - operative complications after surgery for ibd are needed before definitive treatment recommendations can be made . the decision of whether or not to perform staged surgery for ibd patients on ifx needs to be made on an individual basis , based on the clinical context and other known risk factors , such as hypoalbuminemia and high - dose corticosteroid treatment .

conflicting data exist regarding the association between pre - operative monoclonal anti - tumor necrosis factor - alpha antibody therapy with infliximab for crohn disease and chronic ulcerative colitis , and the occurrence of post - operative complications . this report reviews the current literature that supports and refutes this association .

brain - derived neurotrophic factor ( bdnf ) is a member of the neurotrophin family , which includes nerve growth factor , neurotrophin-3 , and neurotrophin-4/5 [ 14 ] . the role of bdnf in cell differentiation , neural growth , synaptic connectivity , and maintenance of target neurons is well established ; it has also been implicated in synaptic plasticity of brain function , such as learning and memory [ 5 , 6 ] . in addition to the role of bdnf in neurological disorders , recent studies reported that peripheral injection of bdnf exerts hypophagic and hypoglycemic effects on obese hyperglycemic animals but not on normal animals , pointing to antiobesity and antidiabetic effects [ 710 ] . bdnf mutant mice developed mature onset obesity , characterized by an increase in body weight . in the same study , the mutant mice displayed elevated serum leptin , insulin , glucose , and cholesterol . data from animal experiments and human studies suggested that bdnf may contribute to glucose metabolism and have a pathogenic role in the development of type 2 diabetes mellitus ( t2 dm ) in humans . according to recent studies , bdnf is associated with systemic inflammatory conditions , such as diabetes , acute coronary syndrome , and atherosclerosis [ 13 , 14 ] . the aim of this study was to evaluate the relationship between serum bdnf levels and various metabolic parameters and inflammatory markers in patients with t2 dm . this study was performed at the gop taksim education and research hospital outpatient department of internal medicine . it included 88 patients ( 38 males and 50 females ) and 33 control subjects ( 17 males and 16 females ) . the presence of diabetes was based on a previous diagnosis of t2 dm or a random plasma glucose level of 200 mg / dl or higher , together with classical features of dm , such as polyuria , polydipsia , polyphagia , and weight loss , or a fasting blood glucose level of > 126 mg / dl or higher or a hba1c level of 6.5% or higher . exclusion criteria were the presence of systemic diseases : neoplastic , inflammatory , and infectious diseases . the study protocol was approved by gop taksim research and education hospital ethics committee , istanbul . informed written consent was obtained from all the participants ( patients and controls ) after receiving a full explanation about the study and its purpose . hypertension was defined as antihypertensive drug use or systolic blood pressure 140 mmhg and/or diastolic blood pressure 90 mmhg . body mass index ( bmi ) was obtained using the formula weight ( kg)/height ( m ) . serum cholesterol , triglyceride , and high - density lipoprotein cholesterol ( hdl - c ) were measured by enzymatic colorimetric methods with commercially available kits ( cobas 311 , roche diagnostics gmbh , mannheim , germany ) , and low - density lipoprotein cholesterol c ( ldl - c ) was calculated according to the friedewald formula . serum glucose measures were determined enzymatically using the hexokinase method ( roche diagnostics gmbh , mannheim , germany ) . blood hba1c was determined with a cobas 311 analyzer using the particle - enhanced immunoturbidimetric method ( roche diagnostics , mannheim , germany ) . final results were expressed as percent hba1c of the total hb according to the protocol of the diabetes control and complications trial / national glycohemoglobin standardization program ( dcct / ngsp ) . the particle - enhanced immunoturbidimetric method with a behring nephelometer bn-100 ( behring diagnostic , frankfurt , germany ) was used to measure c - reactive protein ( crp ) . white blood cell ( wbc ) levels were measured with an automatic hematology analyzer ( beckman coulter , brea , ca , usa ) . the erythrocyte sedimentation rate ( esr ) was determined with the westergren method using an established normal range of 020 mm/1 hr . ferritin was measured with an electrochemiluminescence immunoassay ( roche hitachi modular e 170 ) . the insulin level was determined with an electrochemiluminescence immunoassay ( roche diagnostics , mannheim , germany ) on an automated roche cobas e 411 ( roche diagnostics ) . the homeostasis model assessment of insulin resistance ( homa - ir ) index was calculated from the fasting blood glucose and fasting serum insulin concentrations by the formula : homa - ir = fasting serum insulin ( u / ml ) fasting blood glucose ( mmol / l)/22.5 . serum bdnf was quantified using an elisa ( chemikine bdnf sandwich elisa kit , cyt306 ; millipore bioscience research reagents ; usa and canada ) following the instructions of the manufacturer . the intra - assay and interassay coefficients of variation were 3.7 and 8.5% , respectively . number cruncher statistical system ( ncss ) 2007 and power analysis and sample size ( pass ) 2008 statistical software ( utah , usa ) programs were used for the statistical analysis . descriptive statistical methods ( mean , standard deviation , frequency , ratio , minimum , and maximum ) were used to evaluate the study data . mann - whitney u test was used for quantitative parameters that were not normally distributed . values of p < 0.05 , p < 0.01 , and p < 0.001 were accepted as statistically significant . there were no significant differences in the age and gender or triglyceride , total cholesterol , hdl , ldl , wbc , and ferritin levels between the t2 dm patients and control subjects . the t2 dm patients had a significantly higher bmi , waist circumference , systolic pressure , diastolic pressure , fasting plasma glucose , fasting insulin , homa - ir , hba1c , esr , and crp levels than the control subjects ( p < 0.001 ) . among the patients , 64% were using oral antidiabetic drug only , 18% were using oral antidiabetic drug plus insulin , and 18% were using insulin only . on the whole , 30% of patients were taking lipid lowering drug , 61% of patients were taking antihypertension drug , and 9% were taking none . mean diabetes duration for enrolled subjects is 4.03 3.38 ( not shown in table ) . the serum bdnf levels showed a positive correlation with homa - ir ( r = 0.28 ; p < 0.05 ) , the triglyceride level ( r = 0.265 ; p < 0.05 ) , and the wbc level ( r = 0.35 ; p < 0.001 ) in t2 dm as shown in table 2 . in the logistic regression analysis , age ( p < 0.05 ) , bmi ( p < 0.05 ) , crp ( p < 0.05 ) , and bdnf ( p < the serum bdnf levels were significantly higher in the t2 dm patients compared to the healthy controls ( 206.81 107.32 pg / ml versus 130.84 59.81 pg / ml , p < 0.001 ) . a receiver operating characteristic curve ( roc ) analysis and diagnostic screening tests were used to determine the cut - off point for bdnf ( table 4 ) . patients , who had a bdnf level more than 137 pg / ml , were catching study group levels with a sensitivity of 71.79% , a specificity of 68% , a positive predictive value of 87.5% , and a negative predictive value of 43.59% . the area under the roc and the standard deviation were 71.8% and 5.6% , respectively . hba1c was the best predictor , followed by fasting blood glucose and homa - ir ( figure 1 ) . in this study , we investigated changes in the plasma bdnf level and correlations between bdnf and clinical and biochemical parameters in t2 dm patients . we found that the serum bdnf of t2 dm patients was significantly higher than that of control subjects . a previous study showed that serum bdnf levels were elevated in newly diagnosed female t2 dm patients compared to healthy subjects . similarly , a recent study reported that serum bdnf was significantly elevated in t2 dm patients compared to healthy controls . there are various potential mechanisms that link bdnf and development of type 2 diabetes . in animal experiments , it is shown that , by suppressing ppar - alpha and fibroblast growth factor 21 , bdnf might facilitate insulin resistance and dyslipidemia and thus has antidiabetic and lipid lowering effects . furthermore , some researchers claim that bdnf treatment reported to lower blood glucose in diabetic models . similarly , yamanaka et al . demonstrated that treatment with bdnf prevents age - related increase in blood glucose and development of diabetes in prediabetic db / db mice . together with the data derived from animal experiments , authors suggest that exogenous bdnf administration shows its antidiabetic and antilipidemic effects similar to thiazolidinediones . however , the diabetic patients in our study were not newly diagnosed ; thus , the duration of diabetes alone could not explain the elevated level of serum bdnf . on the other hand , fujinami et al . reported that serum bdnf levels were significantly lower in patients with advanced t2 dm compared to control subjects . plasma bdnf levels were decreased in humans with t2 dm and were independent of obesity in a study by krabbe et al . . in the same study , plasma bdnf levels were inversely associated with fasting plasma glucose . . this situation may be the result of ethnic differences . in a previous study , bdnf heterozygous knockout ( bdnf + / ) mice showed obesity and insulin resistance . it is possible that the increase recorded in bdnf in the current study of t2 dm may compensate for hyperinsulinemia and insulin resistance . reported a relationship between fasting blood glucose , triglycerides , and homa - ir and serum bdnf values . a previous study found that serum bdnf was associated with fasting insulin and homa - ir in t2 dm . our study demonstrated that serum bdnf levels were significantly positively correlated with homa - ir and triglyceride . other studies showed that bdnf improved hepatic insulin resistance in diabetic animals and that bdnf was positively correlated with triglyceride , total cholesterol , and ldl - c in humans . some authors observed that bdnf treatment of obese and diabetic animals has a positive effect on glucose and lipid metabolism , with one study demonstrating that subcutaneous administration of bdnf reduces food intake and ameliorates impaired glucose tolerance in diet - induced obese mice . the results of these studies show that bdnf may have a role in the treatment of diabetes and dyslipidemia . previous studies described an association of plasma bdnf with inflammatory conditions [ 25 , 26 ] . some studies demonstrated increased bdnf expression in inflamed bladder tissue and in airway epithelium during allergic airway inflammation [ 27 , 28 ] . shin et al . showed that the plasma bdnf level was positively correlated with inflammatory cytokines in hemodialysis patients , suggesting that plasma bdnf might reflect uremic inflammation in patients undergoing hemodialysis . in our study , inversely , a previous study reported a correlation between bdnf and crp in t2 dm patients . the increase in bdnf levels may be associated with protecting neurons from inflammatory injury . in the logistic regression analysis , bdnf was independently associated with t2 dm , irrespective of age , bmi , and crp . patients who had a serum bdnf level more than 137 pg / ml were a predictive value like hba1c for the diabetes diagnosis with a sensitivity of 71.79% and a specificity of 68% . it had a positive predictive value of 87.5% and a negative predictive value of 43.59% . this novel research suggests that serum bdnf may be used as a prediction data for t2 dm like hga1c in future . more importantly , the standardization and cut - off values of serum bdnf had been controversial in previous studies . for the first time , current study suggests a cut - off point for serum bdnf of type 2 diabetes mellitus . first , this was a cross - sectional study of a relatively small number of patients . third , we measured levels of wbcs and crp to determine the association between bdnf values and inflammation but not those of other serum inflammatory markers , such as interleukin-6 or tumor necrosis factor . in conclusion , serum bdnf level was higher in patients with t2 dm , and the cut - off predictive value of bdnf was 137 pg / ml . the findings of this study suggest that bdnf may contribute to glucose and lipid metabolism and inflammation .

background and aim . studies have suggested that brain - derived neurotrophic factor ( bdnf ) plays a role in glucose and lipid metabolism and inflammation . the aim of this study was to evaluate the relationship between serum bdnf levels and various metabolic parameters and inflammatory markers in patients with type 2 diabetes mellitus ( t2 dm ) . materials and methods . the study included 88 t2 dm patients and 33 healthy controls . fasting blood samples were obtained from the patients and the control group . the serum levels of bdnf were measured with an elisa kit . the current paper introduces a receiver - operating characteristic ( roc ) generalization curve to identify cut - off for the bdnf values in type 2 diabetes patients . results . the serum levels of bdnf were significantly higher in t2 dm patients than in the healthy controls ( 206.81 107.32 pg / ml versus 130.84 59.81 pg / ml ; p < 0.001 ) . they showed a positive correlation with the homeostasis model assessment of insulin resistance ( homa - ir ) ( r = 0.28 ; p < 0.05 ) , the triglyceride level ( r = 0.265 ; p < 0.05 ) , and white blood cell ( wbc ) count ( r = 0.35 ; p < 0.001 ) . in logistic regression analysis , age ( p < 0.05 ) , body mass index ( bmi ) ( p < 0.05 ) , c - reactive protein ( crp ) ( p < 0.05 ) , and bdnf ( p < 0.01 ) were independently associated with t2 dm . in roc curve analysis , bdnf cut - off was 137 . conclusion . the serum bdnf level was higher in patients with t2 dm . the bdnf had a cut - off value of 137 . the findings suggest that bdnf may contribute to glucose and lipid metabolism and inflammation .

a 48-year - old female patient visited our cardiovascular outpatient department for treatment of a mass - like dilated neck vein as a procedure concomitant with thyroid cancer surgery . we could detect the gross engorgement of the neck mass in the supine position or by using the valsalva maneuver when the patient was in an erect position ( fig . preoperative contrast - enhanced computed tomography ( ct ) of the neck showed a venous dilatation , similar to a cystic mass ( size : 2.52.2 cm ) communicating with the left external jugular vein ( fig . 2 ) . after thyroidectomy under general endotracheal anesthesia , an additional separate skin incision ( length : approximately 2.5 cm ) along the neck dermatome was made because of the distance from the collar incision ( approximately 5 cm ) . we accomplished aneurysmectomy by the division of both ends of the external jugular vein and a tributary of the aneurysm in the subcutaneous layer ( fig . in contrast to focal thinned media with thickened intima by fibrous tissue in a varicose vein , the vascular wall thickness of a venous aneurysm is relatively homogenous with thickened media and localized thickened intima ( fig . acquired venous aneurysm in the neck area is a very rare disease and requires a differential diagnosis including enlarged cervical lymph node , tumor of the adjacent organs , laryngocele , and various cystic formations . according to the incidence rate , the internal jugular vein is a more frequent site of aneurysm development than the external vein , but the anterior jugular vein is the least frequent site . fusiform venous dilatation is frequently diagnosed in children with a congenital etiology and right - side predominance but appears in adults as an acquired form with left - side predominance ; the suggested mechanism in adults is the patient 's hypertensive aorta compressing the left innominate vein , resulting in venous dilatation . in addition , the etiology of an acquired venous aneurysm can involve tumors , inflammation , trauma , or spontaneous development . of the aneurysms resulting from iatrogenic causes , pseudoaneurysm at the internal jugular vein appears most frequently ; a case at the external jugular vein has also been reported . however , the patient in the present case had no previous neck procedures or trauma history . clinically , although painful swelling is associated with intraluminal thrombus , saccular aneurysm appears with painless swelling . the valsalva maneuver , performed by moderately forceful attempted exhalation against a closed airway , usually performed by closing one 's mouth and pinching one 's nose shut while pressing out as if blowing up a balloon , can induce venous engorgement characteristically . however , by manual compression of an engorged neck mass in the case of the external jugular vein , the valsalva maneuver can not make the swelling prominent . because the patient had already undergone contrast - enhanced ct imaging for thyroid cancer , ultrasonography was not required in this case . cosmetic concerns , painful swelling due to intraluminal thrombosis , or phlebitis of the jugular vein are all motives for surgical treatment . otherwise , reassurance and regular follow - up can be a substitute for prompt treatment of an asymptomatic venous aneurysm . although embolic complications have been reported at a lower incidence rate in jugular venous aneurysms , active treatment can not be neglected . a recent report documented a pulmonary thromboembolism derived from an external jugular venous aneurysm , and large - scale studies are needed to overcome the limitations of rare case reports . surgical resection can minimize the risk of pulmonary thromboembolism as well as aneurismal rupture induced by growth and can confirm the histopathological diagnosis . aneurismal resection is accomplished by excision with ligation in the saccular form , and exclusion via bypass in fusiform aneurysms .

saccular aneurysm of the external jugular vein presenting as a neck mass is very rare . we report the surgical treatment of an external jugular venous aneurysm in a 48-year - old female patient due to the cosmetic problem of neck engorgement , concomitant with thyroidectomy for cancer .

the analysis of dna sequence is increasingly being used to guide the discovery of natural product biosynthetic gene clusters capable of encoding for novel metabolites . when studying cultured bacteria , it is possible to fully sequence a bacterial genome and therefore carry out detailed analyses of complete biosynthetic gene clusters to identify those likely to encode for novel metabolites . similar full gene cluster analyses are not usually possible when exploring complex environmental microbiomes because the immense size of most metagenomes precludes full sequencing and assembly of complete gene clusters . to circumvent this limitation , we and others have explored the use of the detailed phylogenetic analysis of individual , highly conserved natural product biosynthetic genes as markers for guiding the discovery of functionally novel natural product biosynthetic gene clusters from complex metagenomes . in this approach , individual biosynthetic genes are pcr - amplified from environmental dna ( edna ) and sequenced in bulk . the resulting amplicon sequences ( i.e. , natural product sequence tags ) are then aligned with reference sequences from functionally characterized gene clusters . in previous studies , we have shown that sequence tags that are only distantly related to any known sequence ( e.g. , sequence tag a in figure 1 ) can guide the identification of gene clusters that encode structurally novel bioactive natural products . we have also shown that sequence tags that are very closely related ( e.g. , sequence tag b in figure 1 ) to known genes can guide the discovery of gene clusters that encode congeners of known molecules . while the utility of sequence tags that reside at the phylogenetic boundaries is now clear , many sequence tags fall at a more ambiguous intermediate distance from known sequences ( e.g. , sequence tag c in figure 1 ) . sequence tags , especially those that fall between clades containing sequences known to encode for structurally distinct subclasses of natural products , might be useful for guiding the discovery of gene clusters that both encode novel bioactive metabolites and provide insights into the evolution of natural products structural diversity . here , we explore these concepts through the comparison of the genotypes ( i.e. , gene content ) and chemotypes ( i.e. , molecules encoded by ) of edna - derived aromatic polyketide ( pk ) pentangular polyphenols ( pps ) . aromatic pks are a large group of secondary metabolites generated by iterative ( i.e. , type ii ) pks biosynthetic systems . the initial step in aromatic pk biosynthesis involves the action of a minimal polyketide synthase ( min - pks ) . the min - pks is composed of the three proteins ks , ks and acp , which together catalyze the iterative condensation of malonyl - coa into polyketides ranging from 16 to 30 carbons in length . pps are constructed from the largest nonreduced pk precursors known ( 2430 carbons in length ) and are , therefore , potentially the most structurally diverse subclass of metabolites in this family . ( a ) phylogeny - guided strategy for the discovery of new natural products . ( b ) az , ab and tx denote amplicon sequences derived from arizona , california and texas desert soil edna libraries , respectively . a number of edna - derived pp ks sequence tags fall between clades associated with pps derived from polyketide precursors of different lengths ( c24 and c26 ) . the specific group of intermediate edna sequence tags used in this study are highlighted in orange . the highly conserved ks and ks genes from the min - pks have proved particularly useful as sequence tags for guiding the discovery of new aromatic polyketides from metagenomes . while edna ks and ks sequencing studies suggested the presence of large numbers of unique pp clusters in the environment , the biosynthetic landscape of pps from culture - based studies remained quite limited . in this study pp gene clusters associated with edna - derived ks and ks sequence tags that fall between well - defined clades containing genes from the biosynthesis of pradimicin and xantholipin type pp metabolites were recovered from archived soil edna libraries . the molecules encoded by these pp gene clusters were accessed through heterologous expression in streptomyces albus and found to encode new bioactive pentangular polyphenols with potent antiproliferative and antibacterial activities . by comparing the gene cluster genotypes and chemotypes across this expanded pentangular polyphenol biosynthetic landscape , we gain specific insights into pentangular polyphenol biosynthesis and general insights into potential limitations of the evolution of natural product structural diversity . to identify novel gene clusters encoding pp pks , archived soil edna libraries were screened with degenerate primers targeting either the ks or ks genes of the min - pks . ks and ks phylogenetic trees were constructed with the resulting edna amplicon sequences and sequences from previously characterized type ii pks gene clusters deposited in genbank . in this phylogenetic analysis we identified two sequence tags of interest ( ks tag ab1692 and ks tag ab1414 ) from our anza borrego desert ( ab ) edna library . both sequence tags fall between well - defined clades containing ks genes from gene clusters that encode structurally distinct pp subfamilies , pradimicins and xantholipin ( figure 1b ) . sequence tags ab1692 and ab1414 were used to guide the recovery of edna cosmid clones containing pp biosynthetic gene clusters associated with these ks sequences . the recovered cosmids were pgm ( personal genome machine)-sequenced and this sequence data was subsequently used to guide the recovery of additional cosmid clones overlapping each end of the two min - pks containing cosmids . sequencing and bioinformatics annotation ( tables s3 and s4 , supporting information ) of each set of three overlapping cosmid clones revealed a biosynthetic gene cluster flanked by collections of genes predicted to encode primary metabolic enzymes , suggesting that both gene clusters were recovered in their entirety on three overlapping cosmids . to access the metabolites encoded by the ab1692 and ab1414 pp gene clusters , each set of three overlapping edna cosmid clones ( ab1692 , ab916 and ab170 ; ab1442 , ab1414 and ab561 ) was assembled into bacterial artificial chromosomes ( bac - ab1692/916/170 and bac - ab1442/1414/561 ) using pathway specific ptara ( e. coli : yeast : streptomyces ) shuttle capture vectors and transformation - associated recombination ( tar ) in yeast ( figure 2 ) . these approximately 90 kb bac constructs were pgm - sequenced to ensure correct assembly and transferred into streptomyces albus by intergenic conjugation and integrated into the s. albus genome using the c31 integrase . for heterologous expression purposes , the resulting strains , s. albus bac - ab1692/916/170 and s. albus bac - ab1442/1414/561 , were grown at 30 c in r5a liquid media for 9 days . lc ms analysis of etoac extracts derived from these cultures showed the presence of one and two major clone - specific peaks , respectively ( figure 2 ) . the three clone - specific metabolites were purified from the extracts of 1 l cultures using , in each case , two rounds of c18 reversed - phase hplc . this gave calixanthomycin a ( 1 ) ( 12 mg / l ) from s. albus bac - ab1692/916/170 , and arenimycins c ( 2 ) ( 4.6 mg / l ) and d ( 3 ) ( 2.5 mg / l ) from s. albus bac - ab1442/1414/561 . heterologous expression and the structures of calixanthomycin a ( 1 ) , and arenimycins c ( 2 ) and d ( 3 ) . two pp gene clusters were recovered from california desert soil edna library on three overlapping edna cosmid clones ( ab1692/916/170 and ab1442/1414/561 ) . they were each reconstructed into bac clones using tar . the heterologous expression of these two pp gene clusters by s. albus led to the production of three clone - specific metabolites , compound 1 from the strain s. albus bac - ab1692/916/170 , and compounds 2 and 3 from the strain s. albus bac - ab1442/1414/561 . detailed hplc trace analysis can be found in the supporting information ( figure s2 ) . key 2d nmr correlations used for determining the structures of calixanthomycin a ( 1 ) and arenimycin c ( 2 ) . the structures of compounds 13 were determined by spectroscopic methods , including hresims , and 1d and 2d nmr ( figure 3 , see supporting information discussions s1 and s2 for structure elucidation details ) . the structure of calixanthomycin a ( 1 ) features a xanthone - containing pp core structure . the polycyclic xanthone core is tailored by oxidation , o - methylation and glycosylation . rare structural features include the f ring lactone , which is more commonly seen as a lactam in xanthone - containing pps , and the ortho - dimethoxy functionality on the a ring . calixanthomycin a ( 1 ) is most closely related to ib-00208 from an unsequenced marine actinomadura sp . they differ by the position of methoxy groups around the a ring , the presence or absence of the double bond in the d ring and the oxidation state of the c ring . the structures of arenimycins c ( 2 ) and d ( 3 ) produced by s. albus bac - ab1442/1414/561 feature a highly oxidized benzo[a]naphthacene quinone core ( rings a through e ) . this core structure is appended with a disaccharide ( ome - l - rhamnose - ome - l - olivose ) in 2 and a monosaccharide ( l - rhamnose ) in 3 via a rare n - glycosidic linkage . these structures share an aglycone with the sf2446s from a soil actinomycete streptomyces sp . sf2446 , and the recently reported arenimycins a and b from a marine actinomycete salinispora sp . calixanthomycin a ( 1 ) is extremely toxic to hct-116 cancer cells ( ic50 0.43 nm ) ; however , it shows no antibacterial activity against mrsa ( methicillin - resistant s. aureus ) and vre ( vancomycin - resistant enterococcus faecium ) at the highest concentrations tested ( 50 g / ml ) , and weak activity against b. subillis ( mic = 3.1 g / ml ) . this is quite interesting as most reported xanthone - containing pps display a broad spectrum of activity with both potent human cell cytotoxicity and antibacterial activities . arenimycin c ( 2 ) shows potent gram - positive antibacterial activity ( mic = 98 ng / ml against mrsa ; 1.5 ng / ml against b. subtilis ) and moderate cytotoxicity against hct-116 cells ( ic50= 0.17 m ) . the biosynthesis of compounds 13 can be rationalized based on the predicted functions of the biosynthetic genes found on each producing bac construct ( figure 4 ) . bac - ab1692/916/170 is predicted to harbor the 50 kb clx ( calixanthomycin ) gene cluster ( genbank km881706 ) containing 47 genes involved in the biosynthesis , regulation and resistance of calixanthomycin a ( 1 ) . in our biosynthetic proposal , the predicted min - pks ( clx911 ) , cyclases ( clx8 , 30 and 31 ) and a ketoreductase ( clx1 ) are responsible for generating a benzo[a]naphthacene quinone intermediate using an acetate starter unit and 12 malonyl coa extension steps . the quinone could then be transformed to a xanthone via an oxidative rearrangement catalyzed by the predicted bayer - villiger oxidase ( bvo ) clx27 ( 51% identity to pnxo4 from the fd-594 gene cluster ) . the final tailoring steps in our proposed calixanthomycin a ( 1 ) biosynthesis scheme involve formation of the ortho - dimethoxy functionality in the a ring and glycosylation in the e ring . formation of the rare ortho - dimethoxy functionality would require reduction at c-13 and hydroxylation at c-12 followed by two o - methylations . the mechanism of c-13 reduction is not clear at this point , but the hydroxylation at c-12 is predicted to be catalyzed by the cytochrome - p450 hydroxylase clx28 due to its sequence similarity to pnxo5 ( 61% sequence identity ) from the fd-594 gene cluster . methylation of the ortho - hydroxyl groups could then occur by the action of the predicted o - methyltransferases clx2 and clx43 . in our proposed biosynthesis , the resulting hexacyclic xanthone aglycone is appended with a d - quinovose sugar moiety by the predicted glycosyltransferase clx40 . proposed biosynthesis of calixanthomycin a ( 1 ) , and arenimycins c ( 2 ) and d ( 3 ) ( in boxes ) based on the predicted gene function and pfam domain analysis . the presence of arenimycin b in the culture extract was confirmed by lc ms . detailed gene annotation tables for the clx and arn gene clusters appear in tables s3 and s4 . bac - ab1442/1414/561 harbors the 40 kb arn ( arenimycin ) gene cluster ( genbank kj440489 ) containing genes predicted to be involved in the biosynthesis , regulation and resistance of arenimycins c ( 2 ) and d ( 3 ) . in our proposed biosynthetic scheme the benzo[a]naphthacene core is synthesized via a min - pks ( arn31 , 32 and 36 ) , an aromatase ( arn21 ) , two cyclases ( arn19 and 20 ) and a ketoreductase ( arn33 ) ( figure 4 ) . on the basis of their high sequence identity to pdmh from the pradimicin gene cluster , arn 22 and 37 are predicted to oxidize c-6 and c-10 to form the two 1,4-benzoquinone moieties ( a and c rings ) seen in 2 and 3 . arn 17 , which shows 54 and 43% sequence identity to grho8 from the griseorhodin gene cluster and xano5 from the xantholipin gene cluster , respectively , is likely to be involved in hydroxylations at the two angular positions ( c-5 and c-18 ) . finally , this aglycone is predicted to be glycosylated by two glycosyltransferases arn11 and arn14 , completing the biosynthesis of 2 and 3 . the mechanism of n - glycosidic bond formation is not clear at this point . different pp chemistries are organized according to the phylogenetic analysis of ks gene sequences ( figure 1 ) . we have designated these scaffolds pp - a , pp - b , pp - c and pp - d . each different pp scaffold and the arn gene cluster harbors all genes predicted to be required for the biosynthesis of the sugar moieties ( rhamnose and olivose ) found in 2 and 3 ( figure 4 ) . interestingly , it also contains three additional predicted sugar biosynthesis genes , whose functions could not be assigned to a specific transformation in the biosynthesis of the sugar moieties found on 2 or 3 . these include genes predicted to encode for an n , n - dimethyltransferase ( arn6 ) , an aminotransferase ( arn7 ) and a 3,4-dehydratase ( arn8 ) . these observations along with the recently reported structure of arenimycin b , which contains the dimethylated amino deoxysugar forosamine in place of the ome - l - olivose seen in 2 , led us to re - examine culture broth extracts obtained from bac - ab1442/1414/561 by lc the selective ion chromatogram for m / z = 809 revealed the presence of a minor clone specific compound with a mass corresponding to that of arenimycin b ( figure s9 ) . it appears that the edna - derived arn gene cluster has the potential to encode a number of different glycosylated compounds , with arenimycin c ( 2 ) being the major product . the annotated genome of salinispora arenicola cnb527 , the arenimycin b producer , was recently made publicly available ( genbank : nz_azxi01000002 ) . a comparison of the arenimycin b and the arn gene clusters reveals that these two gene clusters are very closely related , showing 90 to 95% sequence identity between most biosynthetic genes . in total , 12 pp biosynthetic gene clusters have now been sequenced and functionally characterized , including eight clusters from culture - based studies and four from culture - independent studies . in addition to the edna - derived clx and arn clusters described here , we previously reported edna gene clusters that encode for fasamycin and arixanthomycin type pps . ks and ks sequence tags associated with these four edna clusters were selected by us for detailed analyses because they all fall between well characterized ks clades ( i.e. , intermediate sequence tags ) . in the following analyses , we explore this closely related collection of biosynthetic gene clusters to gain insights into the evolution of natural product structural diversity . the individual proteins that makeup min - pks ( ks , ks , and acp ) are highly conserved across type ii pks gene clusters . ks and ks phylogenetic trees show very similar topologies , both of which correlate closely with differences in the core polyketide structure ( e.g. , chain length and cyclization pattern ) encoded by the gene cluster from which the min - pks arises . ks and , to a lesser extent , ks genes have thus proved to be useful phylogenetic markers for predicting differences in polyketide core structures encoded by type ii pks gene clusters . while pps have so far been considered a single class of type ii polyketides , based on both ks phylogeny and natural product structure , it appears that they arise from four distinct min - pks lineages . these lineages differ in starter unit selectivity ( acetate or hexanoate ) and the number of malonyl coa chain extension steps they carry out ( 11 or 12 ) . pp polyketide precursors range from 24 to 30 carbons in length and generate four unique pp scaffolds that we have designated pp - a ( c24acetate and 11 extensions ) , pp - b ( c26acetate and 12 extensions ) , pp - c ( c28hexanoate and 11 extensions ) and pp - d ( c30hexanoate and 12 extensions ) ( figure 5 ) . the 30-carbon d scaffold is the largest polyketide chain observed in aromatic polyketide biosynthesis . with more than 30 predicted b scaffold - based natural products reported in the literature , this 26-carbon scaffold , which arises from an acetate starter unit and 12 malonyl coa extension steps is responsible for most of the structural diversity seen in known pps ( figure 5 ) . the only reported gene clusters predicted to encode the a , c , and d scaffolds are the pradimicins ( pp - a ) , arenimycins ( pp - a ) , benastatins ( pp - c ) , fd-594s ( pp - c ) and fredericamycins ( pp - d ) . although both ks and ks gene phylogenies indicate that the four pp scaffolds ( a c ) have evolved independently from a common ancestor , similar tailoring modifications are observed across scaffolds from different lineages . this is especially interesting in light of the fact that some of modifications seen in multiple different pp lineages ( e.g. , the xanthone core and the gem - dimethyl functionality ) are in fact quite rare outside of pp biosynthesis . this suggests that functionally successful horizontal transfer of the tailoring genes responsible for these modifications has not occurred globally throughout bacterial secondary metabolism but instead locally within the limited universe of pp biosynthesis . to evaluate evolutionary relationships between the genes responsible for the common modifications found across different pp scaffolds , we carried out detailed phylogenetic analyses of the four common tailoring genes seen in pp gene clusters including baeyer villiger oxidase , geminal bis - methyltransferase , asn - synthetase homologue and glycosyltransferase ( figure 6 ) . the most notable tailoring modification observed in pp biosynthesis is an oxidative rearrangement of the pp scaffold to generate either a xanthone or a spiroketal . these oxidative rearrangements involve the action of fad - dependent baeyer villiger oxidase ( bvo ) . bvos are also found in some angucycline gene clusters ( giloi , jadh and urdm ) and in the mithramycin gene cluster ( mtmoiv ) . to determine the phylogenetic relationship between these genes , a maximum likelihood phylogenetic tree was generated using full - length bvo gene sequences ( figure 6 ) . in this analysis , the pp bvo genes form a monophyletic clade that is distinct from other bvo genes , suggesting that the pp bvo gene was acquired once in pp biosynthesis and diverged to enable the production of distinct xanthone and spiroketal functionalities . interestingly , the bvo genes clx27 and pnxo4 from the calixanthomycin a and fd-594 gene clusters , respectively , show the highest sequence identity even though their ks genes belong to distinct pp lineages ( figure 6 ) . this suggests that once the bvo gene successfully entered the pp biosynthetic universe it was not only passed down vertically through the pp - b lineage , but also horizontally transferred between pp lineages . curiously , while it appears that this bvo gene was capable of horizontal transfer between pp lineages with related core structures , similar , more global transfer to numerous other sequenced type ii pks gene cluster families is not observed , as the bvo genes found in other type ii pks gene clusters appear to be of distinct phylogenetic lineages . geminal bis - methylation is another rare tailoring reaction that is seen in pp biosynthesis . the introduction of this functional group is known to occur through the action of a single , sam - dependent methyltransferase ( geminal bis - methyltransferase , gbm ) via two rounds of c - methylation . known aromatic polyketides with this functionality include two pps , the fasamycins and the benastatins , a pentacyclic polyketide resistomycin , and the tetracyclic quinone tetarimycin . in resistomycin biosynthesis , two methyl transferases are predicted to be required to introduce the dimethyl functionality , neither of which is closely related to the pp gbms . the three tetracyclic structures upon which the pp - like gbms act ( fasamycins , benastatins and tetarimycins ) are closely related ( figure 6 ) , once again suggesting that horizontal transfer of these tailoring genes has been limited to interchange between gene clusters that encode closely related core structures . ( a ) phylogenetic relationships between ks , ks , baeyer villiger oxidase , asn synthase homologue , geminal bis - methyltransferase and glycosyltransferase genes from pp biosynthetic gene clusters are reconstructed from the maximum likelihood phylogenetic trees to highlight the relationships between genes from clusters of different pp lineages . ( b ) the parent maximum likelihood phylogenetic trees for bvo genes , gbm genes and asn synthase homologue genes are shown . for both bvo and gbm tailoring genes , it appears that functionally successful horizontal gene transfer has been limited to gene clusters that encode metabolites with closely related core chemical structures . this would explain their presence in different pp lineages and few other gene clusters outside of pp biosynthesis and suggest that for many tailoring enzymes the challenge of changing substrate specificity ( or limited substrate promiscuity ) may have restricted their more extensive spread throughout natural product biosynthesis by horizontal gene transfer mechanisms . the terminal carboxylate in pp structures undergoes a variety of reactions including o - methylation , esterification / cyclization , alanine addition , serine addition / cyclization and amidation / cyclization . among these , amidation in general and , more specifically , transamination carried out by asn synthetase homologues is the most commonly seen transformation . in the phylogenetic analysis of pp asn transaminase genes , we once again see what appears to be horizontal transfer between different pp lineages . we also see what appears to be horizontal transfer between different polyketide classes as the closest relatives of the llpa and xana genes from the lysolipin and xantholipin gene clusters , respectively , are found in tetracycline - type gene clusters . one of the most common tailoring steps found in aromatic polyketide biosynthesis is glycosylation . to investigate relationships between pp glycosyltransferases , a phylogenetic tree was constructed using 40 glycosyltransferase genes found in sequenced pp gene clusters and additional glycosyltransferase genes found in gene clusters that encode a diverse collection of non - pp aromatic polyketides ( figure s1 ) . this maximum likelihood phylogenetic tree shows that clades do not correlate strongly with aromatic polyketide aglycone substrate structures or donor sugar types . among pp glycosyltransferse genes , clx40 , arx9 , arx3 , llpu and xanp do form a monophyletic clade , suggesting that they have been vertically transferred through pp scaffold b gene cluster evolution . other pp glycosyltransferases , including arn11 , arn14 , pnxgt1 , pnxgt2 , pdms and pdmq , do not clade based on their respective pp min - pks lineages , suggesting that they have likely entered the pp biosynthetic gene clusters in distinct horizontal gene transfer events . for tailoring genes that to date are exclusively found in secondary metabolism ( e.g. , bvo and gbm ) , we see limited introduction of these genes into pp biosynthesis . within the examined set of gene clusters , both bvo and gbm genes appear to have entered pp biosynthesis only once . however , for tailoring gene families that are common across the global metagenome ( e.g. , asn - synthetase homologue and glycosyltransferase genes ) , we see many more introduction events resulting in their more varied use in pp structural diversification . the more frequent introduction of asn - synthetase homologue and glycosyltansferase genes into pp biosynthesis is likely due to both their more frequent appearance in the environment and their more relaxed substrate specificity . soil metagenomes represent the products of nature s ongoing combinatorial biosynthetic efforts . here , we show that sequence tag - based metagenomic screening methods provide an opportunity to identify collections of related natural product gene clusters that not only encode novel metabolites , but can also provide insights into the natural combinatorial biosynthetic process . more specifically , we show that the functional characterization of gene clusters whose sequence tags fall between clades associated with two related but distinct chemotypes ( intermediate sequence tags ) can be used to increase the chemical and the biosynthetic diversity associated with a targeted class of natural products . on the basis of the differences in min - pks gene phylogeny and the pp chemical structures our phylogenetic analysis of pp tailoring genes indicates that the horizontal transfer of at least a subset of pp tailoring genes has likely been restricted to gene clusters that encode closely related chemical structures . if true , this would suggest that nature has sampled only a fraction of the natural product - like chemical space that can theoretically be encoded by secondary metabolite tailoring genes . these observations provide a more detailed picture of the nature s combinatorial biosynthetic process , which can help guide future laboratory - based combinatorial biosynthetic efforts and help ensure the construction of a set of molecules that is orthogonal to those produced naturally . in particular , our study suggests that combinatorial biosynthetic experiments focused on widely distributed enzymes ( e.g. , glyocsyl- or amino - transferases ) are likely to yield metabolites already sampled by evolution . on the other hand , experiments focused on increasing the substrate promiscuity of tailoring genes that are unique to secondary metabolism ( e.g. , bvo or gbm ) would be much more likely to yield metabolites not yet encoded by natural biosynthetic pathways . all recovered cosmids and tar constructs were sequenced using iontorrent personal genome machine ( pgm ) . optical rotations of isolated compounds were measured on a jasco p-1020 polarimeter and ir spectra were recorded on a bruker tensor27 ir spectrometer . all nmr data used for structural characterization were obtained on a bruker avance dmx 600 mhz nmr spectrometer equipped with a cryoprobe . h and c nmr chemical shifts were referenced to the dmso - d6 solvent signals ( h 2.50 and c 39.51 , respectively ) . hresims data was acquired on the lct premier time - of - flight mass spectrometer . our ab ( california ) , az ( arizona ) and tx ( texas ) desert soil edna libraries were used for screening with ks and ks degenerate primers . each library contains > 10 clones , which is predicted to approach saturation of the genetic diversity present in soil microbiomes . these libraries are arrayed as 5000 membered subpools to facilitate screening and gene cluster recovery studies . dna aliquots from the 5000 membered subpools found in each arrayed library were screened using degenerate primers designed to amplify partial ks and full - length ks genes . each 25 l pcr reaction contained 50 ng of cosmid dna , 2.5 m of each primer , 2 mm dntps , 1x thermopol reaction buffer ( new england biolabs ) , 0.5 units taq dna polymerase and 5% dmso . pcr was conducted using the following touchdown protocol : denaturation ( 95 c , 2 min ) , 8 touchdown cycles [ 95 c , 45 s ; 65 c ( 1 c per cycle ) , 1 min ; 72 c , 1 min ] , 35 standard cycles ( 95 c , 45 s ; 58 c , 1 min ; 72 c , 1 min ) and a final extension step ( 72 c , 2 min ) . approximately 500 bp of ks or 600bp of ks gene ( corresponding to nucleotides 5621061 of bena or 25621 of benb ) fragments from each amplicon were aligned using clustalw . the edna clones from which the ab1692 and ab1414 amplicon sequences were amplified were recovered from the ab1692 and ab1414 subpools using serial dilution method . briefly , overnight cultures of the subpools were inoculated into 96-well microtiter plates at a dilution of 50 cells / well . after overnight shaking at 37 c , the diluted cultures were screened by whole - cell pcr using specific primers designed to recognize the ab1692 and ab1414 amplicon sequences . pcr - positive wells from the second round were plated onto lb agar media to give distinct colonies that were screened individually using colony pcr to identify the clones ab1692 and ab1414 . the ab library was screened for overlapping edna clones using clone - specific primers designed to recognize the sequence at each end of the edna clones ab1692 and ab1414 . hits were identified in subpools ab916 for the ab1692 clone , and ab1442 and ab561 for the ab1414 clone . the overlapping edna clones were recovered from these subpools using the serial dilution method outlined above . all three cosmids were pgm - sequenced and annotated using metagenemark , in combination with blast search and pfam domain analysis . annotation results indicated that the biosynthetic gene cluster associated with the ab1414 ks sequence was completely captured in three overlapping clones , ab1442 , ab1414 and ab561 , but the gene cluster associated with ab1692 ks sequence was not complete in the ab916 , therefore the additional overlapping clone was recovered using the same method described above . sequencing of the additional overlapping clone ab170 suggested the complete recovery of the ab1692 ks sequence - associated pp gene cluster over three overlapping edna clones , ab1692 , ab916 and ab170 . the three overlapping edna cosmid clones ( ab1692 , ab916 and ab170 ; ab1442 , ab1414 and ab561 ) that are predicted to contain the ab1692 and ab1414 amplicons - associated pentangular polyphenol gene clusters were assembled into bac clones in yeast using tar . the ab1692 and ab1414 pentangular polyphenol pathway - specific tar capture vectors were constructed using infusion cloning methodology ( clontech ) . five hundred bp upstream ( ups ) and downstream ( dws ) homology arms were amplified from the ab1692 and ab170 cosmids , and the ab1442 and ab561 cosmids , respectively , using the primers listed in the supplementary protocol 1 . gel - purified amplicons ( qiagen ) and bmti / sphi linearized ptara vector were added into a standard infusion cloning reaction ( clontech ) to yield the pathway - specific capture vectors . for tar assembly , 200 ng of each drai - cut cosmid ( ab1692 , ab916 and ab170 ; ab1442 , ab1414 and ab561 ) were mixed with 100 ng of the hpai - digested pathway - specific capture vector and then transformed into 200 l of saccharomyces cerevisiae cry12 spheroplasts prepared according to published protocols . transformed spheroplasts were mixed with synthetic complete ( sc ) top agar ( 1 m sorbitol , 1.92 g / l synthetic complete uracil dropout supplement , 6.7 g / l yeast nitrogen base , 2% glucose and 2.5% agar ) and overlaid onto sc dropout plates without uracil . plates were incubated at 30 c for approximately 72 h , until colonies appeared . twelve yeast colonies were picked and cultured overnight in sc dropout liquid media without uracil . dna was isolated using a zymolyase lysis protocol ( zymo research ) and screened by pcr with primers designed to recognize sequences from each cosmid . a bacterial artificial chromosome ( bac ) clones that amplified with all the primer sets were electroporated into e. coli epi300 ( epicenter ) . dna isolated from the resulting e. coli epi300 was pgm - sequenced to confirm the correct reassembly of the three overlapping cosmids into approximately 90 kb insert bac clones ( bac-1692/916/170 and bac - ab1442/1414/561 ) . bac - ab1692/916/170 and bac - ab1442/1414/561 were transformed into e. coli s17.1 and transferred into s. albus via intergenic conjugation to generate the strains s. albus bac - ab1692/916/170 and s. albus bac - ab1442/1414/561 ( apramycin selection ) . calixanthomycin a ( 1 ) , and arenimycins c ( 2 ) and d ( 3 ) were isolated from 1l cultures ( 200 rpm at 30 c ) of s. albus bac - ab1692/916/170 and s. albus bac - ab1442/1414/561 , respectively , in 125 ml baffled flasks , each containing 50 ml of r5a media . the cultures were harvested after 9 days , extracted with ethyl acetate [ 1:3 ( v : v ) , ethyl acetate to the culture ] and dried in vacuo . calixanthomycin a ( 1 ) , and arenimycins c ( 2 ) and d ( 3 ) were isolated from the resulting ethyl acetate extracts using two rounds of reversed - phase hplc ( c18 column , 10 mm 250 mm , 3.5 ml / min ) . the first round of hplc ( isocratic , 60% acetonitrile for 1 ; 40% acetonitrile for 2 and 3 ) yielded crude samples , from which 13 were purified . the second rounds of hplc used 60% aqueous methanol for calixanthomycin a ( 1 , 6.0 mg / l ) , and 70 and 60% aqueous methanol with 0.1% trifluoroacetic acid for arenimycins c ( 2 , 4.6 mg / l ) and d ( 3 , 2.5 mg / l ) , respectively . yellow brownish powder ; [ ]d 68 ; uv ( meoh ) max 226 , 295 , 329 nm ; ir ( neat ) max 3393 , 2927 , 2736 , 1679 , 1604 , 1557 cm ; h and c nmr , cosy , and hmbc data , see table s1 ; hresims m / z 695.2327 [ m + h ] ( calcd for c36h39o14 , 695.2340 ) . dark reddish powder ; [ ]d 77 ; uv ( meoh ) max 216 , 256 , 421 , 477 nm ; ir ( neat ) max 3384 , 3070 , 2929 , 2856 , 1682 , 1620 , 1511 cm ; h and c nmr , cosy , and hmbc data , see table s2 ; hresims m / z 812.2770 [ m + h ] ( calcd for c40h46 no17 , 812.2766 ) . dark reddish powder ; [ ]d 133 ; uv ( meoh ) max 215 , 254 , 416 , 474 nm ; ir ( neat ) max 3392 , 3072 , 3050 , 2931 , 2858 , 2738 , 1686 , 1641 , 1619 cm ; h and c nmr data , see table s2 ; hresims m / z 654.1826 [ m + h ] ( calcd for c32h32no14 , 654.1823 ) . minimal inhibitory concentrations ( mic ) for calixanthomycin a ( 1 ) , and arenimycins c ( 2 ) and d ( 3 ) against staphylococcus aureus usa-300 ( mrsa ) , bacillus subtilis rm125 , enterococcus faecalis ( vre ) and escherichia coli drc39 were determined by liquid dilution methods . each bacterial strain was grown overnight in either brain heart infusion ( mrsa and vre ) or lb ( b. subtilis rm125 and e. coli drc39 ) liquid media . overnight cultures were diluted 1000-fold and transferred to 96-well microtiter plates ( 75 l / well ) . compounds were dissolved in dmso , added to the media in the first well of row to give a concentration of 50 g / ml and serially diluted 2-fold / well across the plate . these solutions ( 75 l ) were then added to the corresponding well in an assay plate and incubated at 30 c overnight without shaking . bacterial growth was visually inspected and the last well with no bacterial growth was reported as the mic . the antiproliferative activity of calixanthomycin a ( 1 ) , and arenimycins c ( 2 ) and d ( 3 ) was evaluated in duplicate using the colon carcinoma cell line hct-116 ( atcc ; ccl-247 ) . hct-116 cells were grown in mccoy s 5a modified medium ( gibco ) supplemented with 10% ( v / v ) fbs . trypsinized cells were seeded into 96-well plates ( 1000 cells / well ) and incubated overnight at 37 c in the presence of 5% co2 . compounds 13 ( in dmso ) were sequentially diluted in culture media ( 2 or 3-fold dilutions starting at 50 g / ml ) across a 96-well plate and 100 l was transferred to the appropriate well in an assay plate . the plates were incubated at 37 c for 3 days and then evaluated for viability using a crystal violet - based colorimetric assay . cell viability was recorded based on the a590 of stain present in each well relative to no drug dmso control wells .

sequence - guided mining of metagenomic libraries provides a means of recovering specific natural product gene clusters of interest from the environment . in this study , we use ketosynthase gene ( ks ) pcr amplicon sequences ( sequence tags ) to explore the structural and biosynthetic diversities of pentangular polyphenols ( pp ) . in phylogenetic analyses , edna - derived sequence tags often fall between closely related clades that are associated with gene clusters known to encode distinct chemotypes . we show that these common intermediate sequence tags are useful for guiding the discovery of not only novel bioactive metabolites but also collections of closely related gene clusters that can provide new insights into the evolution of natural product structural diversity . gene clusters corresponding to two edna - derived ks sequence tags that reside between well - defined ks clades associated with the biosynthesis of ( c24)-pradimicin and ( c26)-xantholipin type metabolites were recovered from archived soil edna libraries . heterologous expression of these gene clusters in streptomyces albus led to the isolation of three new pps ( compounds 13 ) . calixanthomycin a ( 1 ) shows potent antiproliferative activity against hct-116 cells , whereas arenimycins c ( 2 ) and d ( 3 ) display potent antibacterial activity . by comparing genotypes and chemotypes across all known pp gene clusters , we define four pp subfamilies , and also observe that the horizontal transfer of pp tailoring genes has likely been restricted to gene clusters that encode closely related chemical structures , suggesting that only a fraction of the natural product - like chemical space that can theoretically be encoded by these secondary metabolite tailoring genes has likely been sampled naturally .

the study began in december 2004 and terminated on february 17 , 2007 . a notice sent to all physicians in maritime canada described the study and asked the physicians to submit serum samples from patients with suspected q fever ( febrile illness or pneumonia after exposure to parturient cats or other animals ; outbreaks of pneumonia in a family ) . all samples were sent to the nova scotia public health laboratory for testing for antibodies to coxiella burnetii . physicians of patients with a positive test result were contacted , and they in turn contacted their patients to ask if they would participate in the study . this study was approved by the university of alberta research ethics review board and the capital health research ethics board . during the study period , serum samples from 210 patients suspected of having c. burnetii infection were tested by using commercially available immunoglobulin ( ig ) m and igg elisas ( panbio , brisbane , queensland , australia ) . convalescent - phase samples were collected from those who agreed ; further testing for igg antibodies to phase i and phase ii c. burnetii antigen was conducted by using an indirect immunofluorescence test as previously described ( 3 ) . of the 210 patients , 35 had antibodies to c. burnetii and 13 met the criteria for acute q fever ( positive igm elisa result and a > 4-fold rise in antibody to phase ii antigen between the acute- and convalescent - phase samples ) . phase i titers > 512 , suggestive of chronic q fever , were found for 3 patients . of the 13 patients who fit the case definition of acute infection , 11 agreed to participate in the study , 1 declined , and 1 moved to another country . of the 11 participating patients , 7 were from nova scotia , 2 were from new brunswick , and 2 were from prince edward island ; 6 were male ; and mean age was 54.6 years ( table ) . one case occurred in december 2004 ; 6 in 2005 ; 4 in 2006 , and no cases in the first 6 weeks of 2007 . cases occurred in every month except august , september , october , and february . * within 2 weeks of onset of symptoms , this patient had occupied a small space ( automatic bank teller area ) with 2 farmers who smelled of manure . one patient ( patient 8) was a sheep farmer who had recently had 60 lambs born on his farm , several of which were stillborn in the 2 weeks before the farmer became ill ; 7 patients had > 1 cat as a pet ; and only 2 ( patients 5 and 8) had no pets . in terms of clinical signs , all 11 patients had sweats , fever , and myalgia ; 9 had chills ; 8 had a cough ; 7 were short of breath ; 5 each had nausea , diarrhea , sputum production , and confusion ; 4 had chest pain , which was pleuritic for 2 ; and 2 had abdominal pain and vomiting . of the 7 patients for whom chest radiographs were taken , 6 had acute opacities compatible with pneumonia . patient 11 had diffuse bilateral pneumonia , which required him to be admitted to an intensive care unit to receive ventilatory support ( figure ) . chest radiograph of patient 11 at time of admission to hospital , before intubation , demonstrating extensive bilateral airspace disease . only 4 received initial empiric therapy that would be considered effective against c. burnetii , e.g. , doxycycline ( n = 2 ) , ciprofloxacin ( n = 1 ) , or levofloxacin ( n = 1 ) . four other patients received azithromycin , which may have been effective but has suboptimal in vitro activity against c. burnetii ( 4 ) . acute q fever is still present in maritime canada ; however , the number of cases has diminished considerably from the 1980s and early 1990s . since 2004 , only 45 cases have been reported each year . the passive design of our study may have underestimated the number of cases . however , in the 1980s , a number of q fever outbreaks involved entire families . a typical scenario was exposure to the parturient family cat and her newborn kittens , after which everyone in the family became ill ( 2 ) . some outbreaks involved poker players ( 5 ) or most of the employees of a factory ( 6 ) . for our study , we carefully asked whether family members were ill ; only 2 patients mentioned such illness , and for each , it was a spouse . pneumonia seems to still be the dominant form of acute q fever in maritime canada . major differences in the manifestations of q fever occur in different regions . in maritime canada and in the basque region of spain , the predominant manifestation is pneumonia ( 7,8 ) ; in newfoundland and australia , fever with no apparent localization of infection ( 9,10 ) ; in the canary islands , fever and hepatitis ; and in southern france , hepatitis and pneumonia , although hepatitis is more frequent than pneumonia ( 11,12 ) . the factors responsible for these disparate manifestations are not known . when isolates of c. burnetii from different geographic areas were typed by using multisequence typing , all 7 isolates from nova scotia were identical and shared this type with 2 isolates from france and 1 from the united states ( 13 ) . the reservoirs for human infection with c. burnetii in nova scotia have likely spread from cats and dogs ( 14 ) to the more traditional reservoirs of sheep and cattle ( 12 ) . patient 8 , a sheep farmer , had pneumonia that appeared on radiographs as a rounded opacity in the right middle lobe . rounded opacities are very common in cat - associated cases of q fever but may not be specific for cat - associated infection ( 15 ) . our findings indicate that after c. burnetii is established in an area , it is likely to persist , although the incidence may fluctuate .

since the 1990s , reports of q fever in nova scotia , canada , have declined . passive surveillance for q fever in nova scotia and its neighboring provinces in eastern canada indicates that the clinical manifestation of q fever in the maritime provinces is pneumonia and that incidence of the disease may fluctuate .

accurate diagnosis is the most critical assignment in planning orthognathic surgery , and conventional lateral cephalometry has been the standard for evaluation and orthodontic diagnosis of maxillofacial deformity since the early 1930s1 . various methodologies have been introduced , and normal average value ( norm ) for corresponding analysis has been reported2 . analysis of lateral cephalometry includes the analysis of angle and length of hard tissues and soft tissues of the face and is the most commonly used method3 . however , two - dimensional ( 2d ) lateral cephalometry is associated with many problems . second , it is difficult to clearly reflect the difference between the right and left sides of the face . third , there is a fundamental problem in that a three - dimensional ( 3d ) subject is illustrated in two dimensions7 . in addition , weaknesses of 2d lateral cephalometry have been reported including low concordance with results of actual clinical evaluation8,9 . conventional lateral cephalometry has another limitation in that deficiency in the paranasal and infraorbital areas can not be accurately demonstrated . 3d facial analysis has been presented to solve such shortcomings and can be used to achieve simulation treatment for 3d operation and orthodontic processes10,11,12,13 . systems have been established for normal average values , landmarks , reference lines , and reference planes , all of which can serve as standards of analysis14 . if a landmark is defined for 3d analysis , such landmarks should be used for 2d analysis in most cases . this is because lateral cephalometry analysis has been widely performed , and the massive amount of data accumulated can be effectively used for 3d analysis15 . however , few studies have analyzed whether 3d analysis agrees with the large amount of 2d data . in this regard , this study analyzed the concordance and patterns between eight angles measured using lateral cephalometry as well as 3d computed tomography ( ct ) images . in addition , it also aims to identify a 3d frankfort horizontal ( fh ) plane with a high degree of concordance to the 2d plane by studying four fh planes . the study population consisted of 20 patients who visited the department of oral and maxillofacial surgery , samsung medical center ( seoul , korea ) in 2012 who underwent lateral cephalometry ( planmeca promax ; planmeca oy , helsinki , finland ) and 3d ct ( ge lightspeed vct xt ; general electronic medical system , milwaukee , wi , usa ) imaging . , 20 patients were selected among whom there were 12 males and eight females with an average age of 23.2 years . the area from the nasal bone to the mandible was included in the image , and the axial slice thickness was 1 mm . during ct scanning , the jaw and teeth were maintained in centric occlusion . after completion of ct scanning , the images were saved as digital imaging and communication in medicine ( dicom ) files . ct images were also reconstructed to produce 3d images using simplant pro 2011 ( materialize dental nv , leuven , belgium ) . the following patients were excluded : those with severe congenital malformation , such as hemifacial microsomia , those with pathological lesions of the jaw , and those with severe inflammation . in the reconstructed 3d image , lines , planes , and angles were set as follows , and the fh plane was the horizontal reference plane in this study . the fh plane on lateral cephalometry was defined with the lines passing the orbitale ( or ) and porion ( po ) , and the upper occlusal plane angle ( uopa ) was measured . four fh planes ( table 1 ) were established in the 3d system , and the uopa of each point was measured . the angles measured in 3d and lateral cephalometry were evaluated with regard to intraclass correlation coefficient ( icc ) with ibm spss statistics 21.0 ( ibm co. , armonk , ny , usa ) . ( 1 ) gonial angle ( ga ) , ( 2 ) palatal angle , ( 3 ) mandibular plane angle ( mpa ) , ( 4 ) uopa , ( 5 ) u1 to occlusal plane angle ( u1opa ) , ( 6 ) u1 to fh plane angle ( u1fhpa ) , ( 7 ) sna , ( 8) snb were measured both in lateral cephalometry and reconstructed 3d ct ( table 1 ) and were evaluated with regard to icc . the study population consisted of 20 patients who visited the department of oral and maxillofacial surgery , samsung medical center ( seoul , korea ) in 2012 who underwent lateral cephalometry ( planmeca promax ; planmeca oy , helsinki , finland ) and 3d ct ( ge lightspeed vct xt ; general electronic medical system , milwaukee , wi , usa ) imaging . , 20 patients were selected among whom there were 12 males and eight females with an average age of 23.2 years . the area from the nasal bone to the mandible was included in the image , and the axial slice thickness was 1 mm . during ct scanning , the jaw and teeth were maintained in centric occlusion . after completion of ct scanning , the images were saved as digital imaging and communication in medicine ( dicom ) files . ct images were also reconstructed to produce 3d images using simplant pro 2011 ( materialize dental nv , leuven , belgium ) . the following patients were excluded : those with severe congenital malformation , such as hemifacial microsomia , those with pathological lesions of the jaw , and those with severe inflammation . in the reconstructed 3d image , lines , planes , and angles were set as follows , and the fh plane was the horizontal reference plane in this study . the fh plane on lateral cephalometry was defined with the lines passing the orbitale ( or ) and porion ( po ) , and the upper occlusal plane angle ( uopa ) was measured . four fh planes ( table 1 ) were established in the 3d system , and the uopa of each point was measured . the angles measured in 3d and lateral cephalometry were evaluated with regard to intraclass correlation coefficient ( icc ) with ibm spss statistics 21.0 ( ibm co. , armonk , ny , usa ) . ( 1 ) gonial angle ( ga ) , ( 2 ) palatal angle , ( 3 ) mandibular plane angle ( mpa ) , ( 4 ) uopa , ( 5 ) u1 to occlusal plane angle ( u1opa ) , ( 6 ) u1 to fh plane angle ( u1fhpa ) , ( 7 ) sna , ( 8) snb were measured both in lateral cephalometry and reconstructed 3d ct ( table 1 ) and were evaluated with regard to icc . on lateral cephalometry , the average uopa was 11.38. when the concordance of uopa against the four fh planes on 3d was evaluated , the iccs showed high concordance with 0.745 , 0.751 , 0.755 , and 0.754 , respectively . the icc differences among the four angles acquired in 3d were also not significant different.(table 2 ) concordance of the average of eight angles was verified.(table 3 ) iccs of ga , palatal angle , u1opa , and u1fhpa were low at 0.583 , 0.287 , 0.404 , and 0.617 , respectively . thus demonstrating a low concordance between lateral cephalometry and 3d ct . on the other hand , iccs of mpa , uopa , sna , and snb were high at 0.752 , 0.745 , 0.798 , and 0.869 , respectively . additionally , ga and mpa acquired in 2d were larger than those in 3d for all 20 patients included in this study.(table 4 ) on lateral cephalometry , the average uopa was 11.38. when the concordance of uopa against the four fh planes on 3d was evaluated , the iccs showed high concordance with 0.745 , 0.751 , 0.755 , and 0.754 , respectively . the icc differences among the four angles acquired in 3d were also not significant different.(table 2 ) concordance of the average of eight angles was verified.(table 3 ) iccs of ga , palatal angle , u1opa , and u1fhpa were low at 0.583 , 0.287 , 0.404 , and 0.617 , respectively . thus demonstrating a low concordance between lateral cephalometry and 3d ct . on the other hand , iccs of mpa , uopa , sna , and snb were high at 0.752 , 0.745 , 0.798 , and 0.869 , respectively . additionally , ga and mpa acquired in 2d were larger than those in 3d for all 20 patients included in this study.(table 4 ) two - dimensional cephalometry has been widely used for diagnosis and operation in craniofacial skeletal development and orthodontic and orthognathic surgeries13,16 . the measured values from the patients can be compared with generally known normal values to judge the asymmetry17 . in lateral cephalometry , however , it is difficult to distinguish the overlapping structures ; in patients with an asymmetric face , the accuracy of analysis is low . consequently , it is desirable to also use other radiographic images in order to achieve more accurate analysis . to evaluate the asymmetry of a face , panorama radiography18 and posteroanterior cephalometry panorama radiography is the most familiar technique to most dentists and orthodontists , and it is the representative examination for dental diagnosis and treatment planning . this type of imaging enables dentists to detect cysts and tumors , supernumerary teeth , missing teeth , and bony deformities and to evaluate the asymmetry of the lower jawbone19,20 . updegrave21 , however , reported that an incorrect treatment plan may results from serious asymmetry of the mandible ramus and condyle and coronoid processes on panorama radiography . posteroanterior cephalometry has the same problem in that precise perception is difficult because of many landmark overlaps13 . as such however , no overlapping landmarks are produced in 3d imaging , minimizing the confusion of left- and right - side structures . therefore , with 3d ct imaging , clinicians can diagnose all patients accurately including those with facial asymmetry , easily identify the shapes of craniofacial structures , and recognize the shapes of bones and soft tissues from various angles . according to previous studies measuring length on volume - rendered 3d ct images and in cadavers23 , the difference in length between two groups was minimal , confirming the accuracy of 3d - ct images . in addition , 3d lateral cephalometry analysis abstracted from 3d ct was introduced , and its accuracy has been reported . van vlijmen et al.1 performed comparative analysis on various angles and distances between 3d lateral cephalometry and conventional lateral cephalometry and reported that there was no statistically significant difference in many parts . it is true that lateral cephalometry analysis has been the major method to evaluate facial types for tens of years15,25 . 3d ct analysis , a very useful method in diagnosis , uses the same landmarks as conventional lateral cephalometry15 . however , the concordance between the measurements of lateral cephalometry and 3d ct has not been sufficiently studied . using the relationship and patterns of measurements acquired from two analysis methods , as well as their concordance , 3d ct values the uopa abstracted from the fh plane defined in 3d ct and that from lateral cephalometry were found to be very similar.(table 2 ) similarly , the four 3d fh planes did not show differences in concordance with those of 2d analysis . in such cases , it is desirable to choose landmarks with higher representation and reliability . in a study by yu et al.26 , two observers marked lateral cephalometry landmarks three times and calculated the concordance with respect to the x - axis and y - axis . in this experiment , po showed the lowest concordance among 17 landmarks on the x - axis , while or did not show a significant difference in concordance among observers or among trials of the same observer . additionally , when defining po on 3d ct , a wide curve is produced , making repetition and reproduction difficult.(fig . 1 ) based on these results , it is reasonable to define the fh plane in 3d using two or and one po values . according to the results of this study , ga measured on 2d is larger than that measured on 3d in all patients.(table 4 ) there are three possible explanations . first , the difference may be because of error in menton ( me ) . when marking me on 3d ct ( fig . 2b ) and observing the resulting 3d lateral cephalometry , me marked on the superior position that me on conventional lateral cephalometry.(fig . 2c ) this occurs because anatomic structures near the me are extended in the lower direction on 3d ct.(fig . 2a ) in patients with such structures , the 3d values may be different from those on 2d even when me is accurately marked on 3d ct . additionally , when me is marked at the front of skull , it may be marked in a little superior position ( fig . thus , when me is marked on 3d ct , it is recommended to approach from the bottom of the lower jawbone . second , articulare ( ar ) may have larger x - axis error on lateral cephalometry compared with 3d ct . trpkova et al.7 confirmed through meta - analysis that ar and basion showed the largest x - axis errors on lateral cephalometry . the possibility of lower accuracy of ga can not be excluded because of lower accuracy of the reference points . ar on lateral cephalometry is defined as the point of overlap between the skull base and posterior surface of the lower jawbone condyle , while ar in the present study is defined as the most posterior point of the mandibular condyle . thus , it is difficult to assert that ar in this study is absolutely concordant with ar on 2d . however , it is impossible to define ar in 3d using the same definition of that in 2d . therefore , if ar is defined using the method of this study , ga in 2d is slightly larger than that in 3d ct . mpa , just like ga , was larger in 2d than in 3d in all patients . the angles showing low concordance were palatal angle , u1opa , and u1fhpa.(table 3 ) an angle is made by two lines ; if the lines are short , the measurement is more difficult , and the risk of error increases , which lowers the probability of repetition and reproduction and consequently the probability of concordance . the straight palatal line and u1 line used to define the aforementioned three angles had short distances , which resulted in their lower concordance values . on the other hand , mpa , uopa , sna , and snb were formed by two long lines and so showed higher concordance . for measurement of angles made up of two long lines , the concordance between two methods is high . in this study when me was included in the analysis , it was marked in a different location from that of 2d analysis because of the nature of the measurement points . additionally , the fh plane on 3d ct showed concordance with the fh plane on lateral cephalometry , indicating no difference in concordance no matter what is selected among four fh planes defined on 3d ct . however , it is desirable to define the fh plane on 3d ct with two or and one po values considering the reproduction of or itself .

objectivesthe aim of this study was to verify the concordance of the measurement values when the same cephalometric analysis method was used for two - dimensional ( 2d ) cephalometric radiography and three - dimensional computed tomography ( 3d ct ) , and to identify which 3d frankfort horizontal ( fh ) plane was the most concordant with fh plane used for cephalometric radiography.materials and methodsreference horizontal plane was fh plane . palatal angle and occlusal plane angle was evaluated with fh plane . gonial angle ( ga ) , palatal angle , upper occlusal plane angle ( uopa ) , mandibular plane angle ( mpa ) , u1 to occlusal plane angle , u1 to fh plane angle , sna and snb were obtained on 2d cephalmetries and reconstructed 3d ct . the values measured eight angles in 2d lateral cephalometry and reconstructed 3d ct were evaluated by intraclass correlation coefficiency ( icc ) . it also was evaluated to identify 3d fh plane with high degree of concordance to 2d one by studying which one in four fh planes shows the highest degree of concordance with 2d fh plane.resultsiccs of mpa ( 0.752 ) , uopa ( 0.745 ) , sna ( 0.798 ) and snb ( 0.869 ) were high . on the other hand , iccs of gonial angle ( 0.583 ) , palatal angle ( 0.287 ) , u1 to occlusal plane ( 0.404 ) , u1 to fh plane ( 0.617 ) were low respectively . additionally ga and mpa acquired from 2d were bigger than those on 3d in all 20 patients included in this study . concordance between one uopa from 2d and four uopas from 3d ct were evaluated by icc values . results showed no significant difference among four fh planes defined on 3d ct.conclusionfh plane that can be set on 3d ct does not have difference in concordance from fh plane on lateral cephalometry . however , it is desirable to define fh plane on 3d ct with two orbitales and one porion considering the reproduction of orbitale itself .

the herpes family of viruses includes 8 separate species that infect humans : herpes simplex virus 1 ( hsv-1 ) , herpes simplex virus 2 ( hsv-2 ) , varicella zoster virus , epstein - barr virus , cytomegalovirus , human herpesvirus 6 ( hhv-6 ) , human herpesvirus 7 ( hhv-7 ) and human herpesvirus 8 ( hhv-8 ) . these viruses account for a significant proportion of human cutaneous diseases and they cause similar histological and clinical findings . although herpes labialis , herpes vulvovaginitis , herpetic whitlow and herpes gladiatorum are the most well - known clinical features of the hsv-1 and hsv-2 infections , there are various descriptions of herpesvirus involvement in erythema multiforme , atopic dermatitis , lichenoid dermatitis and seborrheic keratosis [ 2 , 3 , 4 , 5 , 6 ] . lichen simplex chronicus ( lsc ) and acute bilateral carpal tunnel syndrome ( cts ) are unusual complications of herpesviruses , and apparently a few cases of adult patients have been reported previously [ 7 , 8 , 9 ] . we describe an 11-year - old girl with lsc and acute bilateral cts following hsv-1 infection . an 11-year - old girl was admitted with recurrent fever , pain and swelling of her right wrist and proximal phalanx , and relapsing painful , itchy lesions consisting of erythematous papules with excoriations and crusting on the skin with a central hyperpigmentation , mostly distributed on the neck , face , legs and hips . her medical history was significant for a 6-month period of recurrent lesions , always involving the same parts of her body and preceded by severe pain and hyperesthesia on 1 side of her entire body ( sometimes right , sometimes left ) , with a frequency of 3 times a week . she described that both of her wrists , especially the right one and proximal phalanx had become tender and swollen at the same time as the appearance of the skin lesions . up to this time , she had been diagnosed with atopic dermatitis and eczema and treated with various medicines such as systemic antihistamines , local and systemic corticosteroids and/or analgesics and antipsychotics but showed no improvement . on physical examination , she had both old and new skin lesions at the same time on different parts of her body , particularly on the cheeks , neck , extremities and hips . laboratory investigations revealed no abnormality of counter blood cell , erythrocyte sedimentation rate , c - reactive protein and fibrinogen . immunoglobulin levels of iga , igg and ige were within the normal range , but igm showed an elevated pattern . viral hepatitis ( a , b , c ) , epstein - barr virus , cytomegalovirus , hsv-2 , human immunodeficiency virus , parvovirus b19 , lyme serologies , salmonella and brucella agglutinations , organ- and tissue - specific autoimmune antibodies ( especially antinuclear antibodies ) , rheumatoid factor , cryoglobulins and ena test were all negative . antineutrophil cytoplasmic antibodies assay ( anca ) showed a negative pattern for both p - anca ( perinuclear staining ) and c - anca ( cytoplasmic staining ) . the c3 level was 107 mg / dl ( normal 80150 ) and c4 was within the normal range at 22 mg / dl ( normal 2050 ) . specific ige tests involving milk , egg , mold allergy , grass allergy , dog epithelium , house dust allergy , cat - specific ige and weed panel were also negative . cranial mri was performed in order to reveal the reason for her neurologic symptoms but showed no abnormality . u / l ( normal 22240 ) and an electroneuromyographic investigation revealed moderate delay in distal sensory - motor latencies of both median nerves , with delayed median nerve f - responses on both sides . serological tests showed positive hsv-1 specific igm and hsv-1 dna was detected in the patient 's serum by using pcr . a punch skin biopsy specimen from the right upper extremity showed hyperkeratosis with areas of parakeratosis , overlying an acanthotic epidermis with irregular elongation of the rete ridges . chronic perivascular inflammatory infiltrate and fibrosis were also seen in the papillary dermis , but immunofluorescence studies were all negative ( fig . 1 , fig . postherpetic lsc and acute bilateral cts associated with hsv-1 infection were considered and high - dose acyclovir treatment ( 30 mg / kg / day i.v . for 5 days ) , 10 mg / kg / day naproxen sodium and high doses of vitamin b1 , b6 and b12 combinations were started immediately . a dramatic improvement was observed on her neurologic symptoms and skin lesions within a few weeks . during her follow - up , neither adverse effects due to high - dose acyclovir treatment nor any other new lesions were observed . a control pcr was negative and serological tests showed a negative pattern for hsv-1 igm and a positive pattern for hsv-1 igg in the first month of her follow - up . forty - five days after the initial acyclovir treatment she went to the seaside for summer holiday and a few days later she was readmitted with swelling of the proximal phalanx of her right hand and a new skin lesion on her right hip ( fig . 3 ) ; these symptoms were preceded by severe pain and hyperesthesia in the right half of her entire body . a new pcr showed a positive pattern again , but serological tests revealed a negative pattern for hsv-1 igm and a positive pattern for hsv-1 igg . a new viral shedding was thought to be responsible and oral acyclovir ( 30 mg / kg / day ) treatment was given for 2 weeks . afterwards , she had continued the therapy at a dose of 10 mg / kg / day for 2 weeks . the skin lesion on her right hip and swelling of the joints resolved within a few weeks and pcr became negative again . we discontinued the therapy and follow up the patient monthly . thus far , 10 months have passed without any new skin lesions . lsc ( circumscribed neurodermatitis ) is an idiopathic disorder characterized by the presence of 1 or more erythematous , scaling , lichenified plaques with varying degrees of overlying excoriation . in cases of long - standing duration the most common sites are the neck ( sides ) , ankles , scalp , vulva , pubis , scrotum and extensor forearms . the peak of incidence is between 35 and 50 years of age , and women are more affected than men ( f : m = 2:1 ) . severe and intractable itch is the predominant symptom in nearly all patients and is characterized by paroxystical attacks [ 10 , 11 , 12 ] . acute clinical presentation of herpesviridae is often secondary to active virus replication and host immune response , but resolution of symptoms does not herald clearance of the virus . lsc is an unusual complication of herpesviruses ; however , there are a few reports of postherpetic pruritus in adult patients . liddel reported a patient with severe pruritus after a herpes zoster infection , gerritsen et al . reported a patient with lsc following herpes zoster infection of the scalp and darsow et al . described a patient with circumscribed pruritus which started 5 years after herpes zoster in the same dermatome . either clinically or pathologically there were no classical skin lesions of the hsv-1 infection in our patient . but positivity of the serological tests and detection of hsv-1 dna by pcr and the patient 's response to the acyclovir treatment suggested that lsc developed in this patient as a complication of recurrent viral shedding of hsv-1 . it was difficult to distinguish whether the exact cause of the pain was postherpetic neuralgia or increasing stimulation that may produce itch and pain , as these sensations share the same neuroanatomical substrate . we thought that the biopsy taken from the margin of the main lesion and differentiation of herpetic lesions to lsc may be related to the rubbing and scratching . on the other hand , it is known that there is a potential relationship between ultraviolet light and recurrent herpes simplex infections . viruses can reactivate from the latent state in neurons to form recrudescent lesions due to exposure to sunlight [ 14 , 15 ] . the occurrence of a new lesion after the summer holiday in this patient was thought to be the result of exposure to ultraviolet irradiation which led to a new viral shedding . cts is one of the most common peripheral compression neuropathies , but it is rarely seen in children . many aspects of its etiology are not at all clear , and it is often termed idiopathic ; however , it has also been attributed to a variety of underlying disorders and processes and secondary to infectious diseases [ 17 , 18 , 19 ] . childhood cts often has an unusual presentation , with modest complaints and children are often too young to communicate their problem . in our patient , pain and swelling of the wrists and proximal phalanx was thought to be associated with the mechanical entrapment of the median nerves in relation to acute arthritis during the viral shedding of hsv-1 . the rapid response to non - steroid anti - inflammatory drugs and high doses of vitamin b1 , b6 and b12 combinations , tends to support this hypothesis . in conclusion , this is an unusual childhood case of hsv type-1 infection complicated by lsc and acute bilateral cts during viral shedding . the experience with this patient emphasizes the importance of serological tests and pcr as well as the other laboratory techniques for the accurate diagnosis and management of the disease .

we describe an 11-year - old girl presenting with lichen simplex chronicus ( lsc ) and acute bilateral carpal tunnel syndrome ( cts ) following herpes simplex virus type 1 ( hsv-1 ) infection as evidenced by serological data and by detection of hsv-1 dna in the blood with the use of pcr . based on the literature search , this case represents the first childhood case of lsc and acute bilateral cts following hsv-1 infection . the experience with this patient emphasizes the importance of serological tests and pcr as well as the other laboratory techniques for the accurate diagnosis and management of the disease .

cerebral palsy ( cp ) is a non - progressive disease with symptoms that include neurological disorders or developmental disabilities . children with cp have spasticity , musculoskeletal problems , mobility disturbances , and decreased pelvic movements that lead to awkward movement and sitting posture1 , 2 . symptoms also induce unstable posture control , imbalance , and aberrant control of movement . postural control is the ability to control the body position in space , and it has a relationship with the sense of balance3 . hippotherapy and horseback riding have been suggested as interventions for correcting the balance problems of children with cp4 . two types of horseback riding are available : hippotherapy and therapeutic horseback riding ( thr ) . hippotherapy provides better pelvic , hip , and trunk movement and influences childrens posture , balance , and coordination5 . hippotherapy has short - term beneficial effects on the muscle symmetry of the trunk and hip , whereas thr has no effect on muscle tone6 . the effects of these two types of horseback riding have been studied in children with cp , and both types have been demonstrated to improve gross motor function and promote better standing and bipedal balance7 , 8 . a horseback riding simulator ( joba , panasonic inc . , it was developed to overcome the primary limitations of hippotherapy , such as unavailability and high cost . this device offers an indoor experience of horseback riding and mimics the rhythmic movement of horseback riding , thereby promoting muscular strength and improving sense of balance . the objective of this study was to compare hippotherapy to the use of a horseback riding simulator ( joba , panasonic inc . , japan ) with respect to their effects on the static and dynamic balance of children with cp . our results indicate that a horseback riding simulator has beneficial effects as an intervention for children with cp . this study included 26 children with cp who were receiving physical therapy at the h horseback riding center and the n horseback riding center in kyung - ki in korea . the selection criteria for the subjects were a modified ashworth scale ( mas ) grade less than + 1 . children who could perform more than 10 m independent walking and were available for more than 30 min training per day were selected . the parents or guardians of all the participants provided their written informed consent in accordance with the ethical principles of the declaration of helsinki ( table 1table 1 . general characteristics of subjectshippotherapyhrsgender ( m / f)8/59/4age ( year)10.81.610.02.2height ( cm)125.812.6122.614.3weight ( kg)25.26.425.55.7meansd , hrs : horseback riding simulator ) . meansd , hrs : horseback riding simulator the children were randomly divided into two groups : a hippotherapy group that included 13 children , and a horseback riding simulator ( joba , panasonic inc . the two groups participated in 1 hour of exercise per day , 3 times a week , for 12 weeks . the hippotherapy group carried out anterior - sitting , posterior - sitting , and side - sitting exercises for 10 min each while horseback riding at a walking pace ( 6 km / h ) . the course leader held the reins of the horse and two side - walkers held the child s legs to assist the child in sitting in the saddle in order to prevent the child from falling and to help the child to exercise during the horseback riding walk . the horseback riding simulator group exercised with the same protective equipment and were assisted by the same leader and side - walkers . both groups received 20 min of conventional physical therapy before hippotherapy and performed stretching on the horse or horseback riding simulator for 5 min before and after the exercise . three physical therapists , who had more than 3 years of experience in pediatric physical therapy and had received education in the experimental method , participated in this study . the subjects static balance ability was measured using bpm ( software 5.3 , sms healthcare inc . , uk ) as the center of pressure sway length while standing for 30 seconds with the eyes open and looking to the front . statistically significant differences between the measurements obtained before and after the intervention in each group were determined using the independent t - test . the children were divided into two groups and evaluated pre- and post - test using bpm ( software 5.3 , sms healthcare inc . , uk ) and the pbs ( pediatric balance scale ) ( table 2table 2 . comparison of measurement values at pre - test and post - testvariablegroupprepostpbs ( score)hippotherapy35.63.841.24.7*hrs35.84.738.55.3*sway length ( mm)hippotherapy220.127.6135.014.3*hrs219.931.7142.818.8**p<0.05 meansd , pbs : pediatric balance scale , hrs : horseback riding simulator ) . * p<0.05 meansd , pbs : pediatric balance scale , hrs : horseback riding simulator the hippotherapy and horseback riding simulator groups showed significantly decreased sway lengths in the static balance test . comparison of the pre- and post - test sway lengths showed that the hippotherapy group and the horseback riding simulator group showed significant decreases in sway length of 85 mm and 80 mm , respectively ( p<0.05 ) . both groups showed improved static balance , but no significant difference was found between the two groups . dynamic balance was also evaluated pre- and post - test using the pbs ( pediatric balance scale ) . the hippotherapy group showed an increase in score of about 4 points , while the horseback riding simulator group showed an increase of 3 points . both groups showed significant improvements in dynamic balance , but no significant difference was found between the two groups ( p<0.05 ) . balance control is very important for children with cp , and maintaining balance requires three distinct sensory system inputs : visual , somatosensory , and vestibular9 . generally , the dynamic sense of balance depends primarily on vestibular input on unstable surfaces ; however , static balance primarily depends on somatosensory input . many studies have investigated balance and clinical tools for children with cp . a systematic review identified 22 tools that can assess balance10 . hippotherapy and thr are types of exercises that affect balance , coordination , and posture , contributing to the development of sensory and perceptual motor skills6 , 11 . a horse s rhythmic movement , velocity , and variations can facilitate righting and equilibrium actions . during hippotherapy and thr , these facilitations improve muscular co - contraction and postural balance resulting in improvements in the gross motor function of children with cp12,13,14,15 . many studies of hippotherapy and thr in children with cp have demonstrated they have positive effects on postural control and balance . the horseback riding simulator has also been reported to be beneficial for children with cp , improving their postural control and global function , and providing enjoyment16 . haehl et al . found no significant improvement of in hippotherapy and thr but indicated that hippotherapy had positive effects on the postural control and balance of children with cp17 . other studies confirmed that hippotherapy and thr are suitable interventions for children with cp . the horseback riding simulator used in the present study offers several advantages over hippotherapy . the simulator is free from space limitations , has a low price , is easy to handle , and is non - affected by weather conditions16 . use of the horseback riding simulator resulted in improved muscle strength and contraction in elderly people18 . a previous study of children with cp reported that use of a horseback riding simulator significantly improved their postural control and balance16 . our data indicate that both hippotherapy and use of the horseback riding simulator improved the static and dynamic balance of children with cp . however , due to the fast learning characteristics of children , the long - term 12-week exercise program might have been responsible for the similar results seen in balance improvement . many factors remain to be considered regarding exercise for children with cp . therefore , if other tools or exercises were utilized , the results might not match the results presented here . overall , our results indicate that children with cp may respond equally well to the use of a horseback riding simulator as they do to hippotherapy in terms of balance improvement . first , the number of subjects was small , and second , only static and dynamic balance were evaluated . in spite of these limitations , our results show that the use of a horseback riding simulator can be a good intervention for children with cp . further studies incorporating larger sample sizes , various cp types , and full utilization of the programs of the horseback riding simulator might be needed . in conclusion , the horseback riding simulator could be a useful alternative to hippotherapy for improving of static and dynamic balance of children with cp .

[ purpose ] we with respect to their effects on the compared hippotherapy with a horseback riding simulator ( joba , panasonic inc . jp ) static and dynamic balance of children with cerebral palsy ( cp ) . [ subjects and methods ] twenty - six children were randomly divided into two groups : a hippotherapy group that included 13 children , and a horseback riding simulator ( joba , panasonic inc . , japan ) group , which was also composed of 13 children . the two groups participated in 1 hour of exercise per day , 3 times a week , for 12 weeks . the subjects static balance ability was measured using bpm ( software 5.3 , sms healthcare inc . , uk ) as the center of pressure sway length while standing for 30 seconds with their eyes open and looking to the front . dynamic balance ability was measured using the pbs ( pediatric balance scale ) . [ results ] both groups showed significant improvements in static and dynamic balance but significant differences between the two groups were not found . [ conclusion ] the horseback riding simulator could be a useful alternative to hippotherapy for the improvement of static and dynamic balance of children with cp .

it is known that 9095% of all cancers are caused by or closely associated with environmental factors and lifestyle . this includes diet ( 3035% ) , cigarette smoking ( 2530% ) , and alcohol consumption ( 46% ) . cigarette smoking is an important risk factor for heart disease , chronic obstructive pulmonary disease , stroke , and acute respiratory diseases . in addition to all these noncancer diseases , it is also highly associated with human cancer development . the international agency for research on cancer ( iarc ) identified cigarette smoking as the cause of cancer in more organ sites than any other human carcinogens . these include cancers of the lungs , oral cavity , larynx , nasal cavity , esophagus , stomach , pancreas , liver , kidney , urinary bladder , uterine cervix , and bone marrow . there are over 5000 chemical compounds identified in tobacco and 62 of these have been evaluated by iarc as showing sufficient evidence for carcinogenicity in either animals or in humans [ 2 , 3 ] . the major carcinogenic compounds include , but not limited to , radioactive polonium , n - nitrosamines such as 4-(methylnitrosaminao)-1-(3-pyridyl)-1-butanone ( nnk ) , polycyclic aromatic hydrocarbons ( pahs ) ( e.g. , benzo[a]pyrene ( bap ) ) , and benzene . multistep processes of genetic and molecular defects have taken place before the manifestation of cancer . traditionally , there are three basic stages of carcinogenesis : initiation , promotion , and progression . carcinogenesis process is usually accompanied by changes in structure and function of central genomic information coded in the dna leading to various oncogene activations and tumor suppressor gene inactivations . in addition , multiple signaling pathways may also be deregulated during the process of cancer development . cancer growth also requires molecular changes that either affect the tumor cells themselves or alter the interaction between tumor cells and their surrounding stromal environment or the immune system . cigarette smoke components have been reported to promote tumorigenesis by several mechanisms involving all three stages of carcinogenesis . genotoxic agents in cigarette smoke induce dna damage through several mechanisms including gene point mutation , deletions , insertions , recombinations , rearrangements , and chromosomal aberrations . in addition to genotoxic effects , nongenotoxic effects of cigarette smoke are also extremely important . these effects can also act as modulators which alter cellular functions including cell proliferation and cell death . while synergistic effects of genotoxic carcinogens are known to occur , interaction between non - genotoxic ( epigenetic ) factors and genotoxic agents may also synergistically increase the risk for carcinogenesis . the genotoxicity leading to carcinogenesis has been extensively reviewed in recent years [ 911 ] . in this present review , aside from a brief overview on the genotoxic effects of cigarette smoke components , we will provide a more extensive review on the non - genotoxic mechanisms of carcinogenesis by cigarette smoke or its components . step 1 ( initiation of carcinogenesis)carcinogenesis may be the result of chemical or biological insults to normal cells through multistep processes that involves genomic changes ( initiation of cancer development ) . some of the cigarette smoke components can act directly on dna , but many require enzyme conversion before becoming carcinogenic [ 10 , 11 ] . most of such conversions involve metabolic changes via cytochrome p450s ( p450s ) such as p450s 1a2 , 2a13 , 2e1 , and 3a4 to form the electrophilic entities that can bind to dna to form dna adducts . such adduct formation is usually at the adenine or guanine sites of the dna and lead to mutations such as those observed in the kras oncogene in lung cancer or those in the tp53 gene in a variety of cigarette smoke - induced cancers [ 13 , 14 ] . 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone ( nnk ) and n-nitrosonornicotine ( nnn ) are the most potent tobacco - specific nitrosamines in tobacco products and cigarette smoke . these compounds are formed from tobacco alkaloids like nicotine during the curing process of tobacco and are important tobacco carcinogens that can affect different tissues depending on the specific nitrosamines or their metabolites involved [ 5 , 10 ] . nnn has been shown to be carcinogenic to esophagus , nasal cavity , and respiratory tract in laboratory animals . in humans , metabolites derived from nnk and the metabolites of nnk can also be identified in the smoker 's urine .benzo[a]pyrene ( bap ) , one of the pahs , is classified as a group 1 carcinogen to humans . it has been shown to have strong association and tumor - induction potentials in lungs , trachea , and mammary glands . the carcinogenic potency of bap has been demonstrated to be related to its metabolites which form dna adducts with site - specific hotspot mutation in the p53 tumor suppressor gene . positive correlations of such adduct formation and tumor are indeed found in the lung cancer tissues of cigarette smokers . these findings indicate that dna mutations are increased in both tumor and nontumor bearing tissues of smokers . however , it must be pointed out that dna adduct formations induced by cigarette smoke still can not fully represent all the risk factors for cancer development in cigarette smokers . for example , while there is higher incidence of pancreatic cancer in cigarette smokers than nonsmokers . assays for nnk metabolites in pancreatic cancer tissues in humans showed no significant difference between smokers and nonsmokers . thus , it is apparent that nnk - induced dna adducts alone are not solely responsible for the pancreatic cancers in cigarette smokers . nevertheless , nnk and its metabolite , nnal ( 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol ) , are the only environmental carcinogens known to induce pancreatic cancer in animal models . thus , the contribution of nnk to pancreatic cancer in cigarette smokers still can not be ignored . furthermore , it is suggested that , in addition to dna damage , synergistic interactions between dna reactivity and epigenetic actions such as increased cell proliferation induced by nnk or by other chemicals in cigarette smoke may be needed for actual cancer development in such patients [ 23 , 24 ] . there is indication that cigarette smoke carcinogens or cocarcinogen , such as nicotine , may also play a direct role to enhance cancer promotion and progression in human cancers after cancer development . such genotoxic mechanisms for cancer initiation and carcinogenesis by cigarette smoke components are well covered and discussed in several excellent reviews [ 5 , 10 , 11 , 2628 ] . , we will provide more information on the non - genotoxic ( epigenetic ) mechanisms involved in cancer promotion and progression via cigarette smoke . carcinogenesis may be the result of chemical or biological insults to normal cells through multistep processes that involves genomic changes ( initiation of cancer development ) . some of the cigarette smoke components can act directly on dna , but many require enzyme conversion before becoming carcinogenic [ 10 , 11 ] . most of such conversions involve metabolic changes via cytochrome p450s ( p450s ) such as p450s 1a2 , 2a13 , 2e1 , and 3a4 to form the electrophilic entities that can bind to dna to form dna adducts . such adduct formation is usually at the adenine or guanine sites of the dna and lead to mutations such as those observed in the kras oncogene in lung cancer or those in the tp53 gene in a variety of cigarette smoke - induced cancers [ 13 , 14 ] . 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone ( nnk ) and n-nitrosonornicotine ( nnn ) are the most potent tobacco - specific nitrosamines in tobacco products and cigarette smoke . these compounds are formed from tobacco alkaloids like nicotine during the curing process of tobacco and are important tobacco carcinogens that can affect different tissues depending on the specific nitrosamines or their metabolites involved [ 5 , 10 ] . nnn has been shown to be carcinogenic to esophagus , nasal cavity , and respiratory tract in laboratory animals . in humans , metabolites derived from nnk and the metabolites of nnk benzo[a]pyrene ( bap ) , one of the pahs , is classified as a group 1 carcinogen to humans . it has been shown to have strong association and tumor - induction potentials in lungs , trachea , and mammary glands . the carcinogenic potency of bap has been demonstrated to be related to its metabolites which form dna adducts with site - specific hotspot mutation in the p53 tumor suppressor gene . positive correlations of such adduct formation and tumor are indeed found in the lung cancer tissues of cigarette smokers . these findings indicate that dna mutations are increased in both tumor and nontumor bearing tissues of smokers . however , it must be pointed out that dna adduct formations induced by cigarette smoke still can not fully represent all the risk factors for cancer development in cigarette smokers . for example , while there is higher incidence of pancreatic cancer in cigarette smokers than nonsmokers . assays for nnk metabolites in pancreatic cancer tissues in humans showed no significant difference between smokers and nonsmokers . thus , it is apparent that nnk - induced dna adducts alone are not solely responsible for the pancreatic cancers in cigarette smokers . nevertheless , nnk and its metabolite , nnal ( 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol ) , are the only environmental carcinogens known to induce pancreatic cancer in animal models . thus , the contribution of nnk to pancreatic cancer in cigarette smokers still can not be ignored . furthermore , it is suggested that , in addition to dna damage , synergistic interactions between dna reactivity and epigenetic actions such as increased cell proliferation induced by nnk or by other chemicals in cigarette smoke may be needed for actual cancer development in such patients [ 23 , 24 ] . there is indication that cigarette smoke carcinogens or cocarcinogen , such as nicotine , may also play a direct role to enhance cancer promotion and progression in human cancers after cancer development . such genotoxic mechanisms for cancer initiation and carcinogenesis by cigarette smoke components are well covered and discussed in several excellent reviews [ 5 , 10 , 11 , 2628 ] . , we will provide more information on the non - genotoxic ( epigenetic ) mechanisms involved in cancer promotion and progression via cigarette smoke . step 2 ( cancer promotion)cancer promotion is characterized by deregulation of signaling pathways which control cell proliferation , apoptosis , and so forth , . it is believed that although there are various genetic pathways which may lead to cancer development or cancer behaviors , there are certain hallmark capabilities or mechanisms which are commonly shared by all tumors . in the following discussion cancer promotion is characterized by deregulation of signaling pathways which control cell proliferation , apoptosis , and so forth , . it is believed that although there are various genetic pathways which may lead to cancer development or cancer behaviors , there are certain hallmark capabilities or mechanisms which are commonly shared by all tumors . in the following discussion these signals are transmitted into cells by receptors that bind distinct signaling molecules . in cancer cells , receptor overexpression allows cancer cells to become hyper - responsive to low levels of growth factors that generally are not sufficient to trigger proliferation in normal cells . nicotine , a major component of cigarette smoke , is known to be a chemical that plays an important role in carcinogenesis in cigarette smokers . nicotine behaves like those growth factors which exert their biological functions mainly through the nicotinic acetylcholine receptors ( nachr ) , -adrenoceptors ( -ar ) or epidermal growth factor receptor ( egfr ) . the functions of these receptors are cell - type specific and the expression level and receptor sensitivity can be modified by nicotine . obviously , alterations in either the receptor expressions or sensitivity play an important role in cigarette smoke - induced carcinogenesis [ 3436 ] . recent study by lee et al . reported that 9 nachr expression in human breast tumors is elevated in advanced stages of breast cancer and plays important roles in human breast carcinogenesis . nicotine has been shown to mediate 9 nachr signaling and upregulate cyclin d3 expression in breast caner cells and breast cancer tissues . furthermore , it is also found that activation of the expression of 9 nachr by nicotine is through akt signaling and activation of 9 nachr signaling would elevate the phosphorylation status of adhesion molecule which plays a role in cancer metastasis . this enhancement has been shown to be via 7 nachr with activation of erk1/2 cascade as well as induction of nf-b and bad phosphorylation . all these events eventually lead to inhibition of apoptosis and increase of cancer risk . these findings were further supported by wada et al . who observed that nicotine promoted cell proliferation via 7 nachr mediated p44/p42-mapk activation . moreover , in our own study , we also reported that nicotine induced human bladder cells proliferation through erk1/2 and stat3 signaling downstream of 7 nachr and -adrenoceptors ( -ar ) . in sum , all these studies indicate that nicotine , an important ingredient of cigarette smoke , promotes cellular proliferation which plays a critical role in carcinogenesis . other than nicotine , nitrosamines , such as nnk and nnn , also induced cancer cells growth through nachr . nnk induced carcinogenesis by binding to nachr especially for 7 nachr , whereas the biological impact of nnn is mainly modulated by 4/2 nachr [ 8 , 4446 ] . it has been demonstrated that nicotine or nnk stimulated lung cancer cell proliferation via 7 nachr with activations of pkc , raf1 , akt , erk1/2 , and transcription factors such as jun , fos , and myc [ 4749 ] . question has been raised concerning the possibility that specific nachr subunit upregulated by nicotine or nnk may be tissue specific or dependent . for instance , with nicotine or nnk , 7 nachr is the primary nachr subunit which mediates tumorigenesis in lungs giving rise to pulmonary squamous cell carcinoma and mesothelioma . on the other hand , thus , the specific types of nachr expressed in cancer cells may be considered as useful molecular targets for potential clinical therapy . however , most of the nachr present in cancer cells are still not functionally characterized yet . future study will be needed to understand the functions of different nachr subtype in cancer cells and the downstream signal pathways involved in tumorigenesis . in addition to nachr , a number of studies indicated that nicotine and nnk might also exert their biological activities through activation of receptors such as -adrenoceptors ( -ar ) , egfr , or insulin - like growth factor receptor ( igfr ) or transactivation by nachr signaling . for example , nnk can stimulate ht-29 cell proliferation through -ar followed by cyclin amp elevation and cox-2 expression . consistently , nnk stimulates the growth of pulmonary adenocarcinoma in vitro and in vivo via the release of arachidonic acid through cox-2 and 5-lipoxygenase ( 5-lox ) pathways that are mainly regulated by -ar . in another study by schuller and cekanova , nnk is reported to stimulate 2-ar receptor pathway ( including pka , camp , creb ) and transactivate egfr pathway ( such as raf-1/erk1/2 signaling ) in the development of lung cancer . it has also been reported that antagonists of -ar can inhibit the development of nnk - induced lung adenocarcinoma . such antagonists are also found to be effective in reducing the stimulatory effects of nicotine on pkc , erk1/2 activations , cox-2 expression , and gastric cancer cell proliferation . elevation of noradrenaline by nicotine via 7 nachr up - regulation leading to significantly enhanced growth and angiogenesis in both gastric cancer and colon cancer has also been demonstrated . various investigators have also shown increases in neurotransmitters lead to -ar activation , transactivation of egfr , and the release of egf [ 32 , 54 , 56 ] . indeed , our recent investigation provided compelling evidence that chronic nicotine exposure induced release of noradrenaline via 4/2 nachr activation followed by -ar transactivation . our study further demonstrated that blocking of -ar with antagonist reversed the nicotine - induced cellular proliferative and chemoresistance . al - wadei et al . first reported that nicotine contributes to the development of smoking - related pancreatic ductal adenocarcinoma ( pdac ) with elevated levels of stress neurotransmitters ( adrenaline and noradrenaline ) and induction of camp , pcreb , and perk1/2 , and inhibition of -aminobutyric acid ( gaba ) . gaba has been reported to possess tumor suppressor function suppressing both -ar stimulated pdac growth and migration in vitro . however , while gaba is suppressed in pdacs , noradrenaline , pka , p - creb , and perk1/2 in these tissues are overexpressed . these authors suggested that nicotine and nnk may contribute to the development of pdac in smokers by suppression of gaba with induction of stress neurotransmitters . further proposed that nicotine induces the release of stress neurotransmitters through activation of 7 nachr and inhibits release of gaba via inhibition of 4/2 nachr . it is now believed that the stress neurotransmitter released via nachr activation plays an important role in smoking - associated tumorigenesis . however , the precise mechanisms involved in the regulation and the function of neurotransmitter released by nicotine and nnk are still uncertain . future research on this area is encouraged . it has also been shown that nnk can promote -ar - mediated transactivation of egfr followed by erk1/2 phosphorylation leading to an increased proliferation in pancreatic cancer cells . nnk is also reported to induce endogenous igfr which is associated with the development of lung tumors . huang et al . also indicated that both activation of thromboxane a2 ( txa2 ) receptor and synthesis of txa2 play critical roles in nnk - promoted lung cancer cell proliferation . txa2 activates the transcriptional factor creb through both erk and pi3k / akt pathways , which may also lead to pcna and bcl-2 overexpressions and cell proliferation . these studies provide valuable information on the mechanisms which involve in proliferative signaling stimulated by nicotine and nnk through activation of nachr , -ar and other growth factor receptors in cancer cells . triggering such receptors by cigarette smoke would further lead to rapid cell proliferation , cellular migration , invasion , and metastasis . in short , these investigations on the nachr , and nachr transactivated with other receptors represent the pivotal role in regulating multiple cellular cascades in general cell functions and in carcinogenesis . nicotine is also known to influence signal transducers and activators of transcription 3 ( stat 3 ) which is an important signal transducer mediating signaling by numerous cytokines , growth factors , and oncoproteins . findings from our own laboratories indicate that nicotine induces bladder cancer cells proliferation through 7 nachr , 42 nachr , and -ar followed by activation of erk1/2 and stat 3 . stat3 signaling further enhanced nf-b activation , cyclin d1 overexpression , and cell cycle progression . moreover , we also revealed that prolonged stimulation by nicotine upregulated 4/2 nachr and -ar followed with activation of stat 3 leading to significant increase in chemoresistance in cells from bladder cancers . in recent years the biological effects of pahs are mainly mediated via aryl hydrocarbone receptor ( ahr ) . through ahr , other pahs , such as benz(a)anthracene ( baa ) , has also been found to increase dna synthesis and promote g1-s progression in serum deprived mcf-7 cells . bap has been shown to increase incidence of tumors in estrogen - responsive rodents , suggesting that it may also affect er - mediated signaling . some investigators believed that the estrogenic property of pahs may be responsible for the induction of cell proliferation . bap and baa have been reported to act as estrogens that stimulate and initiate the er - mediated transcription and cell cycle progression and enhance er phosphorylation . on the other hand , there is also indication that the estradiol - dependent cell growth of mcf-7 cells can be inhibited by bap and baa [ 71 , 72 ] . thus , the actions of pahs on estrogen - dependent cell proliferation are still controversial . further studies are needed to elucidate more on the roles of pahs in carcinogenicity . in normal tissues , antigrowth signals can block proliferation by forcing the cell cycle progression into the quiescent ( g0 ) state . the cell cycle transition from g1 to s phase is the key regulatory step in the cell cycle and is mainly regulated by cdk4/6-cyclin d and cdk2-cyclin e complexes . nicotine has been reported to induce binding of raf-1 to rb with activation of cyclins and cdks as well as inactivation of rb . via activations of nachr and -ar , nicotine and nnk both exhibit mitogenic properties by inducing cyclin d1 overexpression leading to g1/s transition and increasing cell cycle progression [ 49 , 75 , 76 ] . nnk can also stimulate normal human lung epithelial cells proliferation through nf-b and cyclin d1 upregulation in an erk1/2-dependent pathway . in our own laboratory , we have also demonstrated that nicotine - induced cyclin d1 overexpression is regulated via stat3 , erk1/2 , and nf-b - dependent pathways in bladder cancer cells . other study also shows that pi3k / akt - dependent cellular proliferation is also enhanced in response to nnk . the pi3k / akt pathway is critical in cancer cells because it influences tumorigenesis , tumor growth , and therapeutic resistance . the pi3k / akt activation is documented in both nnk - treated a / j mice and in human lung cancers from smokers . it also plays a role in nnk - induced cell transformation , proliferation , and metastasis . it has been suggested that akt and nf-b may serve as key targets for nicotine or nnk stimulation in the development of lung cancer . west et al . also reported that beas2b cells treated with nnk for eight - week period increased cellular proliferation through activation of pi3k / akt pathways . however , pi3k / akt activation does not always occur in all cancer cells induced by nicotine . our previous study indicates that nicotine induced bladder cancer cell proliferation through stat3 and erk1/2 signalings instead of via akt pathway . all these investigations suggest that nicotine or nnk can activate erk1/2 , stat3 , or akt signaling to interrupt the antigrowth signals leading to enhanced cell cycle progression and cancer promotion . it is important to remember that cigarette smoke components other than nicotine or nnk may also impede on antigrowth mechanisms enhancing cancer development and promotion . apoptosis plays an important role in controlling normal development , homeostasis , and immune defense via elimination of redundant or abnormal cells in the organism . failure in cell elimination ( reduction of apoptosis ) may lead to undesirable cell survival and unchecked cell growths . resistance to apoptosis is often seen in cancers where cancer cells tend to lose their proapoptotic potentials because of gene mutations . nicotine has been shown to inhibit apoptosis induced by tumor necrosis factor ( tnf ) , by ultraviolet ( uv ) , radiation , or by chemotherapeutic drugs such as cisplatin , vinblastine , paclitaxel , and doxorubicin . this antiapoptotic action has been shown to be via pi3k / akt , raf / mekk / erk1/2 , nf-b , bcl-2 , bax , bad , or surviving [ 23 , 8082 ] . west et al . demonstrated inhibition of apoptosis and promotion of proliferation in human bronchial epithelium cells by nnk are induced via activation of 3/4 nachr followed by upregulation of akt , mitogen - activated protein kinase ( mapk ) , and pkc pathways . similar results are also observed by xu and coworkers showing that both akt and survivin pathways are involved in anticisplatin - induced apoptosis by nicotine . indeed , drug - induced enhancements of p53 and p21 expressions are shown to be suppressed by nicotine . our recent study also indicated that long - term nicotine treatment activated 4/2 nachr and -ar leading to reduction of apoptosis induced by cisplatin or paclitaxol . consistently , zhao et al . also reported that nicotine induced up - regulation of mcl-1 phosphorylation though erk1/2 via -ar activation with increased chemoresistance ( anti - apoptosis ) of human lung cancer cells . other investigators also indicate that nnk can prevent cell apoptosis by modulating the anti - apoptotic bcl-2 and c - myc proteins . it is found to be associated with cellular proliferation and is elevated during the developments of certain malignant tumors such as gastric and thyroid cancers [ 1113 ] . comparing the ho-1 in lung tissues of smokers and nonsmokers , li et al . noticed that the expression of ho-1 is significantly increased in both tumor and nontumor tissues of smokers . these studies further revealed that nnk or its metabolites probably induce oxidative stress in lung tissues with elevation on stimulates the expression of ho-1 . such event is through erk and nf-b activation and bad phosphorylation induction leading to eventual apoptosis inhibition [ 11 , 85 ] . cell proliferation and apoptosis can also be modulated by the peroxisome proliferator - activated receptors ( ppars ) . ppar/ is expressed in most tissues and has been reported to be associated with cancer growths , especially those in liver , colon , breast and lungs [ 8789 ] . sun et al . reported a novel mechanism that nicotine increases ppar/ expression through 7 nachr follow by pi3k / mtor activation leading to enhanced lung tumor cells proliferation . in contrast to ppar/ , activation of ppar by its ligands induces apoptosis and inhibits cell proliferation . thus , an intact ppar levels or its activation is needed to reduce cancer risk ( anti - apoptosis and cell proliferation ) . furthermore , a significant reduction in the transcriptional activity of ppar and its endogenous ligands , including 15-s - hydroxyeicosatertraenoic acid ( 15(s)-hete ) and 3-s - hydroxyocatadecadienoic acid ( 13(s)-hode ) , are found reduced in lung tissues of nnk - treated mice . indeed , lung tumors developed in these mice later . further suggested that the reduction of 15(s)-hete and 13(s)-hode may enable lung cells to be more resistant to apoptosis by nnk and facilitate tumor development in the animals . in contrast to nicotine or nnk , pahs induce either apoptosis or antiapoptosis in mammalian cells [ 94 , 95 ] . for instance , bap is known to induce signaling through igfr and increases cell survival through pi3k activation in human mammary epithelial cells . reported that both akt and erk1/2 act as anti - apoptosis signals leading to bad phosphorylation . however , bap can also induce apoptosis through p53 and p21 signaling in the same model . the results suggest that bap is capable in stimulating both apoptosis and anti - apoptosis signals . reported that light - irradiated bap ( lbap ) inhibited apoptosis through production of ros from degraded bap . this anti - apoptotic signal induced by bap in combination with dna damage would increase the possibility of cell survival and producing mutations . thus , while the apoptotic signal of bap induces cell death ( cytotoxicity ) , the anti - apoptotic signals of bap play an important role in cell proliferation and carcinogenesis . the anti - apoptosis mechanisms induced by components of cigarette smoke are obviously quite complex . it is evident that evading apoptosis plays a critical role in cigarette smoke - induced tumorigenesis and chemoresistance . new understandings on the molecular target regulating the apoptotic and anti - apoptosis machineries by cigarette smoke could provide novel strategies for drug development with substantial therapeutic benefits . when a cell population has progressed through a certain number of doublings ( replications ) , they would normally stop growing and enter into a process called senescence . tumor cells , however , appeared to have limitless replicative potentials ( immortalization ) during tumor progression . telomeres , which define the end segments of chromosomes , consist of short , tandemly repeated dna sequences ( ttaggg)n together with associated proteins . small amount of these end dna sequences may be lost during each cell cycle as a result of incomplete dna replication . however , de novo additions of ttaggg repeats by the enzyme telomerase may compensate for this loss . thus , telomerase plays an important role in the maintenance of the telomere ends in normal cells . ectopic expression of telomerase would immortalize the cells . by using human tissue samples , yim et al . reported that there are different distributions of the telomerase activity between smokers or ever - smokers and non - smoker . a strong correlation between telomerase activity and the number of packs years smoked can be established among these subjects indicating that there is an association between tobacco exposure and telomerase activity in the human bronchial epithelium . increased telomerase activity would extend the lifespan of cells and put these cells to be at higher risks for malignant transformation and carcinogenesis . similar finding is reported by targowski et al . that extensiveness of tobacco smoking correlated positively with increases in telomerase activity in tumor cells from patients with non small cell carcinoma of the lungs . all these studies point to the fact that enhancement of the telomerase activity by cigarette smoke certainly underlies the cancer promotion potentials of cigarette smoke . however , which components in cigarette smoke altered telomerase activity are still not known . indeed , there are indications that certain protein synthesis and mitochondria play central roles in neoplastic transformation . it is well known that mtor and map kinase signaling pathways modulate the phosphorylation of transcriptional factors , stability of mrnas , and protein synthesis . jin et al . reported that both nicotine and its metabolite nnk can induce survivin mrna expression through akt - mtor and mediated de novo synthesis of survivin protein in normal lung epithelial cell hbe cells . this induced survivin expression has been claimed to play a role in the malignant transformation of hbe cells by stimulating the survival pathways . cigarette smoke may damage respiratory chain function in mitochondria enhancing oxidative stress leading to mitochondria dysfunction [ 103 , 104 ] . it has also been reported that nicotine exposure resulted in reduced pancreatic mitochondrial enzyme activity , degranulation of beta cells , elevated islet oxidative stress , and impaired glucose stimulated insulin secretion in rats . continued exposure to ros and free radicals from such mitochondrial stress may lead to mitochondria dna ( mtdna ) mutation which may play an important role in carcinogenesis . analyzing clinical samples , . also showed that tumor cells with mtdna mutations grow faster then cells without mitochondrial mutation . hence , it is apparent that cigarette smoke would induce oxidative damage to the mtdna leading to more aggressive tumor growths . step 3 ( cancer progression)the malignancy of a tumor is usually evaluated by its ability in invasion and metastasis as well as in the associated angiogenesis . there are ample evidence which indicate that cigarette smoke participates in the processes of angiogenesis , invasion , and tumor metastasis . the malignancy of a tumor is usually evaluated by its ability in invasion and metastasis as well as in the associated angiogenesis . there are ample evidence which indicate that cigarette smoke participates in the processes of angiogenesis , invasion , and tumor metastasis . angiogenesis , the development of new blood vessels from endothelial cells ( ecs ) , is a critical event which allows the cancer cells to receive adequate nutrients and oxygen . angiogenesis involves mature vascular changes , including detachment of pericytes , degradation of extracellular matrix , endothelial cells remodeling , proliferation , migration , and formation of new endothelial cells into tubular structures . survival and proliferation of vascular endothelial cells are often stimulated by tumor - derived mitogens , and vice versa . tumor cells are known to activate angiogenesis by changing the balance of angiogenic inducers such as vegf ( vascular endothelial growth factor ) and bfgf ( basic fibroblast growth factor ) , and by countervailing inhibitors such as thrombospondin-1 . it has been shown that nicotine can induce angiogenesis both in vitro and in vivo and contributes to the growth of tumors [ 30 , 110 ] . similar to the fgf , nicotine is found to have the ability to promote migration , proliferation , tube formation and nitric oxide ( no ) production of endothelial cells . no is a well - known vasodilator and angiogenesis mediator , and nicotine has been reported to enhance the expression of endothelial nitric oxide synthetase and promote no release . nicotine is also found to induce expression of endothelial growth factors such as vegf , bfgf , pdgf , tgf- , and tgf- in endothelial cells and smooth muscle cells [ 112 , 113 ] . enhanced bfgf release and increases in metalloproteinase expression with degradation of ecm have been demonstrated with nicotine [ 114 , 115 ] . moreover , nicotine is found to induce secretion of prostacyclin which is a vasodilating molecule associated with endothelial cell proliferation , survival and migration . these effects are believed to be associated with cigarette smoke - induced hyperplasia of the intima in the blood vessels and other vascular wall lesions . 7 nachr is important in both physiological and pathological angiogenesis [ 110 , 118 ] . 7 nachr in endothelial cells needs to be sensitized or activated by hypoxia or ischemia in order to induce angiogenesis . indeed , specific antagonist of the 7 nachr ( -bungaratoxin ) is shown to inhibit nicotine - induced angiogenesis ( new vascular tube formation from endothelial cells ) [ 25 , 114 ] . interestingly enough , it is apparent that the akt pathway is found to be not involved in either angiogenesis or vegf release induced by nicotine . suggested that inhibition of erk1/2 , p38 mapk , and pi3k / akt can completely block and prevent endothelial tubule formation induced by nicotine - triggered 7 nachr activation . consistent with heeschen 's study , zhang and coworkers reported that nicotine apparently increases angiogenesis and invasion by activating pkc , pi3k / akt , erk1/2 , mtor , and src in human nsclc . excellent reviews on angiogenesis induced by nicotine were recently published [ 120 , 121 ] and will not be further discussed here . interaction between nachr and the growth factor - mediated angiogenesis occurs at signaling and transcription levels . nicotine - induced expression of vegf has been shown to be regulated by egfr transactivation and via the erk1/2 pathway in smooth muscle cells . phosphorylation of the vegf receptor kdr by nicotine activates vegf and increases its activity . additionally , nicotine can also upregulate the expression of vegf receptor vegfr2 during angiogenesis in certain cancer cells . recent study further indicated that nicotine can synergistically promote the proangiogenic effect of estradiol in nonsmall lung cancer . induction of angiogenesis in colon cancer by nicotine via -ar followed by arachidonic acid pathway has also been reported [ 32 , 125 ] . in sum , 7 nachr subtype has been linked to angiogenic process induced by nicotine leading to tumor vascularity , inflammation , and ischemia . nevertheless , whether nicotine or nnk acts specifically via nachr or -ar receptors or both or whether it is controlled in a cell - specific manner needs further study . other components present in cigarette smoke that may also contribute to angiogenesis remain to be identified . several excellent reviews on the roles of nicotine and nachr in angiogenesis exist [ 117 , 120 , 121 , 126 ] . the ability of invasion and metastasis allows cancer cells to escape from the primary tumor mass to new terrains in the body . the genetic and biochemical determinants as well as the molecular mechanisms involved are still poorly understood . many evidence indicate that cigarette smoking not only increases proliferation of cancer cells but also promotes metastasis . clinical and epidemiological studies suggest that smokers have more rapidly progressing tumors and cancer metastasis than non - smokers . these processes are now known to be dependent on cellular and stromal interactions and on extracellular matrix degradation . e - cadherin is a cell - to - cell interaction molecule expressed on epithelial cells . a loss of e - cadherin is seen in epithelial to mesenchymal transition ( emt ) , which is a major pathologic event in cancer metastasis . chronic treatment of nicotine downregulated the expression of ecm proteins such as e - cadherin and -catenin with concomitant increases of fibronectin and vimentin in lung cancer cells . wei et al . also indicated that nnk enhanced colon cancer cell migration with downregulation of e - cadherin . this author also found that the expressions of snail and zeb1 , 2 major transcription repressors of e - cadherin , were also induced by nnk in colon cancer cell cultures . its upregulation is significantly linked with tumor progression , metastasis and poor prognosis in lung cancer patients . it has been shown that nnk can upregulate contactin-1 via 7 nachr / erk activation and enhances invasiveness of lung cancer cells . breakdown of the extracellular matrix ( ecm ) through a family of enzyme called matrix metalloproteinases ( mmps ) is needed for tumor cells to invade adjacent tissue and to metastasize . reported that nicotine enhanced the invasiveness of esophageal squamous carcinoma cells ( te-13 ) by up - regulating the expressions and activity of mmp-2 , and cox-2 . nicotine is found to enhance the activity of mmp-2 , and mmp-9 as well as activation of plasminogen activators in a cox-2 and vegf - dependent manner . it serves as a good marker for pdac ( pancreatic ductal adenocarcinoma ) metastasis especially in cigarette smoking population . in a recent investigation , lazar et al . demonstrated that nicotine contributes to pdac metastasis through the induction of mmp-9 and vegf mediated by opn . pahs , including bap , are also found to play a role in the promotion of cancer metastasis . through augmented cox-2 expression and pge2 production via activated ahr pathway , bap induces breast cancer cell invasions . bap and pahs mixture has also been demonstrated to induce cancer cell invasions and metastasis through upregulating the expressions of mmps , proteinase - activated receptor-2 , fibronectin , migration stimulating factor , and bcl-2 protein in lung adenocarcinoma . the importance of fgf-9 and its up - regulation by bap in lung cancer invasion and metastasis has been proposed . indeed , recent study by ueng et al . demonstrated that bap increases the invasive potential of lung cancer cells in vitro . such process involves the up - regulation of fgf-9 mrna expression via the p38 and erk1/2 pathways . during metastasis , the cancer cells co - opt signals that control leukocyte trafficking and chemokines - mediated cell migration . among these chemokines , cxcr4 and its natural ligand cxcl12 serve as key mediators for tumor migration and metastasis . nicotine has been shown to increase the expressions of several cxc chemokines receptors such as cxcr2 , cxcr3 , and cxcr4 as well as ccl12 in sclc cells suggesting the nicotine would stimulate cancer cell migration and eventual metastasis . although epidemiology studies have long demonstrated the relationship between smoking and cancer metastasis , the molecular mechanisms of metastasis influenced by cigarette smoke or its components remain very limited . in this paper , we have reviewed the recent investigations concerning cigarette smoke and cancer development , promotion and progression . while chemicals with carcinogenic potentials in cigarette smoke are many ( over 62 ) , most research efforts have been devoted to three components of cigarette smoke : nicotine , nnk , and pahs . while pahs are common chemicals in the environment , nicotine and nnk are considered to be tobacco specific . these three important components of cigarette smoke , especially nicotine and nnk , therefore , are targeted as the major compounds of focus in this review . many previous reviews have devoted to the interrelationship between cigarette smoke and lung carcinogenesis or the genotoxicity of cigarette smoke or its components . in this review , we are focused on the mechanistic information on tumorigenesis , especially those involving epigenetic or non - gentoxic effects . aside from lung cancer , it is our hope that this review will summarize the vast information cumulated in the literature and provide valuable reference resource for those who are interested in tobacco - related carcinogenesis . the overall mechanisms on carcinogenesis cancer promotion and progression are complex involving many molecular targets which include receptors , cell cycle regulators , mitogen - activated protein kinases , apoptosis mediators , angiogenic factors , and invasion , and metastasis mediators . among the receptors , nachr , -ar , and ahr probably are the most important and have the closest association with cigarette smoke - induced carcinogenesis . overexpression or activation of these receptors may result in the release of neurotransmitters and growth factors that participate in apoptosis inhibition , cell proliferation , angiogenesis , cancer cell invasion and metastasis . it should be noted that the importance of nachr in cancer may be cell - type - dependent or specific and their sensitivity and expression can be also be modified by various environmental factors such as insecticide organophosphates . as shown in figure 1 , signaling pathways , pi3k / akt , stat3 , and erk1/2 play important roles in the carcinogenesis processes . they are also common paths affected by the cigarette smoke components , including nicotine , nnk , and pahs . in addition , pkc , akt , erk , and cox-2 signaling pathways are involved in both promotion and progression stages by cigarette smoke . it is suggested that these molecules could be utilized the potential targets for future developments in cancer diagnoses or therapies . avoidance of cigarette smoke remains to be the best way of prevention for cigarette - related cancer . however , in view that tobacco smoke is legalized and smokers are still abundant , understanding on the health impacts by tobacco smoking still constitutes important public health concern . understanding the disease process and the mechanisms involved is the first step to solution . the emerging understanding on the molecular mechanisms in the development and progression of caners induced by cigarette smoke provides novel inspirations and approaches for potential measures on early diagnosis , reduction in progression and metastasis , and therapy of cancers . many dietary supplements , foods , or herbal medicines might significantly attenuate the proliferative effects by cigarette smoke . , the natural compound pterostilbene could induce apoptosis and autophagy in chemoresistant bladder cancer cells derived from nicotine exposure . future research on natural compounds may help to provide additional novel chemopreventive or chemotherapeutic possibilities in reducing cancer risks or other health impacts of cigarette smoke . this review has also discussed the various molecular mechanisms and paths involved in carcinogenesis induced by cigarette smoke . however , there are still many mysteries in the carcinogenic process by cigarette smoke . , most research efforts were focused on the proliferative and antiapoptosis mechanisms induced by cigarette smoking . as tumors are the results of multiple and interactive genetic abnormalities , study of cancers induced by cigarette smoke should assess more than one or two acquired alterations or paths . explorations of other paths or mechanism other than those popular ones are needed . those molecular pathways which are significantly activated by cigarette smoke are probably the most important ones involved in cigarette smoke - induced tumorigenesis . these pathways include nachr signaling ( such as 7 nachr , 9 nachr , or 4/2 nachr ) , -ar signaling , pi3k / akt signaling , erk1/2 signaling , stat3 signaling , vegf , and mmps pathways , and so on . targeting to modulate these pathways via dietary factors or therapeutic drugs may reduce cigarette smoking induced tumorigenesis significantly . studies on the non - genotoxic ( epigenetic ) effects of cigarette smoke components are few and need more efforts . the epigenetic effects of cigarette component must be evaluated to include both upstream and downstream pathways . carcinogenesis is often species or cell - type specific and can be influenced by many factors or cofactors . the same factor which is highly oncogenic to certain cell type or individuals may not be oncogenic to others . moreover , some cell type may become susceptible to a carcinogen only in the presence of certain factor(s ) , co - factor(s ) , genetic predisposition , or immune depression . specific mechanism for carcinogenesis for the same carcinogen may also vary in different tissues . synergistic interaction between cigarette smoke components and other environmental toxicants or carcinogens , such as arsenic or dioxin , on cancer development has been demonstrated both epidemiologically and in animal studies [ 143145 ] . traditionally , most past investigations focused only on single compound or one cigarette smoke component . the synergistic interaction between otherwise safe level of environmental chemical and low level of cigarette smoke or its component ( via either active or secondhand smoking ) for carcinogenesis raised novel public health concerns and challenging questions . we have provided an overview on the major concepts and insights on the molecular mechanisms involved in cigarette smoke - induced cancers . it is hoped that these mechanistic insights can be translated into practical applications for the prevention and treatment of cigarette smoke - related cancers . we also hope that these suggestions will be helpful to those who are interested in this area of cancer research .

cigarette smoking is one of the major causes of carcinogenesis . direct genotoxicity induced by cigarette smoke leads to initiation of carcinogenesis . nongenotoxic ( epigenetic ) effects of cigarette smoke also act as modulators altering cellular functions . these two effects underlie the mechanisms of tumor promotion and progression . while there is no lack of general reviews on the genotoxic and carcinogenic potentials of cigarette smoke in lung carcinogenesis , updated review on the epigenetic effects and molecular mechanisms of cigarette smoke and carcinogenesis , not limited to lung , is lacking . we are presenting a comprehensive review of recent investigations on cigarette smoke , with special attentions to nicotine , nnk , and pahs . the current understanding on their molecular mechanisms include ( 1 ) receptors , ( 2 ) cell cycle regulators , ( 3 ) signaling pathways , ( 4 ) apoptosis mediators , ( 5 ) angiogenic factors , and ( 6 ) invasive and metastasis mediators . this review highlighted the complexity biological responses to cigarette smoke components and their involvements in tumorigenesis .

many benign tumors can affect the orbit and , if symptomatic or cosmetically disfiguring , most can be removed via various cutaneous and bony approaches . many of these approaches involve removal of the lateral orbital wall , with replacement after tumor excision [ 17 ] . this approach can produce excellent operative exposure of the lesion and allow for removal with minimal manipulation of the orbital contents . with interest in minimizing surgical risk , postoperative recovery and scarring , recent surgical trends favor minimally invasive techniques and local anesthesia where possible [ 1 , 2 ] . traditionally , orbital tumors located in the middle to posterior orbit are approached from a variety of soft - tissue incisions incorporating a bony lateral orbitotomy ( marginotomy ) of the zygoma [ 17 ] . the bony flap increases exposure of the deep orbit and provides the surgeon with added maneuverability for tumor removal [ 6 , 7 ] . however , the bony marginotomy can be associated with longer operative time , increased postoperative pain , recovery time , temporalis muscle wasting , and external scar . using modern imaging modalities in conjunction with minimal incision surgical techniques , we have found that bony marginotomy is rarely needed in order to access presumed benign tumors of the middle and deep orbit . in this study , we present our surgical experience in the removal of orbital tumors using a combination of soft - tissue approaches , without bony marginotomy , under monitored local anesthesia and general anesthesia . in this retrospective case series , electronic medical records of all patients with orbital tumors removed through the oculoplastics clinic of the jules stein eye institute between 1992 and 2013 were reviewed . the institutional review board at the university of california , los angeles approved the study protocol , and the tenets of the declaration of helsinki were followed . data including snellen visual acuity , ocular motility , tumor size and type , follow - up duration , recurrence , and intra- and postoperative complications were recorded . imaging was used to classify tumor location as posterior or anterior based on confinement to the anterior half of the orbit or extension into the posterior half , respectively . surgeries were performed under general anesthesia . however , if the case was amenable to completion under local anesthesia , an array of orbital and regional nerve blocks was utilized . after adequate anesthesia is achieved , the appropriate incision is chosen based on location of the tumor within the orbit . tumors inferior to the optic nerve are generally approached with a conjunctival incision 4 mm below the inferior tarsal margin through the lower eyelid retractors and into orbital fat ( figures 1 and 2 ) . the incision can be extended medially or laterally as necessary . when extending the incision laterally , the lateral canthal tendon can be loosened slightly by blunt spreading with scissors , avoiding complete release from the orbital rim . for tumors located superiorly , an incision is made through the upper eyelid crease 810 mm above the lid margin . the incisions were customized medially or laterally based on the location of tumor and the site needs to be exposed . an incision through the septum with medial or lateral displacement of the levator fibers allows blunt dissection through the intermuscular septum . the levator can be safely distracted medially or laterally and we have not found a vertical lid splitting procedure necessary to access the superior orbit . working towards the anticipated 3-dimensional location of the tumor predicted by imaging studies , stevens scissors and peanut or cotton tipped applicators can be used to dissect fatty soft tissue off the face of the tumor . once the face of the tumor is within view , the surgeon can assess the gross surgical pathology and confirm the diagnosis of a benign lesion . a half - circle needle can now be passed through the face of the tumor if appropriate , which if compressible will cause partial exsanguination and a decrease in tumor size . an additional pass of the needle is utilized to create a whip suture that will provide better control and forward traction . in the case of cavernous malformations , this stitch also aids in exsanguination of the lesion , decreasing its overall size for delivery anteriorly . some tumors such as dermoid cysts or schwannomas can be additionally decompressed by suctioning the contents through a small anterior incision into the tumor . with the tumor partly exsanguinated or decompressed ( if possible ) and suture in place , blunt dissection ( e.g. , with a freer elevator ) can now be carried back toward the posterior edge of the tumor with simultaneous forward traction . as the attachments are dissected , the tumor will move towards the surgical opening , revealing posterior attachments , which can be gently lysed with blunt and sharp dissection ( figure 3 ) . there are usually no major vascular stalks associated with cavernous hemangiomas , but any bleeding vessels are controlled with bipolar cautery ( figure 4 ) . small conjunctival or eyelid crease incisions allow a relatively quick recovery with minimal ecchymosis and minimal visible scar ( figure 5 ) . median age was 48.5 ( range 3187 ) years old and median follow - up was 24.5 ( range 4375 ) weeks ( table 1 ) . the average standard deviation tumor size , measured in its longest dimension based on preoperative imaging , was 22.6 6.5 mm ( range 1051 ) . twenty - nine ( out of 30 ) tumors were located in the posterior half of the orbit . surgeries were performed through eyelid crease ( 17 eyes ) , conjunctival ( 9 eyes ) , lateral canthal ( 2 eyes ) , and transcaruncular ( 2eyes ) approaches . six cases were performed with monitored anesthesia care and local block , and 24 were performed under general anesthesia . two patients ( # 16&29 ) had mild decrease in visual acuity at the last follow - up , which was related to ocular surface changes . confirmed surgical pathology revealed several tumor types including 15 cavernous hemangiomas , 5 pleomorphic adenomas , 4 solitary fibrous tumors , 2 neurofibro- mas , 2 schwannomas , and 1 orbital varix . middle to deep orbital tumors are most commonly removed through a bony lateral marginotomy [ 17 ] . this technique was first described by kronlein in 1889 for the removal of dermoid cysts , providing a relatively wide field of view to search for retrobulbar tumors . with the advent of modern imaging modalities and ability to distinguish tumor character and location preoperatively , other less invasive soft - tissue approaches have been described . all documented series have reported surgical excision under general anesthesia and typically involve a lateral canthotomy or rectus resection for exposure . both kiratli et al . and cheng et al . presented series of intraconal orbital cavernous hemangiomas removed via transconjunctival approach [ 10 , 11 ] . their series were limited to tumors touching or near the globe and necessitated removal of the medial rectus for larger tumors . another 2004 paper by yan and wu presented results of removing 139 ( out of 214 ) orbital cavernomas through an anterior orbitotomy . the remaining 75 tumors ( out of 214 ) were removed successfully by standard lateral orbitotomy with bony marginotomy . the authors stated that traditional lateral orbitotomy must be used in cases where tumor dimensions exceed 3 cm , or the tumor extends to the orbital apex , or has imaging inconsistent with cavernous hemangioma . in another series limited to cavernous hemangiomas , pelton and patel excised 5 medial intraconal cavernomas through a superomedial eyelid crease . the authors concluded that the superomedial lid crease approach to the medial intraconal space has a number of advantages over the medial transconjunctival and lateral orbital approaches , including ease of dissection , incision - to - nerve distance , and angle of approach to the optic nerve . with appropriate screening and intraoperative flexibility , we believe the most benign intraconal tumors can be removed through one of many soft - tissue approaches , often under local anesthesia . if the lesion is compressible , for example , cavernous hemangioma or dermoid cyst , large size is not a contraindication to a small incision approach . . the anticipated pathology should be consistent with a benign tumor . additionally ct or mri imaging modalities should demonstrate a well - defined mass , without tethering , infiltration into surrounding tissue or growth into bone or the sinuses . these features suggest that the tumor is likely benign in nature and distinct from the surrounding tissues making it possible to be completely resected . tumors located in the inferior orbit are best approached through an inferior conjunctival incision . most often a canthotomy is not required , but for more exposure , the canthus can be loosened with a small internal incision , leaving the skin and orbicularis intact . occasionally a deep medial orbital tumor adjacent to the periosteum may be more easily accessed using a caruncular incision . superiorly positioned orbital tumors are removed through an upper eyelid crease incision with appropriate medial or lateral orbital entry ( working around the levator aponeurosis ) . most benign tumors of the deep orbit can be safely reached through careful dissection with any of these incisional approaches , although tumors at the deepest apex , for example , those extending through the superior orbital fissure , generally require bone removal for optimal exposure . this is achieved through careful examination of fine cut orbital ct or mri images preoperatively in combination with adequate lighting and the use of intraoperative landmarks such as the bony rim , globe , and rectus muscles . using this spatial knowledge along with palpation and ballottement of the tumor during dissection allows the surgeon to accurately focus the blunt and sharp dissection through the relatively small opening . once the dissection has reached the face of the tumor , the gross pathologic appearance should be assessed . if visual inspection and palpation suggest an infiltrative process , the surgeon must be flexible enough to change the operative plan , for example , to biopsy the lesion or convert to a larger access procedure under general anesthesia . none of our cases necessitated conversion from monitored anesthesia care to general anesthesia or bone removal . the next critical point is the pass of the half - circle needle through the face of the tumor . in the case of a cavernous venous malformation ( cavernous hemangioma ) , the resulting exsanguination of the tumor , which can be easily suctioned , shrinks the tumor in size and allows more space to maneuver . additional passes of the needle create a whip suture , allowing forward traction for adequate dissection and expression of the tumor through the small soft - tissue incision . local anesthesia is preferred by many patients as it shortens the operative time , avoids the discomfort and risks of general anesthesia , and allows the patient to return home sooner . we have found that benign orbital tumors can be removed under monitored local anesthesia in some patients . patients who are overly anxious will do better under general anesthesia although moderate anxiety will respond to common anxiolytic agents used for monitored anesthesia [ 6 , 7 ] . in our series , those who underwent monitored anesthesia did well with common anxiolytic agents . intraoperatively , the orbit should become adequately anesthetized superficially and deep with blocks of the zygomaticotemporal , zygomaticofacial , infraorbital , and supraorbital nerves as well as along the orbital floor back to the level of the superior orbital fissure . it is important to maintain communication with the patient and provide more local anesthetic when necessary . more local anesthetic is often necessary in the central and medial orbit when dissecting down to the orbital rim , as there are many sensory nerves in this region . the retrobulbar block may dampen or eliminate the physiologic pupillary reaction of the ipsilateral eye . this should not affect operative decision - making : the surgeon always uses maximal care to minimize traction and pressure on nerves in the orbit . pupillary dilation is more often related to the efferent nerves to the pupil and is not a reliable sign of optic nerve compression . optic nerve function can be better assessed , if needed , by looking for a reverse afferent papillary defect in the opposite pupil . this includes a detailed history , physical exam , and careful study of appropriate imaging modalities . combining this data with a thoughtful orbital differential diagnosis , appreciation for nuances of intraoperative gross surgical pathology , and readiness to convert to an open procedure if necessary allows the surgeon to safely approach most orbital tumors with the techniques described above . the desire to perform minimally invasive surgery should not be pursued to the extent that adequate exposure or patient safety is compromised in any way . however , within the constraints of good judgment and safety , it is appropriate to try to minimize the invasiveness of orbital surgery . in our experience , with appropriate preoperative evaluation and a creative flexible surgical approach , many benign orbital tumors can be safely approached through a minimally invasive soft - tissue approach , avoiding a bony marginotomy .

to present our experience of removing middle to deep orbital tumors using a combination of minimally invasive soft tissue approaches , sometimes under local anesthesia . methods . in this retrospective case series , 30 patients ( 13 males and 17 females ) underwent tumor removal through eyelid crease ( 17 eyes ) , conjunctival ( nine eyes ) , lateral canthal ( two eyes ) , and transcaruncular ( two eyes ) approaches . all tumors were located in the posterior half of the orbit . six cases were removed under monitored anesthesia care with local block , and 24 were under general anesthesia . results . the median ( range ) age and follow - up duration were 48.5 ( 3187 ) years old and 24.5 ( 4375 ) weeks , respectively . visual acuity and ocular motility showed improvement or no significant change in all but one patient at the latest followup . confirmed pathologies revealed cavernous hemangioma ( 15 cases ) , pleomorphic adenoma ( 5 cases ) , solitary fibrous tumor ( 4 cases ) , neurofibroma ( 2 cases ) , schwannoma ( 2 cases ) , and orbital varix ( 1 case ) . none of the patients experienced recurrence . conclusions . creating a bony marginotomy increases intraoperative exposure of the deep orbit but adds substantial time and morbidity . benign orbital tumors can often be removed safely through small soft - tissue incisions , without bone removal and under local anesthesia .

science facilities projects require the collaboration of many different parties , and an effective coordination between these individuals leads to successful outcomes . architects with experience in designing laboratories bring expertise and creativity as they provide their clients with a variety of issues and examples to consider as decisions are made about optimal space configurations . the success of the project is dependent upon the effective teamwork between the client , the architects , and the construction management staff . in the design phase of the process , there is a recursive aspect to the exercise , as concepts get refined and translated to actual construction documents . although the campus planning , physical plant and administration ( e.g. , dean of the faculty or financial affairs ) staff are critical representatives of the institution , it is also important to have faculty perspectives heard as they pertain to the evolution of the program for the building . as described below , a faculty shepherd is a desirable addition to the team . this person represents the faculty and is charged with maintaining the integrity of the program as the design and construction go forward . the opportunity to provide input into the development of new spaces comes with the responsibility to provide the lab designers with as much information as possible . descriptions of how teaching and research are accomplished , expectations of new equipment or staffing changes , and needs and desires to have shared spaces are all important issues to raise . it is important to balance the vision for new spaces with a flexibility to seriously consider suggestions from the architects , who have the benefit of knowing designs that have worked well at other institutions . for any project , one of the first considerations is how the objectives fit into the campus curricular and strategic plans . at some campuses , there might be a master plan indicating how renovations or expansions of science facilities contribute to the strategic objectives of the institution . in some cases , specific curricular programs are considered important to develop as focal points for the science offerings . in others , increases in science enrollments have occurred or buildings that were effective years ago no longer support modern science , both in terms of the increased demand on technology or laboratories that need to be renovated for safety concerns . one common driving force for renovation of science facilities is that science education has changed over the decades . one of the main changes at many institutions is the incorporation of student research as a focal point in the way in which students learn science , either in laboratory courses or in conducting independent studies . in other cases , the changes in teaching style might not match the spaces that were appropriate for teaching in a different era . for example , in some disciplines , there is less division between laboratory and lecture ; new spaces are constructed to allow for a seamless transition to both types of teaching within the same spaces . extensive faculty discussions are critical in developing an optimal set of perspectives regarding the needs and the priorities of the spaces to be developed . a self - study serves the purpose of identifying where the opportunities and needs lie . the discussions of how to build on strengths and how to deal with weaknesses have the potential to alter the educational environment for years to come . if possible , faculty discussions across departments are helpful , particularly when incorporating the opinions of younger faculty who will benefit the most from the new spaces . benchmarking data can be helpful to the administration and trustees as they decide on the magnitude of a project . in the case where there is a possible these data are particularly relevant to faculty when there are multiple departments involved in the project . departments have different needs , and comparative data provide the information to enable appropriate allocation . once the appropriate determinations of total space have been made , departments might decide to partition the space in different ways . one way to accomplish this successfully is to have a faculty member serve as the shepherd of the project . ideally , this person would be involved in all meetings with the administration , architects , and construction professionals to represent the faculty and to be responsible for ensuring that the academic program is maintained as the many decisions occur throughout the duration of the project . as a central figure , this person serves to keep all parties informed of the progress and issues that arise . faculty shepherds become so familiar with the different faculty and departmental needs that they can be critical in keeping important program considerations at the forefront . they provide an effective way for architects to communicate with the faculty and staff , and they assist the deans in dealing with committees and meetings that would otherwise be organized by an administrator from the dean s office or physical plant . particularly in large projects , the constituencies that need to be involved thus , several committees are helpful to have in place , each with a specific charge . for example , a general building committee would bring together a larger number of people to review progress of the design , but a health and safety committee might be responsible for reviewing and revising the policies regarding storage of and access to chemicals . in our project at hamilton , one committee consisted of representatives of the departments or areas included in the project , and this group played a fundamental role in coordinating the deliberations within the different departments regarding their teaching and research spaces . other items require the input from other members of the campus community who will have a significant impact on the operation of the building , including those in the maintenance , registrar , information technology , and food service areas . if there are classrooms that will serve other departments , then conversations should take place with faculty from non - science departments . it is desirable to include students on many of these committees to provide their perspective on the important and desirable features of new spaces . many faculty members are so heavily involved in their own work that they have not had the opportunity to examine the best practices and spaces at other institutions . it is well worth the investment of time and money for them to see the most interesting projects that have been constructed . when the faculty members visit these buildings , they become aware of different approaches and come up with creative ideas of their own . although each project has its own unique features , some general characteristics can be seen in many modern science buildings . one of the trends at small colleges has been to bring several disciplines ( and sometimes all of the sciences ) under one roof . this serves interdisciplinary programs particularly well , but also recognizes the fact that the traditional disciplines have blurred boundaries between each other . these arrangements benefit neuroscience programs in particular , since those students typically take courses in biology , chemistry , and psychology , and the faculty and student research sometimes requires access to equipment that is housed in different departments . modern undergraduate science education is exciting to watch , with students engaging in laboratory work , collaborative activities , and oral presentations with attractive slides ( see figures 1 and 2 ) . buildings are energized when the activity in the classrooms and laboratories is visible to visitors passing through the building . the presence of glass in doors and walls also provides an element of safety by allowing students to be seen from hallways . of course , there are times when visibility is counterproductive to a particular laboratory experiment or demonstration . at hamilton , a few faculty members have covered the glass windows to their laboratories with shades . in the behavioral neuroscience testing rooms , when we are conducting experiments that require privacy , these panels are easily inserted for the times when testing is taking place . at hamilton college , offices are located near laboratories to let faculty members always be in close proximity to the student research . the teaching laboratories are also in the same areas , which makes those spaces available for use for research during summer months when they are not being used for classes . when possible , shared prep rooms and support spaces allow for efficient shared access to equipment and chemical storage . over time , faculty members will change , curricular programs will evolve , new areas of inquiry will develop , and student course selections will differ . over many years , the distribution of faculty might change from discipline to discipline . as part of the design process , it is useful to ask how the configuration of spaces would accommodate an additional faculty member or a faculty member with a different specialty area . one objective might be to include expansion spaces that are shared and thus not designated for any particular department . as needs change and some programs become smaller and other new ones are formed , these spaces can serve different departments at different times . flexibility can also be incorporated into the way individual spaces are arranged . for example , for wet lab bench work , it might be useful to have a shared student - faculty research lab that serves more than one faculty member . in this way , during a semester when one faculty member needs more space and another one less , their use of the area in the laboratory can expand and retract accordingly ( see figure 3 ) . for other situations , all other things being equal , a larger number of small spaces might be more helpful than fewer spaces that are larger in size . some neuroscience teaching laboratories have incorporated both laboratory and seminar areas in the same space , and others have had adjacent rooms configured for wet lab and seminar or computer work , thus making it easy to have students engage in both types of activities in the same class period ( see figures 4 and 5 ) . if built - in benches are not required , then sturdy but moveable tables can be considered . in teaching laboratories , this can make it easy to have different configurations from semester to semester or even from week to week ( see figure 6 ) . for those classes where reconfigurations are frequent , casters might be placed on the legs of the tables . tables with built - in outlets and umbilical cords to connect to floor boxes keep sight lines free . behavioral neuroscience laboratories often require lighting , curtains , and cameras that are sometimes suspended from the laboratory ceilings . they are connected to the steel structure of the building , can be configured to provide power , and have tracks to make the position of equipment easy to adjust ( see figure 7 ) . finally , although the teaching and research areas are the focus of the attention of faculty members , there are other types of spaces that create a successful academic building . when students are asked about the spaces that are the most important for them , they place good study areas at the top of the list ( see figure 8) . similarly , a science center that has a caf and attractive classrooms is used by students and faculty from all disciplines and ensures that the building serves the entire campus . modern undergraduate science education is exciting to watch , with students engaging in laboratory work , collaborative activities , and oral presentations with attractive slides ( see figures 1 and 2 ) . buildings are energized when the activity in the classrooms and laboratories is visible to visitors passing through the building . the presence of glass in doors and walls also provides an element of safety by allowing students to be seen from hallways . of course , there are times when visibility is counterproductive to a particular laboratory experiment or demonstration . at hamilton , a few faculty members have covered the glass windows to their laboratories with shades . in the behavioral neuroscience testing rooms , when we are conducting experiments that require privacy , these panels are easily inserted for the times when testing is taking place . at hamilton college , offices are located near laboratories to let faculty members always be in close proximity to the student research . the teaching laboratories are also in the same areas , which makes those spaces available for use for research during summer months when they are not being used for classes . when possible , shared prep rooms and support spaces allow for efficient shared access to equipment and chemical storage . over time , faculty members will change , curricular programs will evolve , new areas of inquiry will develop , and student course selections will differ . over many years , the distribution of faculty might change from discipline to discipline . as part of the design process , it is useful to ask how the configuration of spaces would accommodate an additional faculty member or a faculty member with a different specialty area . one objective might be to include expansion spaces that are shared and thus not designated for any particular department . as needs change and some programs become smaller and other new ones are formed , these spaces can serve different departments at different times . for example , for wet lab bench work , it might be useful to have a shared student - faculty research lab that serves more than one faculty member . in this way , during a semester when one faculty member needs more space and another one less , their use of the area in the laboratory can expand and retract accordingly ( see figure 3 ) . for other situations , all other things being equal , a larger number of small spaces might be more helpful than fewer spaces that are larger in size . one example of this might be for spaces used for behavioral testing . some neuroscience teaching laboratories have incorporated both laboratory and seminar areas in the same space , and others have had adjacent rooms configured for wet lab and seminar or computer work , thus making it easy to have students engage in both types of activities in the same class period ( see figures 4 and 5 ) . if built - in benches are not required , then sturdy but moveable tables can be considered . in teaching laboratories , this can make it easy to have different configurations from semester to semester or even from week to week ( see figure 6 ) . for those classes where reconfigurations are frequent tables with built - in outlets and umbilical cords to connect to floor boxes keep sight lines free . behavioral neuroscience laboratories often require lighting , curtains , and cameras that are sometimes suspended from the laboratory ceilings . they are connected to the steel structure of the building , can be configured to provide power , and have tracks to make the position of equipment easy to adjust ( see figure 7 ) . finally , although the teaching and research areas are the focus of the attention of faculty members , there are other types of spaces that create a successful academic building . when students are asked about the spaces that are the most important for them , they place good study areas at the top of the list ( see figure 8) . similarly , a science center that has a caf and attractive classrooms is used by students and faculty from all disciplines and ensures that the building serves the entire campus . effectively designed science facilities produce long - term benefits , not only in teaching and research , but also in admissions and in the success of the institutional mission . although the examples and descriptions mentioned above have been in the context of large renovation and expansion projects , there are lessons that can easily be applied to small projects . it is remarkable how providing appropriate furnishings to a room can change the effectiveness of the space . regardless of the magnitude of the projects and their eventual design features , the process is critical . when all parties become involved in the discussions , then they can all contribute constructively in the difficult choices that sometimes have to be made because of budgetary limitations . the increases in the effectiveness of the spaces and in the morale of the users can be impressive .

science facility renovation and expansion projects provide opportunities for faculty members to play a role in working with architects and administrators to produce effective spaces for teaching and research . this article summarizes information regarding the process and design features that have proved beneficial for many recent science facilities projects . although the context focuses on large projects , the general principles pertain to smaller changes in spaces that individual departments might be pursuing .

paragangliomas in the mediastinum are rare tumors originated from the neuroendocrine cell . about 90% of paragangliomas exist in adrenal medulla ( commonly called pheochromocytoma ) . the other 10% of tumors are extra - adrenal ( paraganglioma ) , and 90% of these are intra - abdominal , most commonly arising from chromaffin cells near the aortic bifurcation or near the kidney . other sites include the paravertebral sympathethic ganglia , the urinary bladder , other automatic ganglia ( celiac , mesenteric ) , the thorax ( mediastinum , the heart , and paracardic regions ) and the neck ( in sympathetic ganglia , carotid body , cranial nerve or glomus jugulare ) . as far as we reviewed , there are no reports of ectopic acth syndrome with mediastinal paraganglioma in korea . we experienced a case with acth - secreting paraganglioma in the anterior mediastinum who was admitted because of cushing s syndrome . a 51-year - old - female was admitted to our hospital with 5 months duration of generalized edema and weakness . two years later , hypertension was detected and she had been taken antihypertensive medications intermittently . the patient had not any symptoms such as cough , sputum , fever and chest pain , except mild exertional dyspnea . her blood pressure was 170/100 mmhg , but other vital signs were stable . on physical examination , she was obese and had a moon face , buffalo hump and purplish abdominal striae . a 22 cm - sized hard and fixed the serum ast , alt , alp and ldh level was 50 , 140 , 131 and 1,065 u / l , respectively . the renal function test , including serum electrolytes , were within normal limits , except that potassium level was 2.5 fasting glucose was 472 mg / dl , hemoglobin a1c was 11.8% and spot urine glucose was above 1,000 mg / dl , but proteinuria was not found . chest x - ray ( figure 1 ) and chest ct scan ( figure 2 ) revealed a 1210 cm - sized well - defined and partially capsulated and lobulated homogeneous attenuated mass in the anterior mediastinum . there was no suppression of the plasma cortisol level ( over 50 g / dl ) after a high - dose dexamethasone suppression test . the plasma acth level was also elevated to 278 pg / ml , but pituitary mri scan did not reveal any evidence of abnormality , therefore ectopic acth syndrome was suspected . to find out the other focus of ectopic acth secretion , except mediastinal mass , abdominopelvic ct was performed , but there was no evidence of abnormalities , including adrenal gland , pancreas and ovary . the 24h - urine metanephrine , vanillymandelic acid ( vma ) and homovanilic acid ( hva ) levels were slightly elevated to 3.4 , 11.4 and 13.9 mg / day , respectively , which are not typical ranges of pheochromocytoma . however , epinephrine and norepinephrine levels were within normal ranges . the 24h - urine 5-hiaa was elevated to 11.3 mg / day but serum calcitonin and parathyroid hormone levels were within normal ranges . the gross appearance was a soft , nodular and well - encapsulated 13106 cm - sized mass . light microscopic sections showed a predominantly diffuse pattern of monotonous neoplastic cells arranged in organoid cell nests ( classic zellballen pattern ) that are characteristics of paraganglioma ( figure 3 ) . tumor cells were polygonal with a clear or finely granular eosinphilic cytoplasm and a round or ovoid nucleus . immunohistochemical stains revealed a diffuse and strong cytoplasmic immunoreactivity for chromogranin in about 90% of the tumor cells . immunoreactivity for s-100 protein , confirming the presence of sustentacular cells which are characteristics of paraganglioma , was detected in scattered supporting cells , mostly at the periphery of the tumor cell nests(figure 4 ) . she was confirmed as rarely occurring mediastinal paraganglioma secreting acth , cushing s syndrome and secondary diabetes . unfortunately , she died on the 22th postoperative day because of septic shock and respiratory failure due to uncontrolled mediastinitis with combined pneumonia . paragangliomas are unusual neuroendocrine cell tumors arising from paraganglia tissue which are widely dispersed groups of specialized neural crest cells . it has been known historically by a variety of names , including glomus tumor , chemodectoma , non - chromaffin paraganglioma , carotid body and tympanic body tumor , and receptoma . the frequent location of paraganglioma in the mediastinum is controversial . in a report of cesar et al . , twelve tumors were located in the posterior mediastinum , and only three were anterior . in addition , tumors arising in an anterior mediastinum are claimed to be more often associated with older age , larger size and lower incidence of functionality , and are less amenable to surgical resection . mediastinal paragangliomas usually cause no symptoms . occasionally , however , they do present with varying degrees of hypertension , diabetes and hypermetabolism . vma and hva are the chief urinary excretion products but epinephrine and norepinephrine may also be secreted in the urine . the word however , because of potentially serious consequences of a catecholamine crisis during manipulation of a functioning tumor , evaluation of patients should include screening for symptoms and signs and the measurment of appropriate blood and urine product . the tumors may produce functioning peptides that can cause cushing s syndrome like this case , secretory diarrhea and polycythemia vera . in the thorax the 24h - urine metanephrine , vma , hva and serum serotonin levels may also be incerased , although slightly in this case . paraganglioma in association with other neoplasms ( part of multiple endocrine neoplasia syndrome , men ) and reports of multicentricity have been well documented . therefore , care should be exercised in differentiating between multicentric neoplasms and metastasis from a malignant tumor . malignancy is rare and typically defined by the existence of metastasis , rather than cellular characteristics . lung and bone were the usual sites of metastasis , whereas other sites included lymph nodes , liver , spleen , heart or kidneys . because paraganglial tissue is not usually found there , its presence in any of these organs constitutes metastasis . some patients with known lung and bone metastses can live as long as 25 years after the lesions are discovered . there was no evidence of men , but supraclavicular lymph node metastasis was found in this case . large masses may be visible on the chest radiograph but , in most patients , ct scans are necessary to visualize the tumors . the technetium - methoxyisobutylisonitrile ( mibi ) scan is also helpful to detect ectopic acth - producing tumors undiagnosed with ct and mri . when the tumors are located in the anterior mediastinum , the most important differential diagnosis is with thymic carcinoid . however , the presence of bands of connective tissue , nuclear pleomorphism with bizarre forms and absence of festfoons or rosettes are histological features more in favor of paraganglioma . in addition , the positive immunohistochemical reaction for keratin in the tumor cells is a feature that has been more often documented in carcinoid tumor . paraganglioma located in the posterior mediastinum should be differentiated from metastatic tumors , such as melanoma , renal cell carcinoma and alveolar soft part sarcoma . however , the patient should first undergo alpha blockade with penoxybenzamine for 1 week and then beta blockade with metoprolol or propranolol . the blood supply of paragangioma is usually rich . when surgery is planned , preoperative embolization of the main arterial supply and the tumor bed is helpful for safe and less morbid removal of large tumors the decision to operate or irradiate should be based on a formula that considers tumor size and location , patient age and health , symptoms or signs , potential morbidity and the expertise and availability of those involved in treatment . mediastinal paraganglioma , because of their unusual anatomic site , may pose problems in diagnosis . a detailed history , hormone study and immunohistochemical studies play an important role in separating these tumors from other neoplasms .

paragangliomas are unusual neuroendocrine cell tumors arising from paraganglia , of which acth - secreting cases in the mediastinum are exremely rare . a 51-year - old woman was admitted for generalized edema and weakness which began 5 months ago . chest x - ray and ct scan revealed a tumor mass in the anterior mediastinum . the plasma cortisol and acth levels were very high . other sources secreting acth , except mediastinal mass , were not found . surgical excision of mediastinal mass and left supraclavicular lymph node was performed . the postoperative microscopic finding and immunohistochemical staining revealed organoid tumor cell nests ( zellballen ) and s-100 protein positive sustentacular cells which are characteristics of paraganglioma . this was thus a case of cushing s syndrome resulting from ectopic acth production in anterior mediastinal paraganglioma .

spider silks are renowned for their mechanical properties , but many aspects of how spiders produce these high - performance materials remain elusive . a spider s abdomen contains numerous silk glands that are differentiated into morphologically and functionally distinct types . most gland types express members of a spider - specific gene family that encodes fiber - forming structural proteins called spidroins , a contraction of spider fibroins . after synthesis , spidroins are stored in the lumen of silk glands as part of a liquid spinning dope . as needed , the dope travels through ducts leading to external spigots on the spider s spinnerets . during extrusion , the mechanical properties of fibrous spider silks are related to the amino acid sequences of their spidroin components in that primary structure largely determines secondary and higher - level protein structures . spidroins consist of iterated repeat units that vary in length and complexity depending on spidroin type . the significance of particular spidroin repeat sequences to fiber - specific mechanical properties has been demonstrated through a variety of structural studies . because of the direct relationship between spidroin amino acid sequences and the physical attributes of silk , characterization and recombinant expression of spidroins have been a main focus of spider silk research . despite extensive spidroin research , knowledge of the gene expression and protein profiles that contribute to silk synthesis is far from complete . to further explore how spiders synthesize distinct silk types the western black widow spider , latrodectus hesperus , produces six fibrous silk types from six corresponding silk gland types ( major ampullate , minor ampullate , aciniform , flagelliform , pyriform , and tubuliform ) . here , we focus on major ampullate , minor ampullate , and tubuliform silks . we chose these silk types because of the overlapping functions of major ampullate and minor ampullate silks in orb - weaving spiders , the similar shapes of the major and minor ampullate glands , and evidence that the major ampullate and tubuliform silk glands share expression of some spidroin genes . major ampullate silks emerge from major ampullate glands , which have a long tail that synthesizes proteins , an ampule - shaped sac that both synthesizes and stores proteins , and a z - shaped duct that connects to an external spigot ( figure 1a ) . minor ampullate silks are produced from glands that are morphologically very similar to major ampullate glands but that are smaller , with a shorter tail and lower capacity sac ( figure 1b ) . the combination of a long tail , ampule - shaped lumen , and z - shaped duct are unique to the major and minor ampullate silk glands . bright - field images of l. hesperus silk glands : ( a ) major ampullate gland , ( b ) minor ampullate gland , and ( c ) tubuliform gland . ( a , b ) anterior tail is at the top right , ampule - shaped lumen is to the left , and posterior z - shaped duct is at the bottom right . anterior tail is pale and to the right ; posterior duct is darker and to the left . l. hesperus major ampullate glands predominantly express the genes for two major ampullate spidroins ( masp1 and masp2 ) , and these proteins are the main components of l. hesperus dragline fibers . minor ampullate silk glands express minor ampullate spidroin ( misp ) genes , and misps are the primary proteins found in minor ampullate silks . minor ampullate silks are generally thought to be used by orb - weaving spiders as the auxiliary spiral during web construction and as support for major ampullate silks in draglines , web radii , and web scaffold . in l. hesperus , which builds a cob - web instead of an orb - web , minor ampullate silks have been found in a composite of silk types used in prey wrapping . cob - webs are a derived type of orb - web , and whether minor ampullate silks have any function during cob - web construction remains unknown . their similar gland morphology and related silk functions suggest that major and minor ampullate spidroins have a close evolutionary relationship , but phylogenetic analysis of spidroins is inconclusive . for example , in garb et al . , misps tended to , but did not always , form a sister group to a large clade of masps . in contrast to major and minor ampullate silks , the thick fibers that wrap l. hesperus egg cases are produced by glands that are unlike any other silk glands in shape : the elongated , worm - like tubuliform glands ( also known as cylindrical glands ; figure 1c ) . the dominant transcripts in tubuliform glands encode tubuliform spidroins ( tusp1 ) and a family of silk proteins unrelated to spidroins , egg - case proteins ( ecps ) . despite having different shapes and producing functionally distinct silks , major ampullate and tubuliform silk glands are known to have overlapping spidroin gene expression . major ampullate glands are dominated by masp1 and masp2 gene expression , but a 3 tag profiling study of transcripts from l. hesperus major ampullate glands found expression of the tubuliform spidroin gene , tusp1 . the converse is also true : masp1 and masp2 transcripts were present in l. hesperus tubuliform silk gland cdna libraries . it remains unknown , however , if the presence of nondominant spidroin transcripts in silk glands unequivocally indicates their translation and whether the protein products are incorporated into silk fibers . tubuliform and major ampullate silk glands each express nonspidroin silk - related genes . ecps are members of a nonspidroin gene family that were initially identified in black widow tubuliform glands and fibers and are thought to cross - link with tusp1 . further research has also found ecp homologues in a spider species without tubuliform glands , suggesting that ecps have functions across multiple silk types . the recently characterized low - molecular - weight cysteine - rich proteins ( crps ) are another category of nonspidroin silk constituents . crps appear in l. hesperus major ampullate glands and fibers and are thought to cross - link with masp1 and masp2 to increase silk fiber toughness . other nonspidroin proteins that are vital to the process of synthesizing and assembling spidroins must exist in each gland type , but these are currently unknown . deep transcriptome sequencing studies have made it possible to explore the gene expression profiles of silk glands and to address the dearth of nonspidroin , silk - related genes . a substantial advance to understanding spider silk synthesis was the identification of 647 silk - gland specific transcripts ( ssts ) from l. hesperus by clarke et al . ssts are transcripts that were found to be significantly differentially expressed and at least 630 times more abundant in silk glands compared to that in nonsilk gland control tissues . hence , ssts are promising candidates for roles in spidroin production and fiber assembly . previous mass spectrometry studies of protein extracts from l. hesperus egg case wrapping , attachment discs , and gland luminal contents has demonstrated the translation of seven types of silk structural proteins encoded by ssts ( aciniform spidroin , ecps , minor ampullate spidroin , major ampullate spidroins 1 and 2 , tubuliform spidroin , and pyriform spidroin ) . it remains unknown whether all or only some of the remaining ssts are translated into proteins . in the present study , we use peptide sequencing to generate protein profiles for the major ampullate , minor ampullate , and tubuliform silk glands as well as the major ampullate fiber of l. hesperus . from these proteomes , we identify proteins arising from the ssts . are from the combined set of l. hesperus silk glands , which makes their localized expression among gland types impossible to discern . knowing which ssts are translated in different glands will identify ssts that are necessary for general silk gland functions ( protein products in multiple gland types ) and ssts that are necessary for the synthesis and storage of specific silks ( protein products restricted to one gland type ) . additionally , if an sst protein product appears in both the major ampullate gland and fiber , then it may have utility in silk fiber assembly or function rather than having exclusive importance in the gland - specific functions of producing and maintaining silk protein reserves . we predict that major and minor ampullate glands will have similar proteomes , consistent with their similar shape and the overlapping functions of major and minor ampullate silks . we also expect that tubuliform glands will contain tusp1 and ecps and that major ampullate glands will contain masp1 , masp2 , and crps . tubuliform glands may also express masp1 and masp2 , which would be consistent with the discovery of masp1 and masp2 transcripts in tubuliform gland cdna , and , conversely , major ampullate glands may express tusp1 , consistent with the detection of tusp1 transcripts in major ampullate glands . comparing major ampullate glands to major ampullate fibers , we expect that only a small subset of the sst protein products identified in major ampullate glands will appear in the fiber . the goal of this research is to combine hypothetical gene products predicted by de novo assembled rna - seq data with proteomic data to gain a broader understanding of how spiders generate silk in vivo . this analysis will extend the framework on how silk dope and fibers are generated by spiders and of gene products that are exceptional candidates for involvement in the synthesis of spider silk . adult l. hesperus females were collected on the university of california , riverside , campus and were fed commercially available crickets . prior to dissection , individuals were not fed for 2 days to minimize contamination from gut contents . spiders were individually anesthetized using carbon dioxide gas , and their abdomens were dissected in 1 ssc . in l. hesperus , major ampullate , minor ampullate , and tubuliform glands are easily distinguished during dissection because of their distinctive morphologies and spatial arrangement in the abdomen ( figure 1 ) . silk glands were visually inspected to ensure that there were no breaks or punctures . if damage was detected , no silk glands from that individual were used to avoid cross - contamination . major ampullate , minor ampullate , and tubuliform glands were dissected into separate microfuge tubes and flash frozen in liquid nitrogen or immediately used for protein extraction ( below ) . major ampullate silk fibers were manually drawn from spiders as described in the literature . major ampullate fibers are extruded from large and distinctive silk spigots , enabling visual verification of major ampullate silk collection during fiber harvesting . fibers were drawn onto spools at a rate of 57 cm per second for a total of 23 min . it was sometimes necessary to use up to three individuals to amass the targeted amount of silk fibers . fibers from each individual were stored on separate spools at room temperature in closed containers . two biological replicates were collected for each gland type and major ampullate silk fibers . replicates consisted of one of the following : two major ampullate glands from one individual , six tubuliform glands from one individual , four minor ampullate glands from two individuals , or the combined silk spools of up to three individuals . fresh or frozen silk glands were macerated with a sterile pestle in protein extraction buffer ( 10% glycerol , 50 mm tris , ph 7.5 , 5 mm mgcl2 , 2% sds , 150 mm nacl , 0.2% -mercaptoethanol , 0.005 m edta ) supplemented with halt protease inhibitor cocktail ( thermo fisher scientific , waltham , ma ) . silk fibers were cut with a clean razor blade , immersed in protein extraction buffer supplemented with halt protease inhibitor cocktail , macerated with a sterile pestle , and incubated at room temperature overnight . samples were then fractionated on an sds - page gel ( 5% stacking and 6.4% separating ) and visualized with bio - safe coomassie stain ( bio - rad , hercules , ca ) . protein concentrations were estimated from these visualizations , with a target of 500 ng / lane . for each sample , multiple lanes ( 1015 l / lane ; total of 2030 l of sample ) were excised and combined for in - gel digest ( supporting information figure s1 ) . standard protocols for in - gel tryptic digestion from the arizona proteomics consortium ( http://proteomics.arizona.edu/protocols ) were followed . briefly , proteins in acrylamide gels were digested overnight at 37 c with 12.5 ng/l trypsin and 1.25 ng/l chymotrypsin in 50 mm ammonium bicarbonate with 10 mm calcium chloride . digested peptides were extracted from gel pieces with a bioruptor standard sonication system ( diagenode , denville , nj ) for 30 min . after sonication , the supernatant was purified using ziptips c18 pipette tips according to the manufacturer s instructions ( millipore , billerica , ma ) , dried , and stored at 20 c until shipment for analysis at the arizona proteomics consortium . lc ms / ms analysis of trypsin / chymotrypsin - digested proteins was carried out using a ltq orbitrap velos mass spectrometer ( thermo fisher scientific ) equipped with an advion nanomate esi source ( advion , ithaca , ny ) . 2 cm , thermo fisher scientific ) onto an analytical column ( 75 m i.d solvent b ( acetonitrile , 0.1% formic acid ) was used for the following concentrations and times : 5% , 5 min ; 57% , 5 min ; 715% , 45 min ; 1535% , 60 min ; 3540% , 28 min ; 4085% , 5 min ; 85% , 10 min ; a return to 5% in 1 min , and another 10 min hold of 5% solvent b. all flow rates were 400 nl / min . ms / ms replicate runs were also performed using a shorter 125 min rp gradient ( 5% solvent b hold for 10 min ; 520% , 65 min ; 2035% , 25 min ; 35% , 9 min ; 3595% , 5 min ; and finally a 95% solvent b hold for another 5 min ) . data - dependent scanning was performed by xcalibur , v 2.1.0 , software using a survey mass scan at 60 000 resolution in the orbitrap analyzer scanning mass / charge ( m / z ) 4001600 , followed by collision - induced dissociation ( cid ) tandem mass spectrometry ( ms / ms ) of the 14 most intense ions in the linear ion trap analyzer . precursor ions were selected by the monoisotopic precursor selection ( mips ) setting , with selection or rejection of ions held to a 10 ppm window . dynamic exclusion was set to place any selected m / z on an exclusion list for 45 s after a single ms / ms . tandem mass spectra were searched against a protein database made by combining chelicerata proteins downloaded from ncbi on october 17 , 2013 and reduced for redundant entries via cd hit ( available at http://weizhong-lab.ucsd.edu/cd-hit/ ) with common contaminant proteins such as keratins ( ftp://ftp.thegpm.org/fasta/crap/ ) and with a nonredundant longest open reading frame translation of an l. hesperus transcriptome ( clarke et al . ; see above ) . at the time of the search , this combined protein database contained 182 000 entries . all ms / ms spectra were searched against the protein database described above using thermo proteome discoverer 1.3 ( thermo fisher scientific ) considering tryptic / chymotryptic peptides with up to 2 missed cleavage sites . proteins were identified at 99% confidence with xcorr score cutoffs , as determined by a reversed database search . the protein and peptide identification results were visualized with scaffold , v 3.6.1 and v 4.0 ( proteome software inc . , portland , or ) , a program that relies on various search engine results ( i.e. , sequest , x!tandem , mascot ) and that uses bayesian statistics to reliably identify more spectra . proteins were accepted that passed a minimum of two peptides identified at 95% peptide confidence and 99.9% protein confidence by the peptide and protein profit algorithms , respectively , within scaffold . the similarities among the identified protein sets from each sample were quantified in r using the ape package via a binary character matrix , with observed protein presence within a library ( protein identification probability > 95% ) as the positive result . from the matrix , we calculated the distances between protein libraries using simple matching , from which we built a dendrogram via hierarchical clustering . support for the dendrogram was calculated via bootstrapping with 10 000 replications using a custom r script . replicates were collapsed by taking the higher protein identification probability of the two replicates from the scaffold analysis . the list of identified proteins included ssts . because an sst was not always the top protein identified , we further examined the identified ncbi proteins to see if they were associated with ssts . to do this , we used the identified ncbi nr proteins to build a custom blast database . we searched this database with the longest open reading frame nonredundant translation of the l. hesperus transcriptome in a blastp search . up to five results with an e - value less than 1 10 for each query sequence were retained . the most relevant sst from the set of blast hits was determined by searching for presence of peptide sequences . crps were identified with a blastp search against the longest open reading frame translation of the l. hesperus transcriptome using an e - value cutoff of 1 10 . we identified 842 putative proteins in the major ampullate , minor ampullate , and tubuliform glands of l. hesperus ( scaffold sample report ; supporting information file s1 ) . the protein identifications in tubuliform gland biological replicates clustered together in a well - supported group ( dendrogram bootstrap support = 100% ; supporting information figure s2 ) . major ampullate gland biological replicates also clustered with each other ( dendrogram bootstrap support = 92% ; supporting information figure s2 ) . one minor ampullate gland replicate grouped more tightly with the major ampullate gland replicates ( bootstrap = 88% ; supporting information figure s2 ) than it did with the other minor ampullate gland replicate . the difference between the minor ampullate gland replicates may be due to concentration - based variability in detectable proteins . minor ampullate glands are , at most , approximately one - third the length of the other gland types used in this study , and glands from multiple individuals were combined in a single replicate to achieve target concentrations ( figure 1 ; see materials and methods ) . the detectable proteins between the replicates may differ because of variation in protein expression among individuals . despite differences between the minor ampullate gland replicates , consistent with shared ampullate shapes , both minor ampullate gland biological replicates formed a group with the major ampullate gland replicates ( bootstrap support = 91% ; supporting information figure s2 ) , with tubuliform gland replicates being more differentiated . the subsets of identified proteins that correspond to ssts also show greater similarity between major ampullate and minor ampullate glands than to tubuliform glands . , 48 were predicted by peptide matches in major ampullate , minor ampullate , and tubuliform gland protein extractions ( supporting information file s2 ) . some of the identified proteins were fragments from different regions of the same protein ; therefore , the total number of unique proteins predicted by peptide matches to ssts was 39 ( figure 2 and supporting information file s2 ) . out of these 39 , only 28 had uniprot hits with an e - value less than 1 10 . chart of presence ( red ) or absence ( light orange ) of peptide - predicted proteins encoded by ssts . predicted proteins from l. hesperus major ampullate , minor ampullate , and tubuliform silk glands with best uniprot hits < 1 10 are shown . proteins are arranged alphabetically by name within groups : silk structural proteins , other characterized proteins , uncharacterized proteins , and proteins with no uniprot hits . abbreviations : a2 m , alpha-2 macroglobulin ; ac , aciniform ; asg , aggregate spider glue ; ceh , carboxylic ester hydrolase ; crp , cysteine rich protein ; cuticle , cuticle protein ; ecp , egg case protein ; fmo3 , dimethylaniline monooxygenase ; gt , glutamyltranspeptidase ; hdc , histidine decarboxylase ; hemo_d , hemocyanin subunit d ; lectin , putative lectin ; ma , major ampullate ; mi , minor ampullate ; mur89f , mucin related 89f ; scaffold i d no . , scaffold predicted protein number ; sp , spidroin ; tu , tubuliform . twenty - four unique predicted proteins that matched ssts were identified from major ampullate glands , 24 from minor ampullate glands , and 14 from tubuliform glands ( figure 2 ) . the major ampullate and tubuliform glands had one sst encoded protein in common that was not found in the minor ampullate gland , and minor ampullate and tubuliform glands also had one in common that was not observed in the major ampullate gland . four sst encoded proteins were found in all gland types ( figure 3 ) . in dendrogram analyses of sample replicates , the subset of sst encoded proteins grouped by gland type ( minor ampullate bootstrap support = 85% , major ampullate bootstrap support = 91% , tubuliform bootstrap support = 100% ; supporting information figure s3 ) . minor ampullate gland replicates grouped with the major ampullate gland replicates ( bootstrap support = 88% ; supporting information figure s3 ) . euler diagram showing numbers of unique and shared predicted sst proteins in l. hesperus major ampullate ( purple ) , minor ampullate ( yellow ) , and tubuliform ( blue ) silk glands . ellipses areas and overlap areas are proportional to the number of unique and shared proteins in and between gland types . the higher amount of overlap between the sst protein profiles from major and minor ampullate glands reflects their comparable gland morphologies and silk functions ( figures 13 and supporting information figure s3 ) . these findings are also consistent with phylogenetic analyses of the spidroin family that showed a large clade of masps as most closely related to misps and that tusp1 is more closely related to non - misp prey - swathing silk proteins ( aciniform spidroin , acsp1 ) . thus , multiple sources of evidence ( proteomic profiling of silk glands , morphology of silk glands , and evolutionary relationships of spidroins associated with particular silk glands ) support the close evolutionary and functional relationship of major and minor ampullate spidroins as well as the similar morphologies of major and minor ampullate glands . nine previously characterized silk - related proteins were identified among the 39 sst proteins ( 9/39 ; 23% ; figure 2 ) . as expected , masp1 , masp2 , and crp1 were identified in major ampullate silk glands , misp , in minor ampullate silk glands , and tusp1 and ecp1 , in tubuliform silk glands . misp was identified only from minor ampullate glands , and tusp1 and ecp1 were found solely in tubuliform glands . in contrast , masp1 was identified from all three glands , and masp2 was found in both major and minor ampullate glands . major ampullate silk transcripts and proteins have not previously been documented in minor ampullate silk glands . the expression of major ampullate spidroins in minor ampullate glands could be due to similarities in their genetic programs , mirroring their shape and functional similarities . the discovery of masp1 proteins in tubuliform silk glands is consistent with previous studies that identified masp1 transcripts from tubuliform silk gland cdna libraries . however , contrary to previous observations of masp2 transcripts in tubuliform silk glands and tusp1 transcripts in major ampullate silk glands , we identified neither masp2 proteins in tubuliform silk glands nor tusp1 proteins in major ampullate glands . little is known about regulation of spidroin expression and translation , and our results suggest complicated translational control of silk structural proteins . unexpectedly , we found two other known silk - related proteins , aciniform spidroin and an aggregate spider glue ( acsp1 and asg2 , respectively ) in the major and minor ampullate glands ( figure 2 ) . a previous investigation that used 3 tag profiling of rna transcripts from major ampullate glands did not report the presence of acsp1 or asg2 transcripts . if acsp1 is a rare transcript , then the focus on highly differentially expressed 3 tags in lane et al . l. hesperus acsp1 is an exceptionally large molecule ( > 19 kb ) with a lengthy repetitive region flanked by relatively short amino ( n)- and carboxyl ( c)-terminal regions . in our analysis , 471 ; figure 2 ) has peptide matches to the n - terminal and repetitive regions . rare transcript expression would mean that there are few 3 terminal regions available for tagging , whereas enzyme digested acsp1 protein would result in many protein fragments detectable by peptide analysis . an alternative explanation is that the two individuals in the 3 tag profiling study were not expressing acsp1 transcripts at the time of rna extraction . this explanation suggests that ampullate glands may transiently express acsp1 and that accumulated acsp1 protein could be detectable with proteomic approaches . asg2 is a putatively glycosylated protein secreted as part of sticky droplets involved in prey capture . asg2 transcripts and proteins have not previously been detected in major and minor ampullate glands . our finding may be indicative of the specific uses of ampullate silks by black widows . in orb - web weaving spiders , major ampullate silk is used as dragline and as the web frame and radii , and minor ampullate silks are used as temporary spiral during web construction . in black widows , major ampullate silks are thought to be the core fiber of the gumfooted lines that stick to and entangle prey . after ensnarement , minor ampullate silks are incorporated into a composite of different silk types for prey restraint . given the function of major and minor ampullate silks in l. hesperus , black widow ampullate - shaped silk glands may additionally synthesize prey capture glue proteins such as asg2 . in the major ampullate gland , peptides identified the translation of an sst sequence that we refer to as masp to distinguish it from masp1 and masp2 ( figure 2 , scaffold i d no . the masp peptide matches are unique to an sst with a top uniprot hit to masp1 ( supporting information file s2 ) , but the sequence is distinct from either masp1 or masp2 . the sst that encodes masp appears to be a newly discovered member of the spidroin gene family , and its translation into a protein indicates that masp is functional . other than known silk - related proteins , 11 sst translations with characterized uniprot protein matches were detected in the ampullate glands . four were exclusive to the major ampullate gland , four were identified in the major ampullate and minor ampullate glands , one was found in the minor ampullate and tubuliform glands , and two were in all glands ( figure 2 ) . the four proteins exclusive to the major ampullate gland were three enzymes and a protein that have been described in vertebrates ( abbreviation , sst to uniprot e - value ) : alpha-2 macroglobulin ( a2 m , 0 ) , carboxylic ester hydrolase ( ceh , 6 10 ) , dimethylaniline monooxygenase ( fmo3 , 7 10 ) , and putative triacylglycerol lipase ( ptl , 3 10 ) . because they are exclusive to major ampullate silk glands , a2 m , ceh , fmo3 , and ptl are candidates for having specific roles in the synthesis of major ampullate silks . five uniprot annotated sst proteins beyond silk structural proteins were associated with two gland types ( figure 2 ) . major and minor ampullate glands shared four proteins : putative cuticle protein ( cuticle , 2 10 ) , putative gamma glutamyltranspeptidase-1 ( -gt-1 , 4 10 ) , histidine decarboxylase ( hdc , 0 ) , and mucin related protein 89f ( mur89f , 2 10 ) . minor ampullate and tubuliform silk glands contained one protein in common : scavenger receptor cysteine - rich protein ( srcr ; 1 10 ) . cuticle proteins have previously been described in spider silk gland ducts , but the other proteins are undescribed in spiders . on the basis of the glands in which they were found , these five proteins may be involved in either functions exclusive to ampullate glands ( cuticle , -gt-1 , hdc , mur89f ) or functions that are more general to different silk gland types ( srcr ) . two non - silk - related sst proteins were found in all three silk gland types . these were an oxygen transport molecule and a carbohydrate binding protein : hemocyanin subunit d ( hemo_d , 0 ) and a putative lectin ( lectin , 6 10 ) , respectively . because of their detection in the three silk glands , these proteins may be common to other types of spider silk glands , such as aciniform , pyriform , and flagelliform . analysis of protein extractions from major ampullate glands and fibers resulted in 527 protein predictions ( scaffold samples report is supporting information file s3 ) . of the 527 proteins , 86 were found in the fiber ( supporting information file s3 ) . twenty - nine of the 527 proteins had sst encoded protein matches , with 17 in the fiber ( supporting information file s4 ) . only nine of the 29 were unique to this major ampullate gland and fiber analysis ; the remaining sst encoded proteins overlapped with those identified in the three - gland analysis of major ampullate , minor ampullate , and tubuliform silk glands . some of the 29 sst predicted proteins were fragments of the same protein ; thus , we identified a total of 25 unique putative proteins ( figure 4 and supporting information file s4 ) . these fell into four groups : six matched silk structural proteins , nine were other characterized proteins , three were uncharacterized , and seven had no hits in the uniprot database ( figure 4 and supporting information file s4 ) . crp1 was identified and considered a silk structural protein ( figure 4 , scaffold i d no . chart of presence ( brown ) or absence ( purple ) of peptide - predicted proteins encoded by ssts from l. hesperus major ampullate silk glands and fibers . proteins are arranged alphabetically by name of best uniprot hits < 1 10 within groups : silk structural proteins , other characterized proteins , uncharacterized proteins , and proteins with no uniprot hits . * , protein type has more than one predicted protein . abbreviations : a2 m , alpha-2 macroglobulin ; ac , aciniform ; asg , aggregate spider glue ; ceh , carboxylic ester hydrolase ; crp , cysteine - rich protein ; cuticle , cuticle protein ; fmo3 , dimethylaniline monooxygenase ; gt , glutamyltranspeptidase ; hdc , histidine decarboxylase ; hemo_d , hemocyanin subunit d ; lectin , putative lectin ; ma , major ampullate ; mi , minor ampullate ; mur89f , mucin related 89f ; ptl , pancreatic triacylglycerol lipase ; scaffold i d no . , scaffold predicted protein number ; sp , spidroin . we predicted that major ampullate fiber sst matches would be a subset of the gland sst matches . indeed , of the 25 unique predicted sst proteins , 12 were found in the fiber and the gland , nine were exclusive to the gland , and four were exclusive to the fiber ( figure 4 and supporting information file s4 ) . we did not expect to find any proteins exclusive to the fiber because the fiber is a product of the gland . one of the fiber - exclusive sst proteins matched an uncharacterized uniprot protein , and the remaining three had no hits in the uniprot database . these four fiber - exclusive sst encoded proteins may have been present in the gland , but they were difficult to detect because the gland has a greater variety of proteins than that in the fiber . in addition , the possibility of contamination of major ampullate fibers with products from other silk glands can never be entirely dismissed despite our use of microscopic observation while collecting fibers . consistent with our results from the three - gland analysis , we identified the expected masp1 , masp2 , and crp1 in the major ampullate gland and we also found acsp1 , asg2 , and masp. three of these silk structural proteins were identified in the fiber : acsp1 , crp1 , and masp1 . discovery of acsp1 protein in the fiber confirms the protein matches to acsp1 in ampullate glands ( figures 2 and 4 ) . similarly , the presence of crp1 and masp1 is consistent with previous peptide studies of l. hesperus major ampullate glands and fibers . the nondetection of masp2 may be due to a greater proportion of masp1 in the fiber . the relative proportions of masp1 and masp2 varies in draglines , and previous evidence suggests a more than 2-fold greater abundance of masp1 compared to that of masp2 in l. hesperus major ampullate fibers ( 2.5:1 ratio of masp1 to masp2 ) . alternatively , the lack of masp2 detected in the fiber could be due to poor solubility of this spidroin , hampering its inclusion in the protein digests . of the nine sst proteins with uniprot matches that were not silk - related , four were identified in both glands and fibers and five were exclusive to the gland . the four in both glands and fibers were also previously identified in the three - gland analysis : ceh , cuticle , lectin , and mur89f . the identification of these four proteins in major ampullate fibers suggests that they may be crucial to fiber assembly or function . we identified 48 sst encoded proteins in our three - gland analysis and nine additional sst encoded proteins in the major ampullate gland and fiber analysis ( supporting information files s2 and s4 ) . thus , we provide evidence for translation of 9% ( 57/647 ) of the ssts from clarke et al . these included known silk - related , non - silk - related , and uncharacterized proteins . we show that silk structural genes that are not known to have dominant expression in a given gland type are translated and that some of these nondominant proteins are incorporated into draglines ( e.g. , acsp1 ) . peptide analysis also demonstrates translation of putative enzymes , cuticle , and lectin proteins and 18 predicted proteins encoded by ssts that had no uniprot hit . these findings provide clues about the evolutionary history of spider silk types and suggest new ways that spiders may vary silk properties . this result is consistent with the differentiation of major and minor ampullate glands from a more generalized ampullate gland type . we also show that minor ampullate glands contain masp1 and masp2 and that acsp1 is present in dragline silk fibers . inclusion of masp1 and masp2 in minor ampullate silk fibers and inclusion of acsp1 in draglines could modulate the tensile properties of silk fibers . similarly , the expression of asg2 glue proteins in l. hesperus ampullate glands may improve the utility of ampullate silks for prey capture by altering major and minor ampullate silk properties . overall , our proteomic analysis combined with rna - seq - derived transcript predictions provides greater insight into the evolution and synthesis of spider silks .

spider silk research has largely focused on spidroins , proteins that are the primary components of spider silk fibers . although a number of spidroins have been characterized , other types of proteins associated with silk synthesis are virtually unknown . previous analyses of tissue - specific rna - seq libraries identified 647 predicted genes that were differentially expressed in silk glands of the western black widow , latrodectus hesperus . only 5% of these silk - gland specific transcripts ( ssts ) encode spidroins ; although the remaining predicted genes presumably encode other proteins associated with silk production , this is mostly unverified . here , we used proteomic analysis of multiple silk glands and dragline silk fiber to investigate the translation of the differentially expressed genes . we find 48 proteins encoded by the differentially expressed transcripts in l. hesperus major ampullate , minor ampullate , and tubuliform silk glands and detect 17 sst encoded proteins in major ampullate silk fibers . the observed proteins include known silk - related proteins , but most are uncharacterized , with no annotation . these unannotated proteins likely include novel silk - associated proteins . major and minor ampullate glands have the highest overlap of identified proteins , consistent with their shared , distinctive ampullate shape and the overlapping functions of major and minor ampullate silks . our study substantiates and prioritizes predictions from differential expression analysis of spider silk gland transcriptomes .

from now on , molecular study regarding female sexuality has focused on female sexual hormone , like estrogen receptor ( er ) and er derivatives.12 but flibanserin is an agent of 5-hydroxytryptamin ( 5-ht ) type 1a receptor and an antagonist of 5-ht type 2a and thus a new molecular entity.3 flibanserin was originally developed as antidepressant drug . in clinical stage 2a as antidepressant , flibanserin could not prove its effectiveness , but almost no sexual function disorder was reported in subjects . for this reason , the arizona sexual experiences scale ( asex ) was used for making a comparison of the sexual function effectiveness of flibanserin on antidepressant vs. placebo proven in stage 2b studies among four depression programs.4 stage 2b studies have failed to prove its general effectiveness on depression , but in answering this question " how intense is your sexual desire ? " in the asex , flibanserin turned out to be more excellent in both placebo and active - comparator . since then , drug development has changed its directivity toward being a potential medicine for treating hypoactive sexual desire disorder ( hsdd ) . hsdd is chara or absence of sexual fantasy and sexual desire associated with personal pain in the 4th edition of the diagnostic and statistical manual - text revision ( dsm - iv - tr).5 determining lack or absence is done by clinicians by considering the factors influencing sexual activitie such as age and contexts of personal life . sexual function disorder is not explained well by other axis 1 disorders ( except other sexual function disorders ) and also it is not that it is caused only by direct physiological effects of materials ( i.e. , drug overuse , drug ) or overall medical status . the dsm-5 completed in 2013 addresses new illness called as female sexual interest / arousal disorder ( fsiad ) , which has both properties of hsdd and another illness of dsm - iv known as female sexual arousal disorder . flibanserin underwent new drug application ( nda ) as original drug in 2009 . the two central phase 3 studies ( violet6 , daisy7 ) all used e - diary and did not show big statistical improvement compared to placebo in the pre - specified co - primary efficacy endpoint that assessed daily sexual desire . in both violet6 and daisy7 studies , flibanserin showed an increase in satisfying sexual events ( sse ) and female sexual function index ( fsfi ) and reduction in female sexual distress scale - revised ( fsds - r ) , but did not show a meaningful increase in e - diary scores compared to placebo . in both studies , the most commonly reported adverse effects in women prescribed with flibanserin include drowsiness ( 11.8% ) , vertigo ( 10.5% ) , and fatigue ( 10.3%).67 these studies show a statistically significant improvement in secondary end points that measured sexual desire with other tools known as fsfi compared to placebo . applicants told that the effectiveness of flibanserin against sexual desire is better explained with fsfi , but most advisory committees did not agree to alteration in the e - diary evaluation method set as the one designed to evaluate sexual desire . the safety consciousness expressed by the advisory committees includes adverse effects such as fatigue and drowsiness as well as drug - drug interactions ( ddis ) and flibanserinalcohol interactions . another central stage 3 study is begonia,8 which set sse , fsfi , fsds - r as major evaluation methods rather than using e - diary in the existing viole and daisy studies and reported that flibanserin shows a statistically significant increase in sse and fsfi and reduction in fsds - r compared to placebo . however , many problems with safety were brought up in the second submission , too . vertigo , drowsiness , and vomiting are the most commonly reported adverse effects , and these occurred 2% to 3% , respectively if using placebo during stage 3 placebo - control premenopausal hsdd , whereas occurred close to 11% in subjects who used flibanserin as 100 mg every night at bedtime . events matching central nervous system depression ( fatigue , drowsiness , and vertigo ) occurred close to 21% in subjects who used flibanserin as 100 mg every night at bedtime and this proportion is three times higher than in placebo group . flibanserin is very difficult to endure if taking in combination with fluconazole and strong cytochrome p450 3a4 ( cyp3a4 ) suppressant and this combination may increase the risk of swoon and symptomatic low blood pressure . and ethanol administered in combination with flibanserin greatly increases the risk of drowsiness , fatigue , orthostatic hypotension , and swoon . additionally , these studies were limited to general healthy women who did not take benzodiazepines , sleep aid , narcotics and many other drugs . the effectiveness was not so big in general , and considering these concerns , fda refused to approve it again and recommended that additional safety studies are required . according to the latest fda review , there is no data indicating new efficacy . instead , flibanserin sponsors submitted additional safety data next day including research data proving that there is no obstacle to driving , data comparing the commercialized products and the side effects profile of these products , and analysis clarifying the potential effects of alcohol on side effects.9 despite the trustfulness of highly influential studies , fda product review is not a fundamental comparison , in fact , and so comparison of safety between individual products may be misunderstood . in particular , alcohol interaction study was conducted in 25 healthy volunteers as a sample , among which only two were female . since the refusal from fda in 2013 , a civic group called as even the score was formed to support what was called as " gender equality " in the manner approaching sexual function disorder treatment.10 this group insisted that there is no treatment for women although even 26 items for male sexual function disorder were already approved . this argument was rejected by fda on the ground that there are no approved products for low sexual desire in men and the 26 items for treatment contain various mixtures of testosterone . despite the fact that flibanserin is supported by consumer advocacy groups and consequently not the first product supported by pharmaceutical companies , fda 's argument on gender bias while regulating is worthy of notice in that the range of making efforts against this argument is from campaigns via social media even to letters from the members of the national assembly.11 another noteworthy feature in application for flibanserin to fda is use of the findings on sexual desire reported by patients as primary efficacy variable for approval . sexual desire can be seen by those who experience it and the results reported by patients can be measured without confounding this concept from others . the results reported by patients have become more important in studies and other drugs that have such results as primary end point have been approved ( although none were based on sexual desire ) . among all new molecular entities and biological permit applications approved by fda from 2006 to 2010 , gnanasakthy et al.12 found that among which , 17% ( 20 in 116 ) used the results reported by acknowledged patients as primary outcome in approval labels . the complex element in evaluating flibanserin lies in recalling the frequency of desire and extent of intensity and then measuring with fsfi index four weeks after the daily records of the most intense desires when applying for the primary desire end point first time . fda raised some concerns about optimizing fsfi as desire assessment tool , including the validity of contents and the period of recall , but the recent advisory committees did not focus on changing the primary end points or consulting the validity of fsfi . the final concern with flibanserin is associated with its use in clinical settings and this was an important issue for the committees . despite little reliable estimate of hsdd prevalence , this product will be used obviously in women in a broader sense than had been studied so far like others , many will not satisfy the official diagnostic criteria of hsdd , and many will increase the risk of adverse effects or be administering other drugs . the concerns with its use without labeling were reemphasized again by speakers who argued that they need flibanserin , but at the same time , it was reported that those who were diagnosed with cancer or in menopause should be excluded from treatment with flibanserin by labeling when sold . on june 4 , 2015 , fda called a committee meeting to review the efficacy and safety of flibanserin , and after the meeting , the committee approved flibanserin under the condition that risk management tools should be enforced mandatorily . despite the presence of many problems with flibanserin , fda finally approved it under the condition that risk evaluation and mitigation strategies ( rems ) should be enforced for flibanserin . in the future , flibanserin is expected to gradually increase in amount with changes from off - label to on - label . if prescription with flibanserin is determined as necessary , before using it , it is important to check prescription criterion , possible adverse effects after prescription , and precautions during administration . to get a prescription with flibanserin , the first criteria is premenopausal women aged 18 year or more who met the stages of reproductive aging workshop ( straw ) criteria . the straw criteria are that she has a regular menstrual cycle for 21 to 35 days in the previous twelve months and a normal level of follicle stimulating hormone ( fsh ) . in addition , it should be prescribed if primary diagnosis is hsdd according to dsm - iv - tr criteria or if there is any secondary sexual arousal disorder or female disposition disorder accompanied by hsdd . besides , its prescription is likely to be considered if the fsds - r ( range , 0 - 52 ) score13 used in clinical studies is 15 points or higher . in addition , in case that sexual desire disorder comes from disease , psychiatric problems , and interpersonal problems or in case that it does so from medications or other substances taken in conjunction , these cases are not the diseases for which medicine is efficacious . besides , postmenopausal women and sexual desire decline disorder male patients are not adapted during use and i t is not intended for reinforcing sexuality.1415 the adverse effects most common in prescribing flibanserin are reported as vertigo , fatigue , vomiting , insomnia , and mouth dryness . precautions in prescription include avoiding the use of other substances associated with cyp3a4 and cytochrome p450 2c19 ( cyp2c19 ) which are associated with drug metabolism , for example , administration with ketoconazole as cyp3a4 inhibitor and grapefruit juice and fluconazole as cyp3a4 or cyp2c19 inhibitor , which are likely to increase the risk of orthostatic hypotension side effects . furthermore , selective serotonin reuptake inhibitors and selective norepinephrine reuptake inhibitors ( ssri / snri ) may cause anxiety , drowsiness , fatigue , insomnia , and vertigo , if used . so carefulness is required . alcohol is contraindication because if administered with alcohol , it is highly likely to cause central nervous system depression ( fatigue , drowsiness , and vertigo ) , low blood pressure , and swoon . hormonal contraceptives are known as weak cyp3a4 inhibitors , and likely to cause side effects such as vertigo , drowsiness , and fatigue if coadministered with flibanserin . triptans is used for treating migraine as agonist of 5-ht type 1b and 1d receptor and may cause side effects of drowsiness if administered with flibanserin . in addition , if flibanserin is administered to patients with decline in liver failure , increase in concentration of drug in blood is observed and thus it 's considered that adverse effects are likely to occur . besides , taking it with herb extracts and digesters and gastroprotective drugs requiring no doctor prescription is likely to cause the risk of adverse effects and the use of flibanserin to women who are breast - feeding is contraindication . as we discussed above , flibanserin was refused to approve by fda in 2009 for its adverse effects and discordance of its efficacy from different evaluation methods . most common side effects were central nervous system depression , like fatigue , drowsiness , and vertigo . ddis with fluconazole , ethanol , and many other drugs were mainly discussed and fda refused to approve it again recommending additional safety data . with the additional profile and analysis which can prove their safety compared with other products , on june 4 , 2015 , fda finally approved it on the grounds that rems should be enforced , and soon we can get flibanserin on - label under the strict prescription . flibanserin can be prescribed to premenopausal women aged 18 years or higher who has a regular menstrual cycle , but if liver function fails , if breast - feeding , and if drinking alcohol , the use of flibanserin is contraindication . also , its administration with fluconazole , ketoconazole , ssri / snri , triptans , hormonal contraceptives , herb extracts , and several other drugs requiring no doctor prescription is prohibited because it is likely to increase the risk of adverse effects . we are waiting for patients ' reaction , considering the cost and effectiveness , in countries where flibanserin will be on sale , and more agents like unicenta ( uncnt ) and melsmon , which proved as having a efficacy of menopausal symptoms , have to be studied as treating for female sexual dysfunction.16

there have been several products developed for male sexual dysfunction . however , developing agents for female sexual dysfunction is lagging behind for various reasons . sildenafil citrate ( viagra ) and tadalafil ( cialis ) , which have been prescribed for male sexual function disorders , are known to act on vessels.[1 ] on the other hand , flibanserin is thought to act on brain . flibanserin has been approved by u. s. food and drug administration ( fda ) for treatment of hypoactive sexual desire disorder ( hsdd ) of premenopausal women in 2015 , and is expected to be released in south korea soon . authors wrote this article to acknowledge flibanserin to sexologists for females or physicians for menopausal medicine , so that this agent can be safely used for females who have hsdd .

in cases of acute mesenteric ischemia , 5 - 15% cases are caused by superior mesenteric vein ( smv ) thrombosis.1 in cases of mesenteric vein thrombosis ( mvt ) , intestinal ischemia can occur if there is mesenteric venous blood flow obstruction , resulting in critical bowel wall edema and fluid efflux into the lumen . this can result in diminished arterial blood flow , leading to submucosal hemorrhage and bowel infarction . mvt can be fatal if the diagnosis and subsequent treatment is delayed.2 in the present report , we describe the case of a patient with smv thrombosis who presented with abdominal pain and was treated with systematic anticoagulation therapy for a 6-month period . myelodysplastic syndrome ( mds ) was diagnosed during the work - up that was performed to identify the cause of the smv thrombosis . a 62-year - old woman who was admitted to the hospital presented with a 7-day history of abdominal pain , which had worsened over time and was associated with postprandial bloating . the patient had been diagnosed with rheumatoid arthritis 7 years previously and was taking the following medications : low - dose glucocorticoids such as prednisolone ( 5 mg / day ) and methotrexate ( 15 mg / week ) , as well as the nonsteroidal anti - inflammatory drug celecoxib ( 200 mg / day ) . the patient had no history of surgery or trauma , and there was no family history of venous thromboembolism . on admission , initial laboratory findings revealed pancytopenia ( hemoglobin concentration , 9.8 g / dl ; white blood cell count , 3,100/mm ; and platelet count , 32,000/mm ) . the prothrombin time ( international normalized ratio , 1.43 ) and activated partial thromboplastin time ( 42 seconds ) were prolonged . the levels of fibrinogen and fibrinogen degradation product were 186 mg / dl and > 20 g / ml , respectively , whereas the d - dimer level was elevated by 6 g / ml . ct scan of the abdomen and pelvis showed a moderate amount of ascites with omental and mesenteric infiltration , which was associated with a 4.5-cm thrombus in the smv and its tributary ( fig . in addition , there was no evidence of paroxysmal nocturnal hemoglobinuria , antiphospholipid syndrome , or other autoimmune disease . the protein c concentration ( 46% ) and s activity ( 26% ) were below the normal ranges . anticoagulation therapy was initiated , and the patient was closely monitored for signs of bowel infarction . moreover , low - molecular - weight heparin was initiated . on the third day of admission , the patient 's bone marrow was examined in order to identify the cause of the pancytopenia and the recent thrombotic event . a diagnosis of mds was made , which was believed to involve refractory cytopenia with multilineage dysplasia ( fig . 2 ) . thereafter , the patient experienced sustained hematochezia and vague abdominal pain . on the fourth day , capsule endoscopy was performed to exclude bowel ischemia . through small bowel endoscopy , we identified the presence of diffuse mucosal edema from the distal jejunum to terminal ileum and erythematous patches in the ascending colon , which were compatible with smv territories ( fig . after 6 months of anticoagulation therapy , follow - up ct of the abdomen and pelvis showed complete dissolution of the smv thrombosis ( fig . the patient experienced no recurrence of smv thrombosis during the 2-year follow - up period . acute mvt is a rare but potentially fatal condition , with a mortality rate of 20 - 30%.3,4 however , diagnosis of this condition is difficult because the symptoms are nonspecific . critical symptoms such as hemodynamic instability and peritonitis are important indicators as to whether surgery is required . suspicion of mvt is important for avoiding delays in diagnosis and therapy , particularly in elderly patients . the development of imagining modalities over the past 2 decades has improved the ability to make an accurate diagnosis of mvt . in this patient , the sensitivity of diagnosis by contrast - enhanced abdominal ct is approximately 90%.2 mvt is classified as either primary or secondary . primary smv thrombosis is idiopathic , whereas secondary smv thrombosis can result from various underlying causes , including hypercoagulable states , pregnancy , or recent abdominal surgery.5 after an mvt diagnosis is made , the clinician should attempt to identify the cause of the thrombosis . our patient had been diagnosed with rheumatoid arthritis 7 years prior to the current admission . reports have shown that there is an increased risk of developing deep vein thrombosis in patients with rheumatoid arthritis compared with patients who did not have rheumatoid arthritis.6 however , whether treatment or disease activity can modify the risk of venous thromboembolism in patients with rheumatoid arthritis remains controversial.7 slight decreases in antithrombin iii and protein c levels , and s activity were identified in the present case . after a hematological specialist was consulted , these findings were confirmed to be the result - as opposed to the cause - of the recent massive thrombosis . the pancytopenia that was noted on peripheral blood examination and multilineage dysplasia indicated by the assessment of bone marrow aspirate led to the diagnosis of mds . although myeloproliferative disease and malignancy are the most frequently diagnosed thrombophilic conditions,8,9 other studies have reported the presence of thrombosis in association with mds , as in the present case.10,11 kimura et al.12 found that patients with mds and trisomy 8 were more likely to develop thrombosis and intestinal ulcers . chen et al.11 reported a case of large - vessel thrombosis that was complicated by mds and trisomy 8 . although no definite cause of thrombosis has been identified , thrombosis and hemorrhage complicated by mds most likely occur as a result of quantitative and qualitative platelet anomalies.10 other researchers have noted that in cases of mds , immunological disorders caused by neutrophil dysfunction and inflammatory cytokine overproduction may lead to injury and inflammation of the endothelium.11,13,14 there have been reports of patients with mds and autoimmune disease , including behet 's disease , particularly in japan.15,16 another interesting point of our case is the capsule endoscopic findings . to our knowledge , capsule endoscopic findings have not been reported for smv thrombosis.17 the patient experienced sustained symptoms during initial anticoagulation therapy , and bowel ischemia should be excluded in such cases . therefore , capsule endoscopy can play a major role as a noninvasive procedure in cases where clinicians are determining whether surgery is warranted , particularly in cases involving sustained symptoms or controversial imaging findings . a patient 's clinical status typically determines the initial management of mvt . if the patient is clinically unstable , immediate surgical exploration is warranted . if a patient is clinically stable , conservative management , including systemic anticoagulation , is considered appropriate.18,19 catheter - directed thrombolysis is a safe and effective treatment in cases of main trunk involvement.20 the typical duration of anticoagulation treatment is 6 - 12 months.3 the immediate initiation of anticoagulation with low - molecular - weight heparin and a subsequent 6-month course of warfarin proved to be successful in the present case . this case demonstrates the importance of early detection of smv thrombosis followed by active systemic anticoagulation . thus , we believe that clinicians should adopt a high degree of suspicion in such cases , and this increased awareness along with the early initiation of systemic anticoagulation therapy are essential for successful treatment of mvt .

mesenteric venous thrombosis ( mvt ) is a serious condition due to its potential association with mesenteric ischemia and infarction of the small bowel . symptoms of mvt are often vague , making accurate diagnosis and sufficient treatment difficult . however , increased awareness and new imaging modalities for this condition have improved outcomes for patients with mvt . treatment includes anticoagulation , transcatheter therapy , and surgery . in the present report , we describe the case study of a 62-year - old woman with a presenting diagnosis of superior mvt , who was finally diagnosed with myelodysplastic syndrome . the superior mvt spontaneously dissolved after the patient underwent 6 months of systemic anticoagulation therapy . invasive surgery or bowel resection was not required .

this was a retrospective study designed to analyze the relative merits of various screening techniques and single risk factors for gdm , in terms of diagnostic accuracy ( sensitivity and specificity ) , as part of the atlantic dip research program . universal screening was offered to all pregnant women in five antenatal centers from 2007 to 2009 . screening occurred at 2428 weeks gestation using a 75-g 2-h oral glucose tolerance test , and international association of diabetes in pregnancy study groups ( iadpsg ) criteria were applied for diagnosis . this involved having one or more of fasting plasma glucose 5.1 mmol / l/92 mg / dl , 1-h postglucose load 10 mmol / l/180 mg / dl , or 2-h postglucose load 8.5 mmol / l/153 mg / dl . at the first obstetric visit , 12,487 pregnant women were offered screening and 9,365 ( 75% ) consented . of those who consented , baseline characteristics of age , ethnicity , parity , family history of diabetes in first - degree relatives , bmi , and blood pressure were recorded . data were collected and stored in an electronic database ( diamond ) . if gdm was diagnosed , women received combined antenatal / diabetes care according to local guidelines . once pregnancy was completed , data on maternal ( pre - eclamptic toxemia , pregnancy - induced hypertension , mode of delivery ) and infant ( weight , size for gestational age , apgar scores , hypoglycemia , jaundice , respiratory distress syndrome ) were collected . the screening regimens chosen for analysis were universal screening and selective screening based on the ada , nice , and 2010 irish national gdm guidelines ( table 1 ) . comparison of guidelines for screening for gdm pasw 19 and statspages.org software were used for statistical analysis . the test was used to find the proportion of gdm between the stratified groups of age and bmi . sensitivity , specificity , and 95% cis were calculated for each risk factor , including the stratified groups for age and bmi . descriptive statistics were used to identify the number of gdm women with zero risk factors and with more than one risk factor . classification tree analysis was used to assess the age and bmi at which the risk of gdm was significantly increased . this was a retrospective study designed to analyze the relative merits of various screening techniques and single risk factors for gdm , in terms of diagnostic accuracy ( sensitivity and specificity ) , as part of the atlantic dip research program . universal screening was offered to all pregnant women in five antenatal centers from 2007 to 2009 . screening occurred at 2428 weeks gestation using a 75-g 2-h oral glucose tolerance test , and international association of diabetes in pregnancy study groups ( iadpsg ) criteria were applied for diagnosis . this involved having one or more of fasting plasma glucose 5.1 mmol / l/92 mg / dl , 1-h postglucose load 10 mmol / l/180 mg / dl , or 2-h postglucose load 8.5 mmol / l/153 mg / dl . at the first obstetric visit , 12,487 pregnant women were offered screening and 9,365 ( 75% ) consented . of those who consented , baseline characteristics of age , ethnicity , parity , family history of diabetes in first - degree relatives , bmi , and blood pressure were recorded . data were collected and stored in an electronic database ( diamond ) . if gdm was diagnosed , women received combined antenatal / diabetes care according to local guidelines . once pregnancy was completed , data on maternal ( pre - eclamptic toxemia , pregnancy - induced hypertension , mode of delivery ) and infant ( weight , size for gestational age , apgar scores , hypoglycemia , jaundice , respiratory distress syndrome ) were collected . the screening regimens chosen for analysis were universal screening and selective screening based on the ada , nice , and 2010 irish national gdm guidelines ( table 1 ) . the test was used to find the proportion of gdm between the stratified groups of age and bmi . sensitivity , specificity , and 95% cis were calculated for each risk factor , including the stratified groups for age and bmi . descriptive statistics were used to identify the number of gdm women with zero risk factors and with more than one risk factor . classification tree analysis was used to assess the age and bmi at which the risk of gdm was significantly increased . at the first obstetric visit , 12,487 pregnant women were offered universal screening and 9,365 ( 75% ) consented . of those who consented , most women ( 93% ) were caucasian , with a mean age of 32 5.3 years and a mean bmi of 26.9 5.1 kg / m . the characteristics of women with gdm in the presence and absence of risk factors for gdm are reported in table 2 . in addition , 31% of women required insulin to reach their glucose goals , comprising 10% of women with no risk factors and 21% of women with risk factors . when the nice guidelines were applied , 54% of women ( n = 2,576 ) had at least one risk factor for gdm and would have undergone testing . twenty percent of women ( n = 120 ) with gdm on universal screening had no nice risk factors and would have remained undiagnosed . fifty - eight percent of women ( n = 2,801 ) had at least one risk factor for gdm and would have undergone testing if the irish guidelines were applied . sixteen percent ( n = 101 ) had no risk factors and would have remained undiagnosed . seventy - six percent of women ( n = 3,656 ) had at least one risk factor according to the ada guidelines and would have undergone testing . five percent of women ( n = 31 ) identified with gdm by universal screening had no risk factors and would have remained undiagnosed . the prevalence of gdm among those with no risk factors was 2.7 , 4.9 , and 5.4% using ada , irish , and nice guidelines , respectively ( table 3 ) . numbers diagnosed with screening criteria we analyzed whether using a combination of age and bmi only was effective in terms of diagnostic accuracy . using a classification tree analysis , we found that women who had a low prevalence ( < 1% ) of gdm were those aged < 21 years with a bmi < 25 this can be compared with a prevalence of 7.7% in women aged > 21 years with a bmi 25 kg / m . age ( > 40 years ) is included as a risk factor in the irish guidelines . when applied to our cohort it , equated to a high specificity ( 94% ) for gdm ; however , the sensitivity was very low , at 9% . if age was reduced to 30 years the sensitivity increased to 73% , with a specificity of 34% . there is debate about which bmi cutoff should be considered as part of selective screening criteria . ada recommends 25 kg / m , nice , > 30 kg / m , and the irish guidelines , 30 kg / m . in our cohort , a bmi 30 kg / m was specific ( 81% ) , with intermediate sensitivity ( 48% ) . reducing the bmi cutoff to 25 kg / m , as indicated in the ada guidelines , increased the sensitivity to 80% , with a reduction of specificity to 44% . blood pressure of 140/90 mmhg has a very low sensitivity of 12% , although it is very specific ( 93% ) . of the women screened , 47% stated they had a first - degree relative with diabetes , which had intermediate sensitivity of 47% and specificity of 70% . when ethnicity was assessed , a higher proportion of non - caucasian women ( 28% ) were identified as having gdm compared with their caucasian counterparts ( 11% ) . it is highly specific ( 94% ) but has a very low sensitivity ( 16% ) given that the vast majority of our patients were caucasian . we examined pregnancy outcomes of women who had no risk factors but were diagnosed with gdm on universal screening , defined as the low - risk group of women with ngt ( table 4 ) . these low - risk women with gdm had more hypertensive disorders in pregnancy ( p = 0.029 ) , higher rates of total cesarean section deliveries ( p = 0.0001 ) , and more polyhydramnios ( p = 0.003 ) . infants had a nonsignificant increase in congenital malformations and significantly higher rates of admission to neonatal intensive care units ( nicu ; p = 0.0001 ) , despite treatment . we also compared outcomes in those with gdm who had no risk factors with those who had one or more risk factors . maternal and neonatal morbidities ( hypertensive disorders , previous miscarriages , caesarean section deliveries , admission to nicu , large for gestational age , and premature deliveries 36 weeks ) were higher in those who had at least one risk factor for gdm ( table 4 ) . gdm is common , with a rising prevalence , and is associated with higher maternal and neonatal morbidity . it carries additional long - term health consequences for the mother and her offspring ( 4,5 ) . diagnosis and appropriate treatment of the condition is associated with a reduction in morbidity ( 6,7 ) . given the lack of symptoms associated with this condition and the evidence - base accruing from several studies ( 5,810 ) , screening of asymptomatic patients is considered necessary . however , there is no consensus regarding which women should be screened and whether selective , risk factor based screening or a universal screening approach should be adopted . also , among those groups advocating selective screening , there is still considerable variability in the risk factors that are incorporated into a screening strategy . these uncertainties are based on a lack of randomized controlled trials indicating which approach is superior . our study aimed to apply the available screening strategies to our european , predominantly caucasian population . we set out to analyze whether the current , most commonly adopted national and international selective screening practices are appropriate compared with universal screening using iadpsg criteria ( 11 ) . through unselected screening of all women and recording of risk factors , we were able to evaluate the usefulness of selective screening for the detection of gdm and assess how many women would have remained undiagnosed if selective screening rather than universal screening were applied . we have shown that selective screening misses a significant proportion of women and that outcomes in these pregnancies are worse compared with ngt pregnancies . it is important to note that less than half ( n = 5,500 [ 44% ] ) of those offered screening attended . we have found this to be related primarily to the woman s geographical location relative to the screening site in addition to her socioeconomic status ( 12 ) . we also found that those with risk factors for gdm were more likely to attend for screening , which is a potential confounder in the prevalence rates of gdm . finally the 75-g oral glucose tolerance test was administered at an isolated appointment , which may have affected attendance rates , because the appropriate time for testing ( i.e. , 2428 weeks ) does not coincide with the national routine antenatal schedule . some have argued that universal screening will identify more women with gdm in a low - risk population and that these might have less clinical significance ( 13 ) . also , some of the trials showing improvements in morbidity outcomes used selective screening strategies , and as such , contained higher - risk populations or a high proportion of women of non - caucasian ethnicity , which is different from our european population ( 5,14 ) . contrary to this theory of low - risk gdm , studies have shown similar poor outcomes among young , lean women with gdm ( 15,16 ) . we addressed this by analyzing outcomes in this low - risk cohort with gdm and compared them with women with ngt as well as with women who had gdm and risk factors for the condition . despite diagnosis and subsequent treatment , pregnancy outcomes were worse for the mother and baby in those with the pregnancy outcomes in this group , however , were better than for those with gdm and risk factors . this suggests that although these women carried a lower risk than women with risk factors , they still had poorer outcomes compared with women with ngt . we believe the higher rate of caesarean section and admission rates to the nicu in those with gdm and no risk factors compared those with ngt was partly due to local obstetric practice and the label of gdm rather than to clinical indication . this raises issues regarding appropriate management of these women and education of those involved in clinical care . the current argument for screening lies in several large studies , including the australian carbohydrate intolerance study in pregnant women ( achois ) ( 10 ) trial and the study by landon et al . ( 6 ) , which showed that treating even mild levels of glucose intolerance resulted in a reduction in perinatal mortality and morbidity ; as such , this is accepted to be indirect evidence of the role of screening . of course , levels of glucose intolerance , whether mild or severe , can not be known before actually performing glucose tolerance testing , and instead , risk factors for the condition are accepted as surrogate markers for potential dysglycemia . cohorts such as the achois , however , comprised 25% of higher - risk ethnic minorities , and thus are not directly applicable to our population . the hyperglycemia and adverse pregnancies outcomes ( hapo ) trial ( 5 ) also consisted of 52.7% non - caucasian ethnicities . as previously shown , 18% of this cohort continued to have prediabetes / diabetes in the postpartum period ( 17 ) , and this has further risen to 30% at a median of 2.5 years after the index pregnancy ( 18 ) . of women who progressed to prediabetes / diabetes postpartum , prediabetes / diabetes in the first year postpartum appears to be reduced by breast - feeding , and thus , diagnosis and subsequent encouragement to breast - feed is important , although long - term data in this area are lacking . there are also long - term consequences for the offspring related to fetal hyperinsulinism and -cell hyperstimulation as well as changes in the hypothalamus ( 19 ) . if gdm can be detected and treated in a timely fashion using an appropriate population - specific screening strategy , then hyperstimulation of the fetal pancreas may be avoided , with consequences downstream for the adult health of the infant . during the routine antenatal appointments at our five study centers , the following details were recorded : age , parity , obstetric history , family history of diabetes , bmi , ethnicity , and blood pressure . details such as history of polycystic ovarian syndrome , signs of insulin resistance , lipid panel , physical inactivity , or previous steroid use are not routinely recorded . our practice is representative of that at a national level , and thus , those variables may be of limited usefulness to a screening strategy . also , applying selective screening is time - consuming during routine obstetric assessment and may in fact result in missing gdm in women who do have risk factors because of inadequate recording . because of the lack of international uniformity and financial constraints , we set out to assess the various selective screening guidelines . our analysis suggests that the ada guidelines would result in the highest diagnosis rate with the lowest number of women misclassified as not having gdm . if we are to adjust our current guidelines , we suggest including bmi 25 kg / m rather than the current recommendation of 30 kg / m because this alone will result in correctly diagnosing 80% of women with gdm . we have also shown that women aged < 21 years who have a bmi < 25 kg / m this would incur a cost - saving , but this cohort is small and comprises 2% of the entire group .

objectivethe optimal screening regimen for gestational diabetes mellitus ( gdm ) remains controversial . risk factors used in selective screening guidelines vary . given that universal screening is not currently adopted in our european population , we aimed to evaluate which selective screening strategies were most applicable.research design and methodsbetween 2007 and 2009 , 5,500 women were universally screened for gdm , and a gdm prevalence of 12.4% using international association of diabetes in pregnancy study groups ( iadpsg ) criteria was established . we retrospectively applied selective screening guidelines to this cohort.resultswhen we applied national institute for health and clinical excellence ( nice ) , irish , and american diabetes association ( ada ) guidelines , 54% ( 2,576 ) , 58% ( 2,801 ) , and 76% ( 3,656 ) of women , respectively , had at least one risk factor for gdm and would have undergone testing . however , when nice , irish , and ada guidelines were applied , 20% ( 120 ) , 16% ( 101 ) , and 5% ( 31 ) of women , respectively , had no risk factor and would have gone undiagnosed . using a bmi 30 kg / m2 for screening has a specificity of 81% with moderate sensitivity at 48% . reducing the bmi to 25 kg / m2 ( ada ) increases the sensitivity to 80% with a specificity of 44% . women with no risk factors diagnosed with gdm on universal screening had more adverse pregnancy outcomes than those with normal glucose tolerance.conclusionsthis analysis provides a strong argument for universal screening . however , if selective screening were adopted , the ada guidelines would result in the highest rate of diagnosis and the lowest number of missed cases .

. failed endotracheal intubation and inadequate ventilation with insufficient oxygenation may lead to serious complications , even death . management of an unexpectedly difficult airway consists of laryngeal mask ventilation , gum - elastic bougie and video laryngoscopy - assisted intubation . gum - elastic bougie is the easiest and cheapest tool used in case of an unexpected difficult intubation occurring in the operating room . a 53-year - old male patient with hypogonadotropic hypogonadism presented as an unexpected difficult intubation after the induction of anesthesia . a difficult airway has been described in patients with a variety of endocrine disorders , including pituitary diseases , but not with hypogonadism . there may be an unrevealed relationship between hypogonadism and difficult airway . gum - elastic bougie is still the most attainable and effective tool in the operation room in this situation . anesthesiologists are responsible for maintaining a patent airway in the anesthetized patient . failed endotracheal intubation and inadequate ventilation with insufficient oxygenation may lead to serious complications , as even a few minutes of deoxygenation can result in catastrophic outcome like brain damage or even death.1 anesthesiologists are occasionally faced and challenged with this significant problem in practice . a difficult airway has been described in patients with pituitary endocrine disorders such as acromegaly and dwarfism ; however , airway problems with hypogonadism have not been well identified in the literature.2 a 53-year - old married man ( weight : 85 kg , height : 187 cm , body mass index : 24 kg / m ) presented with a history of nasal obstruction for two years . he had not had any prior operation under general anesthesia , so he did not have any history of difficult intubation , and he did not have any chronic systemic disease . the patient was evaluated for obstructive sleep apnea syndrome ( osas ) with a comprehensive questionnaire on his sleeping habits and medical history ; no complaints or predictors pertaining to osas were identified . the patient s preoperative airway assessment was normal , mallampati class was ii , thyromental distance was 7 cm , inter - incisor gap was 5 cm , and head extension was > 35. his physical examination was characterized by lack of secondary sexual characteristics and presence of fine facial wrinkles . although , as previously indicated , the patient was married , he had had no children . his hormone profile was : testosterone 0.3 ng / ml ( reference range 1.757.81 ) , free testosterone 0.91 ( reference range 4.542.0 ) , prolactin 1.31 ng / ml ( reference range 2.6426.72 ) , luteinizing hormone ( lh ) 0.33 miu / ml ( reference range 1.24103.03 ) . no other pathological finding was obtained as the result of magnetic resonance imaging of the pituitary gland . he was admitted to the operating theater , and following the induction of anesthesia with a dose of 5 mg / kg intravenous thiopental , bag - mask ventilation was barely sustained . fentanyl ( 12 gr / kg ) and , as a muscle relaxant , rocuronium ( 0.6 mg / kg ) were administered . while the patient s head was in the sniffing position , direct laryngoscopy and intubation of the trachea were attempted three times with different sizes of macintosh and miller blades by an assistant professor of anesthesiology with 5 years experience . however , unfortunately , the intubation failed . the lungs were then ventilated with 100% oxygen via a face mask in order to avoid desaturation . glottic visualization was assessed with cook s modification of the cormack lehane classification ; a grade of 3a ( with direct laryngoscopy , only the epiglottis can be visualized ; the epiglottis can be lifted using an introducer or bougie ) was assigned.3 the patient was subsequently successfully intubated with a gum - elastic bougie . after the operation , the patient was extubated successfully without any complication and then examined by otorhinolaryngologists via fexible laryngoscopy . the epiglottis was found to be in a slightly lower than normal position ( figure 1 ) . the overall incidence of unexpected difficult intubation is estimated to be 1%3% , while failed intubation incidence is 0.05%0.35% in europe.1 difficult endotracheal intubation has been described in patients with pituitary diseases like acromegaly , cushing s disease , lh / follicle - stimulating hormone - secreting adenoma , thyroid - stimulating hormone - secreting adenoma , nonfunctional adenoma and prolactinoma.4 difficult airway with dwarfism has also been described . however , difficult airway with a hypogonadotropic patient has not yet been clearly defined . in our case , further , magnetic resonance imaging of the pituitary gland did not reveal any adenoma or mass lesion.2 pulsatile release of follicle stimulating hormone and lh in puberty instigates the development of the male larynx and secondary sexual characteristics . the growth of laryngeal cartilages and maturation of the male larynx is also facilitated by androgen . high - affinity androgen receptors have been found in the human larynx.5,6 in this case , the patient s laryngeal development was compatible with his age and sex . only the epiglottis was located in a slightly lower position than normal , partially leaning toward the laryngeal passage . the patient was infertile and lacked secondary sexual characteristics due to insufficient exposure to androgens . the rate of difficult intubation has been found to be higher in osas patients than the normal , healthy population.7 body mass index , mallampati grading , cormack lehane classification , sternomental distance , and thyromental distance evaluation might be predictive factors for difficult airway in osas patients . as such , anesthesiologists must be aware of this fact and be prepared for difficult intubation in osas patients.7 however , the rate of difficult intubation has not been found to be related to the severity of osas.8 no significant relationship between the apnea hypopnea index and difficult intubation has been found.8 in our patient , no apnea and daytime sleepiness were present in his history . only snoring was present , which might well have been associated with nasal septal deviation . his body mass index was 24 kg / m , mallampati class was ii , thyromental distance was 7 cm , inter - incisor gap was 5 cm , and head extension was above 35. these findings did not suggest any evidence of osas or difficult airway . this multiparameter test reveals the presence and severity of osas.9 in this case , as there was no clinical index of suspicion for osas and to keep costs down , polysomnography was not required by the otorhinolaryngologists . thus , anesthesiologists must take into account difficult airway in endocrinological disorders like hypogonadotropic hypogonadism and be prepared for difficult intubation .

backgrounda critical aspect of safe general anesthesia is providing adequate ventilation and oxygenation . failed endotracheal intubation and inadequate ventilation with insufficient oxygenation may lead to serious complications , even death . anesthesiologists rarely encounter unexpected difficult airway problems in daily routine . management of an unexpectedly difficult airway consists of laryngeal mask ventilation , gum - elastic bougie and video laryngoscopy - assisted intubation . gum - elastic bougie is the easiest and cheapest tool used in case of an unexpected difficult intubation occurring in the operating room.casea 53-year - old male patient with hypogonadotropic hypogonadism presented as an unexpected difficult intubation after the induction of anesthesia . no pathological finding or predictor of difficult intubation was present . in addition , bag - mask ventilation was poor and inadequate . the patient was finally successfully intubated with a gum - elastic bougie.conclusiona difficult airway has been described in patients with a variety of endocrine disorders , including pituitary diseases , but not with hypogonadism . there may be an unrevealed relationship between hypogonadism and difficult airway . gum - elastic bougie is still the most attainable and effective tool in the operation room in this situation .

acute respiratory infection ( ari ) is the major cause of morbidity and mortality worldwide , especially in young children . respiratory viruses such as respiratory syncytial virus ( rsv ) , influenza virus type a and b , parainfluenza virus 1 - 3 and adenovirus are the most common pathogens affecting the respiratory tract of young children ( 1 ) . human metapneumovirus ( hmpv ) is associated with both upper and lower respiratory tract infections among infants and children worldwide ( 2 ) . it was first identified as a novel respiratory virus in 2001 among children with ari in the netherlands . the hmpv belongs to the paramyxoviridae family , pneumovirinae subfamily , and the genus metapneumovirus ( 3 , 4 ) . the rsv is the most common etiological agent of viral lower respiratory tract infection and is considered as the most important cause of viral bronchiolitis in children . respiratory syncytial virus belongs to the paramyxoviridae family , pneumovirinae subfamily , and is placed under the genus pneumovirus ( 5 , 6 ) . the clinical presentation of hmpv infection ranges from a mild upper respiratory tract infection to severe lower respiratory tract disease accompanied by bronchiolitis and pneumonia ( 6 ) . while rsv , influenza virus and parainfluenza virus are considered to be the most common respiratory viruses , the role of other less known viruses such as hmpv in ari children remains largely unknown ( 7 ) . despite studies showing the prevalence of hmpv infection , the actual role of hmpv in children with severe ari in iran molecular techniques such as the polymerase chain reaction ( pcr ) are sensitive methods to define respiratory viruses that may be present in clinical specimens , especially in children suffering from respiratory infections ( 8) . this study had two objectives . first , to determine the hmpv and rsv frequency and their co - infection in hospitalized ari children , aged less than five years old , at one of the children s referral hospitals in iran . second , to describe the seasonal prevalence of hmpv and rsv among this group from march 2010 till march 2013 . this descriptive study was based on the processing of nasopharyngeal or throat swabs from 158 children , aged under five years old , who were admitted to aliasghar children hospital of iran university of medical sciences with acute respiratory infections from march 2010 till march 2013 ; this was done for the determination of the frequency of hmpv and rsv infections and co - infections . the ari children aged less than five years , having tachypnea with or without respiratory distress and cough , were included in this study . the specimens were placed into a vial containing 1 ml of transport media and were sent to the payvand clinical and specialty laboratory , where they were tested by real - time polymerase chain reaction for hmpv and rsv . nucleic acids from specimens were extracted using the qiaamp viral rna extraction mini kit ( qiagen gmbh , hiden , germany ) , according to the manufacturer s recommended protocol . reverse transcription was performed with the use of random hexamers and superscript first - strand ( invitrogen ) , according to the manufacture s instructions . real - time pcr was done using the rotor - gene 6000 from corbet research ( australia ) . the properties of the oligonucleotide primers and probes of hmpv and rsv are shown in table 1 ( 9 ) . the limit of detection for the hmpv and rsv real - time pcr are 100 copies per reaction . there are no cross - reaction between both viruses and other respiratory viruses such as influenza virus a and influenza virus b , parainfluenza virus 1 to 4 , and adenovirus , implying a high specificity . beta-2-microglobulin ( b2-mg ) dna was used as the internal control to monitor nucleic acid extraction . the sequence of the primer and the probe of b2-mg are shown in table 1 ( 10 ) . the pcr conditions were as follows : reverse transcription at 50c for 30 minutes , initial pcr denaturation at 95c for 10 minutes , followed by 45 cycles of denaturation at 95c for 15 seconds , annealing at 58c for 60 seconds . the cycle threshold values of 39.9 were regarded as positive and cycle threshold values of 40 were regarded as negative . the results were discarded for any specimen with a negative internal control . statistical analysis for comparison of relative values this descriptive study was based on the processing of nasopharyngeal or throat swabs from 158 children , aged under five years old , who were admitted to aliasghar children hospital of iran university of medical sciences with acute respiratory infections from march 2010 till march 2013 ; this was done for the determination of the frequency of hmpv and rsv infections and co - infections . the ari children aged less than five years , having tachypnea with or without respiratory distress and cough , were included in this study . the specimens were placed into a vial containing 1 ml of transport media and were sent to the payvand clinical and specialty laboratory , where they were tested by real - time polymerase chain reaction for hmpv and rsv . nucleic acids from specimens were extracted using the qiaamp viral rna extraction mini kit ( qiagen gmbh , hiden , germany ) , according to the manufacturer s recommended protocol . reverse transcription was performed with the use of random hexamers and superscript first - strand ( invitrogen ) , according to the manufacture s instructions . real - time pcr was done using the rotor - gene 6000 from corbet research ( australia ) . the properties of the oligonucleotide primers and probes of hmpv and rsv are shown in table 1 ( 9 ) . the limit of detection for the hmpv and rsv real - time pcr are 100 copies per reaction . there are no cross - reaction between both viruses and other respiratory viruses such as influenza virus a and influenza virus b , parainfluenza virus 1 to 4 , and adenovirus , implying a high specificity . beta-2-microglobulin ( b2-mg ) dna was used as the internal control to monitor nucleic acid extraction . the sequence of the primer and the probe of b2-mg are shown in table 1 ( 10 ) . the pcr conditions were as follows : reverse transcription at 50c for 30 minutes , initial pcr denaturation at 95c for 10 minutes , followed by 45 cycles of denaturation at 95c for 15 seconds , annealing at 58c for 60 seconds . the cycle threshold values of 39.9 were regarded as positive and cycle threshold values of 40 were regarded as negative . statistical analysis for comparison of relative values was performed by the z - test , using the stata software version 8 . from march 2010 to march 2013 , 158 specimens were obtained from hospitalized children under five years old for evaluation of rsv and hmpv infections . the majority of respiratory specimens ( more than 65% ) were from children less than one year old . overall , 31.1% ( 49/158 ) of samples were positive for rsv , and 5.7% ( 9/158 ) of the specimens were positive for hmpv . among the 158 patient specimens , 63.2% ( 100/158 ) were negative for rsv and hmpv ( table 2 ) . ( % ) twenty - seven ( 55.2% ) of the rsv - infected patients were females and 22 ( 44.8% ) were males . five ( 55.5% ) of the hmpv - infected children were male while four ( 44.5% ) were female . all patients with positive results for rsv and hmpv were divided into four age groups : < 6 months , 6 - 12 months , 1 - 2 years and 2 - 5 years . in the < 6 months group , the proportion of rsv cases was significantly greater ( p < 0.05 ) than the proportion of hmpv cases , while in the children with 6 - 12 months , 1 - 2 years and 2 - 5 years , the proportions of rsv and hmpv were nearly the same ( table 2 ) . the seasonal distribution of detected viruses over the three - year study period is shown in figure 1 , during which two peaks of rsv infections occurred annually , first during december 2010 to february 2011 ( 31.4% ) and the second during december 2011 to february 2012 ( 53% ) . the hmpv infections were mainly detected from december 2010 to february 2011 and december 2012 to february 2013 , when 11.4% and 15.4% of infections occurred , respectively . from march 2010 to march 2013 , 158 specimens were obtained from hospitalized children under five years old for evaluation of rsv and hmpv infections . the majority of respiratory specimens ( more than 65% ) were from children less than one year old . overall , 31.1% ( 49/158 ) of samples were positive for rsv , and 5.7% ( 9/158 ) of the specimens were positive for hmpv . among the 158 patient specimens , 63.2% ( 100/158 ) were negative for rsv and hmpv ( table 2 ) . twenty - seven ( 55.2% ) of the rsv - infected patients were females and 22 ( 44.8% ) were males . five ( 55.5% ) of the hmpv - infected children were male while four ( 44.5% ) were female . all patients with positive results for rsv and hmpv were divided into four age groups : < 6 months , 6 - 12 months , 1 - 2 years and 2 - 5 years . in the < 6 months group , the proportion of rsv cases was significantly greater ( p < 0.05 ) than the proportion of hmpv cases , while in the children with 6 - 12 months , 1 - 2 years and 2 - 5 years , the proportions of rsv and hmpv were nearly the same ( table 2 ) . the seasonal distribution of detected viruses over the three - year study period is shown in figure 1 , during which two peaks of rsv infections occurred annually , first during december 2010 to february 2011 ( 31.4% ) and the second during december 2011 to february 2012 ( 53% ) . the hmpv infections were mainly detected from december 2010 to february 2011 and december 2012 to february 2013 , when 11.4% and 15.4% of infections occurred , respectively . the study shows that 31.1% and 5.7% of rsv and hmpv infections , respectively , occurred among iranian children < 5 years of age , hospitalized with acute respiratory tract infections . the prevalence of hmpv infection in iranian children with ari varied from 0.49% to 54.4% , which was not previously reported in iran ( 11 , 12 ) . herein , the proportion of acute respiratory infection of hmpv - positive cases among children under the age of 5 in our study ( 5.7% ) was similar to studies from the middle - east regions such as amman ( 6% ) and other regions such as brazil ( 5.6% ) ( 7 , 13 ) . there are also other reports on the frequency of rsv in hospitalized children in iran . ( 14 ) studied hospitalized children under five years old with acute respiratory tract infection and found a prevalence of 17.2% for rsv , and malekshahi et al . ( 11 ) assessed rsv among children less than six years old with ari and reported that it was the cause of 16.8% cases ( 11 , 14 ) . ( 15 ) observed that among 100 children with acute respiratory infection , 9% were infected with rsv . the above - mentioned results were lower than what was found in this study ( 31.1% ) , which is more similar to the findings of moattari et al . . they found that among 280 children under five years old , 84 ( 30.0% ) were infected with rsv . there are several studies showing co - infection with rsv and hmpv ( 3 , 5 ) . ( 16 ) observed that 10 ( 3.5% ) of the studied patients were infected with both viruses , yet in the current study there was no co - infection with rsv and hmpv , which was similar to the findings of mullins et al.(17 ) . the difference between the prevalence rate of hmpv and rsv infections and co - infection with both viruses in several studies may be described by different groups of patients , methods used for detection of viruses , yearly variation in incidence and other variables , such as age , population density , socioeconomic factor and climate changes . it was also found that hmpv - infected children were older than rsv - infected cases . the rsv was found predominantly in children < 1 year old , while hmpv infection occurred mainly in children aged 1 - 5 years . such finding is similar to other studies on hmpv epidemiology ( 17 - 20 ) . the reason of such difference may be explained by longer lasting maternal immunity to hmpv compared with rsv . the hmpv seasonal distribution was determined by the peaks within the winter and the spring . this finding was observed in other hmpv studies as well ( 8 , 21 ) . in addition , rsv infection occurred largely during the winter months , similar to those previously observed elsewhere(5 , 7 , 22 , 23 ) . in accordance with several studies , the seasonal occurrence of hmpv infection mainly overlaps with rsv infections ( 8 , 24 ) . hence it seems that hmpv is less important than rsv as a cause of ari , especially in children under one year old and the seasonal occurrence of both viruses is the same . large - scale follow - up epidemiological studies are needed to fully explore all respiratory viruses causing ari in iranian young children and assess seasonally patterns .

backgroundacute respiratory infection plays an important role in hospitalization of children in developing countries ; detection of viral causes in such infections is very important . the respiratory syncytial virus ( rsv ) is the most common etiological agent of viral lower respiratory tract infection in children , and human metapneumovirus ( hmpv ) is associated with both upper and lower respiratory tract infections among infants and children.objectivesthis study evaluated the frequency and seasonal prevalence of hmpv and rsv in hospitalized children under the age of five , who were admitted to aliasghar children s hospital of iran university of medical sciences from march 2010 until march 2013.patients and methodsnasopharyngeal or throat swabs from 158 hospitalized children with fever and respiratory distress were evaluated for rsv and hmpv rna by the real - time polymerase chain reaction ( pcr ) method.resultsamong the 158 children evaluated in this study , 49 individuals ( 31.1% ) had rsv infection while nine individuals ( 5.7% ) had hmpv infection . five ( 55.5% ) of the hmpv - infected children were male while four ( 44.5% ) were female and 27 ( 55.2% ) of the rsv - infected patients were females and 22 ( 44.8% ) were males . the rsv infections were detected in mainly < one year old children and hmpv infections were detected mainly in > one year old children . both rsv and hmpv infections had occurred mainly during winter and spring seasons.conclusionsrespiratory syncytial virus was the major cause of acute respiratory infection in children under one - year of age while human metapneumovirus had a low prevalence in this group . the seasonal occurrence of both viruses was the same .

hepatic resection is considered first - line therapy for many primary and metastatic liver tumors . thanks to a more careful perioperative management , the use of highly accurate pre - operative imaging and the refinement of surgical techniques , hepatic resection has been applied more extensively and successfully . post - operative mortality has currently been reduced to less than 5% in almost all groups , whereas 5-year survival after hepatic resection for colorectal metastases , primary tumors or other types of non - colorectal metastases has increased from 30 to 50% . in the past , patients with large tumors involving the inferior vena cava ( ivc ) were not considered candidates for surgical resection . however , these untreated patients had a poor survival rate , less than 12 months , even when using palliative chemotherapy . in recent years , the improvement of surgical techniques with a detailed increase in knowledge of the segmental anatomical structure of the liver has permitted liver resections that were until recently deemed high - risk surgery . innovative and aggressive surgical techniques , principally derived from transplant surgery , such as hepatic vascular exclusion ( hve ) , veno - venous bypass and ex vivo hepatic resection , have been reported in dealing with hepatic tumors involving the ivc . graft prosthetics and autologous or synthetic patches are methods extensively mentioned in the literature for repair of the ivc . in a woman of 67 years in follow - up at our clinic after right hepatectomy performed 3 years earlier , for colorectal hepatic metastasis , a new lesion was detected in the liver . two years before the hepatic resection , the patient had been operated on laparoscopically for left colectomy for adenocarcinoma g2 , t3 , n1 , m0 ( according to tnm , sixth edition ) . quadriphase contrast - enhancement abdomen and chest computed tomography ( ct ) showed a hypodense lesion in liver segment iv , 4 cm in diameter , infiltrating the intrahepatic portion of the inferior vena cava . prior to surgery the patient underwent magnetic resonance imaging ( mri ) with vascular reconstruction in order to evaluate the relationship between the tumor , the vena cava and the hepatic veins in detail . in addition , the patient underwent a stress echocardiogram so as to evaluate the heart function in view of a total hepatic vascular exclusion . the access laparotomy consisted of a bilateral subcostal incision on the previous surgical scar . after extensive and laborious adhesiolysis , since part of the colon and small intestine completely occupied the right upper quadrant , it was possible to expose the liver . an intraoperative ultrasound scan ( ious ) confirmed the preoperative findings and excluded other metastases in liver segments ii iii . the operation began by exposing the inferior vena cava below the tumor and above the hepatic vein ; both parts of the ivc were surrounded by vessel loops . the liver parenchyma was divided along the umbilical fissure ( on the left side ) , by using bipolar forceps . central venous pressure was constantly maintained at or below 5 cm of h2o during the parenchymal transection to minimize the risk of bleeding . during the transection , non - selective we then proceeded with the placement of a vascular clamp on the infrahepatic vena cava and the other clamp on the suprahepatic vena cava ( above the hepatic vein ) , completing the resection of segment iv associated with the excision of retrohepatic vena cava ( fig . the caval reconstruction was via the interposition of a 20 mm ringed - ptfe tube graft , in total vascular exclusion for about 25 min . in the icu , heparin iv anticoagulation was started , maintaining an activated partial thromboplastin time ratio ( aptt ) of 2.53 . on the fifth post - operative day , the infusion of iv heparin was stopped and changed to subcutaneous injection of low molecular weight heparin ( enoxaparin ) ; at the same time , we started the administration of antioral platelet drugs . the postoperative period was characterized by no major surgical complications , although the patient developed right pleural effusion . six months post - operatively , the patient is in good health and tumor - free . hepatic resection for liver mts has been shown to result in better prognosis than other treatments . hepatic resection of tumors located in the middle of the liver and invading the ivc was considered until recently an absolute contraindication to surgery . however , thanks to innovative techniques , these types of liver resections offer a chance of cure to patients who until recently were not considered candidates for any surgical treatment . the benefit of hepatic resection combined with resection and reconstruction of the vena cava is obvious , since it is an attempt to achieve oncological radicality , otherwise impossible . recent papers have clarified the safety and advantages of combining excision of the ivc in continuity with hepatic resection for liver malignancy invading the ivc or hepatic vein . several surgical techniques are reported in the literature in dealing with tumors involving the ivc ( table 1 ) . the type of surgical strategies that can be used depends on the extension of the tumor along the inferior vena cava . when the tumor invades a small portion of the ivc , it is possible to apply a clamp tangentially ; in this case , the ivc can be repaired primarily with a venorraphy or with a patch in enlargement , provided that there is no excessive narrowing of the vascular lumen . caval stenosis , in fact , causes persistent edema of the lower limbs and , in severe cases , renal dysfunction . in cases where tumor invasion is quite extensive , resection of the inferior vena cava requires the interruption of the caval flow . in this case , two different strategies can be used : when the involvement of the ivc is below the hepatic veins , then a clamp is placed above the tumor but below the outlet of the hepatic veins into the ivc ( clamping of the intrahepatic cava ) with the advantage of being able to maintain the blood flow from the liver to the heart while following resection and reconstruction of the ivc . when the clamp is placed on the cava above the hepatic veins , then total vascular exclusion ( hve ) the latter can be performed with or without veno - venous bypass , depending on the hemodynamic stability of the patient . in this case , it is advisable to perform the vascular exclusion test in order to check the stability of the circulation and heart function ; however , bypass is recommended in those patients with heart disease or kidney failure . it has been suggested , in cases of total vascular exclusion , to begin to minimize warm ischemia of the liver before the caval resection . in our case , a total vascular exclusion was achieved as not enough room was available to place the clamp below the outlet of the hepatic vein , since the tumor was close to the hepato - caval junction . however , it is preferable to reduce this time to 60 min in cases of diseased liver . in fact , despite undergoing ischemia for 25 min ( hve ) plus 12 during the pringle maneuver , our patient did not show any signs of postoperative liver failure . in situ and ex situ ( bench surgery ) hepatic resection is a particularly demanding surgical alternative , applicable in those cases in which the resection of the ivc is also associated with the reconstruction of the hepatic veins . in the literature , many varied surgical options are described in the reconstruction of the ivc , when this can not be repaired primarily . autologous veins have been used with success ; however , prosthetic materials are feasible and produce a long patency . an 1820 mm goretx or dacron graft appears superior in terms of patency of that resistance , and this prosthesis is , therefore , the one most frequently used in different surgical settings to reconstruct the cava . the most serious problems that may arise with the use of the prosthesis are infection and thrombosis . the risk of infection after liver resection is a well - documented problem , which varies between 8 and 28% , thus some authors have recommended the use of an omental wrap to protect the caval prosthesis . patency of the caval graft is 70% at 5 years , and systemic heparinization started postoperatively , with subsequent conversion to oral warfarin sodium , is therefore highly recommended . little has been published on the outcome of primary and metastatic liver tumors involving the inferior vena cava , as this surgery is still considered too complex and high risk for mortality and morbidity . in the most important cohorts that appear in the literature , the mortality rate is between 9 and 30% . despite the high risk faced by patients with this tumor , this high - risk surgery offers the only chance of curing tumors that would otherwise be considered non - resectable . however , it is worth noting that 5-year overall survival varies between 22 and 30% , an outcome entirely comparable to the survival of patients undergoing hepatic resection for colon - rectal metastasis . at present , patients with large tumors involving both liver and vena cava may be candidates for liver resection and replacement of the vena cava , achieving long - term survival in selected cases . the surgical techniques used in the resection of these tumors require specialized centers with surgeons experienced in complex processes of hepato - biliary surgery.key learning pointshepatic resection of tumors invading the ivc was considered until recently an absolute contraindication to surgery.innovative and aggressive surgical techniques , such as hepatic vascular exclusion ( hve ) , veno - venous bypass and ex vivo hepatic resection , have been reported in dealing with hepatic tumors involving ivc.patients with large tumors involving both liver and vena cava may be candidates for liver resection and replacement of the vena cava achieving long - term survival in selected cases . key learning pointshepatic resection of tumors invading the ivc was considered until recently an absolute contraindication to surgery.innovative and aggressive surgical techniques , such as hepatic vascular exclusion ( hve ) , veno - venous bypass and ex vivo hepatic resection , have been reported in dealing with hepatic tumors involving ivc.patients with large tumors involving both liver and vena cava may be candidates for liver resection and replacement of the vena cava achieving long - term survival in selected cases . hepatic resection of tumors invading the ivc was considered until recently an absolute contraindication to surgery . innovative and aggressive surgical techniques , such as hepatic vascular exclusion ( hve ) , veno - venous bypass and ex vivo hepatic resection , have been reported in dealing with hepatic tumors involving ivc . patients with large tumors involving both liver and vena cava may be candidates for liver resection and replacement of the vena cava achieving long - term survival in selected cases . written informed consent was obtained from the patient for the publication of this case report and accompanying images . gian piero guerrini ph.d . contributed in data collection , analysis and writing the manuscript . paolo soliani md was the 1st surgeon who performed the hepatic resection and contributed in writing the article .

introductionadvanced tumors of the liver involving the inferior vena cava ( ivc ) have always been considered a contraindication to surgery.presentation of casewe report a case of a patient , who previously underwent right hepatectomy , with recurrence of colorectal liver metastasis invading the ivc . the patient had a liver resection together with replacement of the vena cava using a ringed polytetrafluoroethylene ( ptfe ) graft tube . the operation was carried out in hepatic vascular exclusion ( hve ) without the use of veno - venous bypass . the patient was healthy and tumor - free at 6 months post-surgery.discussionin patients with hepatic malignancy involving the ivc , extended hepatic resection and reconstruction of the ivc is often the prerequisite to obtaining a resection margin.conclusionextended hepatic resection with ivc reconstruction for hepatic malignancy may offer a chance of cure to selected patients who otherwise have poor survival rates .

non - small cell lung cancer ( nsclc ) can exhibit rearranged driver oncogenes , which are possible targets for therapy.1 the most frequently identified driver oncogenes in nsclc leading to targeted therapy are epidermal growth factor receptor ( egfr ) and anaplastic lymphoma kinase ( alk).1,2 the prevalence of these driver oncogenes is significantly influenced by race , smoking habits , and gender.3 the frequency of egfr - mutated nsclc in asian patients is known to be higher than in caucasian patients.3 in contrast , prevalence of alk rearrangements in nsclc is reported to be similar in caucasian and asian patients.3 in recent cohort studies with afro - americans , there is no significant difference in the prevalence of egfr mutations or alk rearrangements with caucasian americans.4,5 about other races , however , less or no data are available.6,7 for obvious reasons , little is known about africa . cancer , however , is a significant health problem in this continent , as it is estimated that there were 715,000 new cancer cases in 2008 , and additional data from southern africa and northern africa already confirm that lung cancer is the leading cause of death related to cancer.8 to our knowledge , there is no literature available about the prevalence of lung cancer and rearranged driver oncogenes in sub - saharan africans . as the latter is influenced by race and smoking habits that may differ from afro - americans ; we have studied the prevalence of egfr mutations and alk translocations in a case series of patients of sub - saharan african ethnicity with nsclc . we retrospectively studied all patients of sub saharan african ethnicity who have been treated for nsclc stage iv in our hospital ( university hospital saint pierre ) in brussels , belgium . the ethics committee of saint - pierre hospital deemed ethical approval not necessary as it was a retrospective study . investigations were performed on biopsies obtained during flexible bronchoscopy and these samplings were immediately fixed in neutral buffered formalin and embedded in paraffin.9 driver oncogenes on biopsy specimens were researched by using the truseq amplicon cancer panel ( illumina , illumina inc . , san diego , ca , usa ) . not only were egfr / alk mutations investigated , but also other mutations included in the panel , such as tumor protein p53 ( tp53 ) , proto - oncogene b - raf ( braf ) and kirsten rat sarcoma viral oncogene homolog ( kras ) . from july 2012 to november 2015 , 6 patients of sub - saharan origin with nsclc stage iv have been treated in our hospital . three patients originated from congo , an ex - belgian colony , 1 from djibouti , lfrom cameroon , and 1 from guinea . egfr mutation was present in 3/6 patients and alk rearrangement was present in 1/6 patients . two egfr mutations were common , i.e. , l858r ( exon 21 point mutation ) and deletion exon 19.10 the significance of the third egfr mutation ( t710i in exon 18 ) is unknown . all egfr mutations and both patients without egfr mutation and alk rearrangement were considered as light smokers ( < 10 pack - years ) . in our small case series , 4 out of 6 sub - saharan patients with nsclc had a driver oncogene , either egfr mutation or alk rearrangement . the prevalence of rearranged driver oncogenes in nsclc is known to be influenced by race , gender , and smoking status.3 differences in prevalence of egfr mutation status in nsclc between asian and caucasian patients have already been extensively studied since the early 2000s.2,3 egfr mutations ( exons 1822 ) are present in ~30% east asian patients with lung cancer , and more specifically in 35%47% of east asian patients with lung adenocarcinoma , whereas egfr mutations are present in ~7% of caucasian patients with lung cancer , and in 13%18% of caucasian patients with lung adenocarci - noma.3,4 even within the asian population , egfr mutation status in nsclc varies.6 the east asian population is the largest asian group studied ( japanese , korean , and chinese patients).3,4,6 in a large cohort of 907 indian patients , an egfr mutation was found in 23% of patients with nsclc and in 26% of lung adenocarcinomas.6 recently , egfr mutations in nsclc in afro - american patients have been investigated.4,5,11 there seems to be no difference in prevalence of egfr mutations in ncslc between caucasians and afro - americans . yamaguchi et al described a prevalence of egfr mutations in nsclc of 18.4% in white patients and in 18.2% of black patients in their patient cohort.4 bollig - fischer et al found a prevalence of 8% of egfr mutations in both black and white patients with nsclc.11 it is not clear whether these results for afro - american patients can be extrapolated to sub - saharan african patients with nsclc . errihani et al performed the only study , to our knowledge , on the prevalence of egfr mutation status in lung cancer patients in the african continent.7 they examined in a moroccan patient cohort the prevalence of egfr mutations in advanced lung adenocarcinoma . a prevalence of 21% egfr mutations in lung adenocarcinoma was found ( 29 out of 137 patients ) , which lies between the prevalence of egfr mutations in caucasian and asian patients with nsclc.7 it is unclear whether the results of the moroccan ( north african ) cohort also apply to sub saharan african patients with nsclc . besides race , the presence of driver oncogenes is influenced by gender and smoking history.3 in this limited series , the role of gender was impossible to assess . finally , smoking status independently influences egfr mutation status in nsclc.2,3,4 the prevalence is higher in never smokers or light smokers . in our study , 3 out of 6 patients had an egfr mutation . this incidence seems high , but interestingly , all our 6 patients were non - smokers or light smokers . in europe , the prevalence of tobacco use among adults is approximately 19% for women and 38% for men.12 in the african continent , at first sight , smoking prevalence does not seem to be different , as among males it ranges from 14% in swaziland to 40% in niger.13 however , according to a recent analysis of the american cancer society , most african countries remain in the early stages of the tobacco epidemic , with tobacco use relatively low compared to the rest of the world.14 on this basis , it is likely that the proportion of lung adenocarcinoma in non - smokers is still high and this may contribute to explaining the frequent egfr mutations in our case series . indeed , egfr mutations in nsclc have been found in 35%40% of caucasian never smokers , and in 55%70% of asian never smokers.3,4 we believe this explanation also applies for the finding of one alk translocation in our small series . the overall prevalence of alk translocation in nsclc is thought to be 3%5% , without ethnic difference , but with a higher prevalence in non - smokers.3,4 even if these results can not be extrapolated due to the small number of patients , the early stages of the tobacco epidemic in africa are probably associated with a high frequency of egfr mutation and alk rearrangement in nsclc . this will probably change with time , as africa is becoming a future epicenter of tobacco epidemic.13 in this small series , 4/6 patients of sub - saharan origin with nsclc presented driver oncogene rearrangements . this may be related to the fact that africa is still in the early stages of the tobacco epidemic , leading to suggest that the prevalence of driver oncogenes is high in this population . as the burden of lung cancer in sub - saharan countries is expected to rise in the next decennia,8 further investigations of nsclc subgroups in this racial group are required .

non - small cell lung cancer can exhibit driver oncogenes , including epidermal growth factor receptor ( egfr ) and anaplastic lymphoma kinase ( alk ) , that are possible targets for therapy . the prevalence of these rearranged driver oncogenes is influenced by race , smoking habits , and gender . most data come from caucasian and asian populations . to our knowledge , there is no literature available about the prevalence of driver oncogenes in sub - saharan africa , where the tobacco epidemic is still in the early stage . in this small case series , 6 patients of sub - saharan african ethnicity with stage iv lung adenocarcinoma are described . egfr mutation was present in 3/6 patients and alk rearrangement in 1/6 patients . this incidence seems high but interestingly , all patients were non - smokers or light smokers . in this series , the high prevalence of driver oncogene was probably related to low smoking habits and these initial data in sub - saharan africans suggest high prevalence of driver mutations for this reason .

cerebral cysticercosis is caused by infection of the central nervous system by the larvae of the pork tapeworm , taenia solium . the parasites can be located in the brain parenchyma , the subarachnoid space and the ventricular system in highly variable combinations . the frequency of the diagnosis has been increasing , presumably as a result of the widespead availability of computed tomography . empty - sella syndrome is the name given by busch to an enlargement of the sella due to bulging of the arachnoid through a defect of the dural diaphragm . pressure and pulsation of the cerebrospinal fluid supposedly act on the walls of the sella turcica so as to flatten the pituitary gland . the primary empty sella syndrome has been a subject of much interest and research in recent years on account of its clinical correlations with headache , visual defect , papilledema and endocrine disorders . in this paper we report a very rare case of total empty sella syndrome associated with cerebral cysticercosis . a 55-year - old korea woman , previously in good health , presented with severe recurrent headache and dizziness . she had no previous parasitic infections and had not ingested any raw or undercooked pork . the headache , which was localized on the frontal and the occipital areas , had a throbbing characteristic . it was not associated with any other specific signs or symptoms and there was also no evidence of migraine . six moths prior to admission , she had once visited a private clinic because of headache . she refused the recommended brain ct scan , but took some medicine prescribed by the clinic . examination of csf showed an opening pressure of 130mmcsf , 9 leukocytes / mm3 , a protein level of 30mg / dl and a glucose level of 49mg / dl . a ct scan showed multiple low - density cystic lesions in the suprasellar and left sylvian cisternae compatible with empty - sella syndrome ( fig . 1 ) . in order to evaluate the communication between the cisternae and the suprasellar cyst , 2 ) . enzyme linked immunosorbent assay ( elisa ) for anti - cysticerci igg in the patient s csf and serum gave titers of 1.14 and 0.95 , respectively , consistent with active infection ( table 2 ) . the reserve capacities were all prompt except for the tsh response which was blunted ( table 1 ) . t4 5ug / dl , ft4 0.86ng / ml , estradiol 25 pg / dl and prolactin 4.31ng / ml . after the patient gave an informed consent , praziquantel therapy was started at 50mg / kg body weight / day in three divided doses for 15 days . on the first day of praziquantel treatment , the patient complained of diffuse headache , and a low - grade fever and signs of mild meningismus developed . the therapy was continued with dexamethasone added to the original regimen from the second day on . she was discharged on an anticonvulsant medication with a tapering course of steroid . a follow - up ct scan , done one month after praziquantel therapy , showed a slight reduction in the size of all intracranial cysts ( fig . the term empty sella was applied by busch to an autopsy material in which the diaphragma sella was incomplete regardless of whether there had been prior surgical or radiotherapeutic interventions . later , weiss and raskind emphasized the need to distinguish primary ( without any prior surgical or - radiotherapeutic procedures ) from secondary ( following such procedures ) cases . because this patient had no past history of operation or radiotherapy , this case would be classified as primary empty - sella syndrome associated with multilobulated cystic lesions in the suprasellar cistern and the left sylvian cistern . the total empty sella syndrome associated with cns cysticercosis has not been reported so far . headache is by far the most frequent clinical manifestation of empty - sella syndrome ; on review of previous case studies , it appears to occur in approximately 80% of cases , while papilledema , visual defects and endocrine disorders occur in no more than 8% , 16% and 25% , respectively . in the case of neurocysticercosis , headache together with seizure is one of the most frequently manifested symptoms . in this patient , however , the origin of the headache might be the combination of the above two conditions , namely the empty sella and the neurocysticercosis , and it was very difficult to confirm whether the one was mainly caused by the other or the two were just incidentally associated with each other . several mechanisms of the pathogenesis of the empty - sella have been postulated : 1 ) rupture of intrasellar or parasellar cyst 2 ) infarction of the sellar contents , 3 ) pituitary hypertrophy and subsequent atrophy , and 4 ) transmission of cerebrospinal pressure through a congenitally defective sellar diaphragm of these etiologic hypotheses , the most commonly accepted one might be the transmission of either normal or elevated cerebrospinal pressure . an incomplete sellar diaphragm is an essential prerequisite to the development of the empty sella according to this hypothesis although it was not confirmed in this particular patient . kaufman called attention to the documented or suspected elevation of csf pressure , but in this patient the elevated csf pressure was not documented . it is speculated however , that a slightly longstanding elevation of csf pressure by the cyst of the pork tapeworm might have resulted in the enlargement and remodeling of the sellar turcica and in the flattening of the pituitary contents against the floor in the setting of the incomplete sellar diaphragm . we do not exclude , however , the possibility of a coincidental association of the total empty - sella syndrome with cysticercosis in this particular case . for the evaluation of pituitary functional reserve , a combined pituitary stimulation test was done . the reserve capacities were all within normal limits , but tsh response by trh was blunted . in the empty sella syndrome , abnormalities of pituitary function have been confined to failure of the growth hormone to increase following insulin induced hypoglycemia , arginine and glucose loads , diminished corticoid response , imparied tsh response , hypogonadotropism and virilization . surgery has been generally disappointing and largely limited to managing the secondary effect of the infection , and it is not helphul for the most common presentation of the disease , namely , diffuse parenchymal infection as observed in our patient . the medical treatment of neurocysticercosis is greatly improved by praziquantel , the first effective drug aganist cysticerocosis . the treatment with praziquantel clearly elicited an immunogenic effect , as evidenced by the devolpment of the cerebrospinal fluid syndrome accompanied by a significant rise in the levels of specific igg that followed treatment . most authors have stressed the importance of simultaneous use of steroid given with parziquantel to prevent the dampening of the inflammatory reaction that often follows the action of the antiparasitic agent on the larvae . early in the course of the praziquantel treatment without dexamethasone , this patient showed symptoms of cerebrospinal reaction syndrome such as headache , meningismus and fever . in this patient there would be no way of determining , retrospectively , if the cerebrospinal fluid reaction was really diminished in its intensity by the steroid therapy , but we were able to complete the praziquantel course in this patient without further complication . in patients with cns cysticercosis , the pituitary gland should be evaluated with a sella ct scan which can diagnose empty sella syndrome . inevitably , the reserve capacity of the pituitary gland should be investigated for the patient with empty sella syndrome .

a 55-year - old woman presented with severe recurrent headache accompanied by dizziness . the brain ct scan showed multiple low - density cystic lesions in the suprasellar and left sylvian cisternae with total empty sell syndrome . the communication between the cisternae and the suprasellar cyst was not verified on the metrizamide ct scan . treatment with praziquantel resulted in headache inilially and a rise in specific igg .

in metazoans , hemoglobin carries oxygen from the lungs , gills or other respiratory organs to peripheral tissues that need the oxygen for efficient metabolism . hemoglobin is composed of one or more globin polypeptide chains , which bind the iron - containing cofactor heme . other organisms use the oxygen - binding and redox capacity of hemoglobin in a variety of other ways . studies of globin genes and their evolution are driven by a desire to understand how this diversity of protein structures and functions was generated . current evolutionary ideas about gene duplication and divergence had their origin in studies of hemoglobin genes and other multigene families . in addition , interest in the hemoglobin genes is fueled by the realization that the most common genetic diseases in humans , such as sickle cell disease and inherited anemias , result from pathological variants in the hemoglobin genes . the quest to understand normal hemoglobin production and function and to learn what goes wrong in hemoglobinopathies should provide insights that could lead to effective therapeutic interventions . the hemoglobins found in erythroid cells of jawed vertebrates ( gnathostomes ) include two -like globins and two -like globins , each bound by a heme molecule . although the hemoglobins produced in embryonic or larval erythrocytes differ from those in adult erythrocytes , each has two copies of a polypeptide related to -globin , such as the embryonic -globin , and two copies of a polypeptide related to -globin , such as the embryonic -globin . the -like and -like globin gene clusters are on different chromosomes in mammals and birds , but one or more pairs of -like and -like globin genes are found in one locus in amphibians and fish ( figure 1 ) . the arrangement of genes in these contemporary species strongly suggested that either -like or -like globin genes moved to a new chromosomal location in the ancestor of birds and mammals . how this occurred , whether by duplication and specific gene loss or by translocation of one type of gene , was far from clear . this mechanism and the effects such a movement had on the regulation of the genes are intriguing questions , and recent work , such as that of patel et al top , gene clusters in contemporary species ; bottom , inferred gene arrangements in the last common ancestor to jawed vertebrates ; middle , some of the possible gene movements mapped onto an evolutionary tree ( thick gray lines ) . genes are indicated by boxes , with those above the line transcribed from left to right and those below the line from the right ; red , -like globin genes ; yellow , -like globin genes ; light blue , or genes ; other colors , other genes ; small orange circles , major regulatory regions . all the known genes encoding hemoglobins expressed in erythroid cells are shown , as well as other genes that are most consistently diagnostic for the loci . numbers to the left of each cluster specify the chromosome on which it is located ; for the frog gene clusters , the scaffold number ( preceded by s ) is given . the greek letter name is specified for hemoglobin genes in human , platypus and chicken , but generic ' -globin ' or ' -globin ' is used for frog and fish because the genes are less well characterized . in the x. tropicalis genome assembly ( version 4.1 ) , scaffolds 733 and 357 are not connected . maps of the gene clusters were derived from a combination of the assembled genomes and recent publications . the gene maps are not complete or to scale ; see or the genome assemblies for more complete information . one globin gene cluster is found in all gnathostomes examined , but frequently , perhaps invariably , it is accompanied by another globin gene cluster at a different genetic locus . the globin genes in the common cluster are flanked by the genes mpg and c16orf35 ' upstream ' of the globin genes , and i will thus refer to this locus as ' mc ' , after these two genes ( figure 1 ) . the major dna region regulating expression of the globin genes ( major regulatory element , mre ) is located in an intron of c16orf35 . frequently , the gene rhbdf1 is adjacent to the mpg gene . in placental mammals and chickens , only -like globin genes ( both embryonic and adult ) are in this locus , whereas in amphibians and fish , the -globin genes are paired with -globin genes ( figure 1 ) . the new analysis of platypus globin genes by patel et al . shows that a -like globin gene , the -globin gene , is closely linked to the -like globin genes , just as it is in marsupials . in addition , a platypus homolog to the gene for globin y ( gby ) , a globin discovered in amphibians , is also in this locus . given its presence in all gnathostomes examined , we can infer with considerable confidence that the mc locus contained globin genes in the last common ancestor ( lca ) of vertebrates ( figure 1 ) . a second locus contains - and -globin genes in the pufferfish fugu rubripes , and examination of the genome assemblies of zebrafish and medaka shows a similar arrangement ( figure 1 ) . the globin genes in this locus are flanked by the genes lcmt1 and aqp8 , and thus i will refer to it as the la locus . the la locus has a similar arrangement of non - globin genes in the genome assemblies of human , platypus , chicken and frog , but no globin genes . given that the presence of globin genes in the la locus appears to be restricted to fish , what is the ancestral arrangement ? the diagnostic features of the mc , ds ( see below ) and la loci are widespread in fish , being present in medaka , pufferfish , tetraodon and zebrafish . following the model of gillemans et al . , figure 1 shows gene arrangements in the gnathostome lca that are similar to those in contemporary fish . this model emphasizes the presence of two different loci containing globin genes in many jawed vertebrates , and proposes that this feature is ancestral . however , it is also possible that the presence of globin genes at the la locus is a derived feature in fish . the -like globin genes are flanked by olfactory receptor ( or ) genes . in placental mammals , hundreds of or genes are in this locus , along with additional multigene families such as trim genes . we therefore have to look several megabases away from the -like globin genes to find single - copy genes that are distinctive for this locus , which are dchs1 on one side and stim1 on the other ; i thus refer to the locus as ds ( figure 1 ) . the new data from platypus reveal a pair of -globin genes flanked by or genes . although the contigs are not sufficiently long to connect these to dchs1 and stim1 , it is likely that the -globin genes in platypus are in the ds locus . a fourth locus for hemoglobin genes is suggested by the gene arrangements in the xenopus tropicalis genome assembly ( version 4.1 released by the joint genomics institute ) . scaffold 733 is annotated with the gene rhbdf1 , several -like globin genes and gby ( figure 1 ) . speculate that the globin gene clusters in scaffolds 733 and 357 are actually linked ; however a more complete assembly and additional analyses are needed to resolve this issue . the genes associated with the ds locus are on short contigs in this assembly , and it is also possible that a future assembly will link the rhbdf1--globin gene cluster with ds ; however , it is notable that no or genes flank the -globin genes , whereas they do flank other genes in the ds cluster . globin genes are present in the mc locus in all gnathostomes examined , and it is therefore very likely that this was true in the lca of jawed vertebrates ( figure 1 ) . the ds locus has no globin genes in fish but it has -like globin genes in amniotes . on the basis of these gene arrangements , patel et al . argue that the -like globin genes transposed from the mc locus and inserted into the ds locus . jeffreys et al . proposed 28 years ago that the separation of the -like and -like globin genes involved either translocation or duplication followed by divergence . identifying a likely source ( mc ) and target ( ds ) means that it is possible to argue strongly that the separation of these two loci involved a specific kind of translocation transposition leading to insertion at a new site . this is because the proposed target locus ( ds ) has no globin genes in fish , but the diagnostic genes flanking the or genes are present . if this were the ancestral arrangement , then it is simpler to invoke transposition of a -globin gene into ds than to invoke a more complex series of chromosomal rearrangements . thus , the transposition model is strongly supported , but we should keep in mind alternative sources and targets . if the model in figure 1 is correct in proposing that gene arrangements in the gnathostome lca were similar to those in contemporary fish , then it seems equally likely that either the la locus or the mc locus was the source of the transposed -globin gene(s ) in the ds locus . of course , if the arrangements of globin genes changed between the gnathostome lca and contemporary fish , then other possibilities arise . for example , the globin genes could have moved from the mc locus into the la locus along this lineage . if that were the case , then one could propose that the gnathostome lca had a single locus containing globin genes , the mc locus . fuchs et al . , using an early assembly of the x. tropicalis genome , propose that one large region contains all the hemoglobin genes and gby . however , in the most recent assembly ( version 4.1 ) , scaffolds 733 and 357 are not connected . if indeed these are separate loci , then -globin genes would have moved from either the mc or la loci into a different locus flanked by rhbdf1 and gby . despite the ambiguities that remain about several details , it is very likely that the -like globin genes transposed , perhaps twice , during the evolution of jawed vertebrates . in the lca of warm - blooded amniotes , the -globin gene moved into a sea of or genes that are expressed exclusively in nasal epithelium . in the ds locus , high - level expression of -like globin genes in erythroid cells requires an enhancer and also an insulator and boundary element . we can expect that the regulatory sequences required in cis to the -like globin genes accompanied them during the transposition ; the only alternatives are that the regulatory regions were already present at the target locus ( but why would erythroid regulatory regions be in a locus expressed in nasal epithelium ? ) or they were formed as a result of the integration of the transposed dna . if the -like globin genes came from the mc locus , then the obvious candidate for the source of the cis - regulatory regions is the mre located in c16orf35 . . did not find evidence for a distal regulator in this locus in pufferfish ; perhaps the transposed -like globin gene carried sufficient proximal cis - regulatory elements to it to maintain erythroid expression . tracing the course of evolution of cis - regulatory regions over such a long phylogenetic distance is a major challenge , because most are conserved , for example , only in placental mammals but no further . detailed studies of additional comparison species in each clade will give more power to this analysis . once the -like globin genes entered the ds locus in the lca of warm - blooded amniotes , their regulatory regions underwent considerable divergence and possibly duplication . they are controlled by multiple regulatory regions in both placental mammals and chickens , but none show clear dna sequence matches in comparisons of entire loci between mammals and birds . in contrast , the -globin mre shows clear sequence alignments between placental and marsupial mammals , and the homologous regions from chickens and fish are active as enhancers . thus , it will be informative to search the platypus noncoding sequences for evidence of distal regulators and see whether they are related to distal regulatory regions in either placental mammals or birds or even neither . what is the fate of genes left at the source loci after transposition ? of course , our ability to detect the genes is much greater if they are still active , and most of the genes included in the diagrams in figure 1 are still functional . jeffreys et al . speculated that a ' fossil ' -like globin gene would remain at the end of the -globin gene complex , and in fact the -like -globin gene is found in exactly that position in marsupials and monotremes ( figure 1 ) . however , it is not a pseudogene , but rather it is transcribed and encodes a functional hemoglobin . the discovery of the -globin gene in marsupials ( and now monotremes ) revealed another important component of the vertebrate hemoglobins , but accurately placing it in a phylogenetic tree has been challenging . if that were true , the -like globin genes of placental mammals would not be orthologous to the avian -like globin genes , which in turn has an impact on the evolutionary analysis of coding and regulatory regions . however , analyses of larger globin gene datasets show that the -globins form a distinct clade separate from all the other -like globins of mammals and birds . furthermore , the comparative gene mapping of patel et al . show convincingly that the -like globin genes of birds and placental mammals are flanked by orthologous single - copy genes ( ds locus in figure 1 ) , and thus these globin gene clusters are indeed orthologous . only two -globin genes are present in the platypus ds locus , which seems similar to the embryonic -globin and adult -globin pair found in marsupials . however , the platypus -globin genes are more similar to each other than to any other -like globin genes . in the homologous gene clusters in every other amniote examined to date , the -like globin gene located at the left end of the cluster ( in the orientation shown in figure 1 ) is expressed exclusively in embryonic erythroid cells . thus , we would expect the gene in the comparable location in monotremes to have a similar expression pattern . indeed , both groups describing this gene cluster call it -globin , but evidence for grouping with therian ( placental mammal and marsupial ) -globin is , at best , inconsistent . both -globin genes are expressed in adult tissues , but no information is available on expression in embryos , so the range of developmental expression has not been determined . on the basis of extensive matches in the flanking regions , opazo et al . propose that the duplication to form the -globin gene pair in monotremes occurred independently of the duplication that formed the ancestral therian -globin and -globin genes . in contrast , patel et al . argue that the gene pair had a common origin in mammals ( including monotremes ) , but the-like globin genes in platypus have become homogenized by gene conversions . as is the case for many evolutionary controversies , examination of homologous gene clusters in additional species , such as another monotreme , the echidna , could shed light on this debate . the diversity of hemoglobins , their crucial functions , their exquisite regulation and the pathological consequences of some mutations make this a fascinating family of proteins and genes . exploration of these genes in many different species continues to illuminate some and challenge other evolutionary models . the recent analysis of the globin gene clusters in platypus , combined with data mining in fish and frog genomes , has strongly supported previous ideas about the relationships of the globin gene clusters in vertebrates , with the - and -globin genes in the mc locus deduced as ancestral . furthermore , -globin genes moved into a cluster of or genes at the ds locus in the lca of birds and mammals , and it will be informative to examine the homologous genes in reptiles . a draft genome sequence of the lizard anolis carolinensis has been released by the broad institute , and it shows at least one -like globin gene in the mc locus , as expected . a full exploration of issues such as those discussed in the platypus globin gene papers will probably require targeted exploration of these loci , with the genome assembly serving as a useful launching pad . likewise , deeper branchings should be resolved by analysis of globin gene loci in jawless vertebrates . a draft genome of the lamprey petromyzon marinus has been released by the washington university genome center . again , the contigs are too short to find instant answers , but the genome assembly provides a great starting point for individual investigators . i predict that more clarity will emerge from such pursuits , as well as more surprises and controversies . this work was supported by nih grants from niddk ( dk65806 , rh ) and by tobacco settlement funds from the pennsylvania department of health . the genome assemblies of the frog xenopus tropicalis and fish medaka are public releases from , respectively , the joint genome institute and a collaboration between the national institute of genetics and university of tokyo , japan .

recent data published in bmc biology from the globin gene clusters in platypus , together with data from other species , show that -globin genes transposed from one chromosomal location to another . this resolves some controversies about vertebrate globin gene evolution but ignites new ones .

systemic and local proinflammatory changes are in focus when investigating the pathophysiology of arteriosclerosis and acute coronary syndromes . in acute myocardial infarction ( ami ) , proinflammatory markers such as c - reactive protein ( crp ) , interleukins , or monocyte - chemoattractant protein ( mcp)-1 are elevated [ 13 ] and their increase is of prognostic relevance for future cardiovascular events [ 46 ] and mortality [ 79 ] . moreover , in healthy persons elevated proinflammatory markers are associated with an increase in cardiovascular risk [ 1012 ] . patients with increased circulating proinflammatory markers in ami present with decreased myocardial salvage after coronary reperfusion therapy . sources of inflammatory response are vascular cells such as activated endothelial cells , which release proinflammatory cytokines such as interleukin ( il)-8 . il-8 is a cxc cytokine that acts as a chemoattractant and agonist for neutrophils , lymphocytes , and monocytes and is found in macrophage - rich atherosclerotic plaques . under flow conditions , il-8 facilitates the arrest of monocytes on endothelium , which is necessary for migration into the intima in evolution of arteriosclerosis . reperfusion injury after ami as well as systemic inflammatory response syndrome can be associated to increased levels of il-8 . in experimental setting , murine il-8 receptor knock - out mice display smaller arteriosclerotic lesions with less macrophages . apart from their contribution to arteriosclerosis , cxc cytokines are also produced by malignant cells and can promote tumor progression of a large variety of malignancies . besides il-8 , many other cytokines such as il-6 take part in inflammatory responses by inducing b - cell differentiation , t - cell activation , and synthesis of acute phase proteins , but also contributing to proliferation of vascular smooth muscle cells ( smcs ) the pathogenesis of proinflammatory changes in acute coronary syndromes as well as the interplay between coagulation and inflammation is poorly understood and is subject to intense research . the mechanisms by which the coagulation system is altered by inflammatory interactions comprise enhanced synthesis and activation of coagulant proteins , decreased synthesis of anticoagulants , and suppression of fibrinolysis . accordingly , increased levels of prothrombin fragment f1 + 2 , fibrinopeptide a and d - dimer , reflecting activation of the coagulation cascade , are also associated with an unfavorable outcome in acute coronary syndromes [ 2527 ] . in this paper we focus on possible mechanisms of the interplay between coagulation and inflammation in acute coronary syndromes . a strict separation between the intrinsic and extrinsic coagulation cascade surely fails to reflect physiologic conditions . nevertheless , in the setting of arteriosclerosis and acute coronary syndromes the extrinsic pathway of coagulation is of particular significance . tissue factor ( tf ) is the most important initiator of the extrinsic coagulation cascade . tf , a 47 kda transmembrane glycoprotein and member of the class ii cytokine receptor family , is the cofactor for the activated plasma clotting factor vii ( fviia ) . the tf - fviia complex catalyzes the activation of factor x and ix , which leads to the generation of thrombin and thus finally of a fibrin clot . under physiologic conditions tf is abundantly expressed only in the adventitia and is induced by several inflammatory mediators such as il-6 , il-8 , and mcp-1 [ 28 , 29 ] . after vascular injury , tf is rapidly augmented in smc of the media and accumulates in the smc of the developing neointima . consequently , tf is highly expressed within atherosclerotic lesions and displays high procoagulant activity suggesting a role in determining plaque thrombogenicity . in atherosclerotic carotid lesions disruption of plaques exposes tf - positive cells within the plaque to plasma clotting factors and initiates local thrombosis with subsequent occlusion of the vessel . furthermore , increased tf expression can be noticed on circulating monocytes and microparticles in acute coronary syndromes and may , thereby , contribute to activation of coagulation [ 3234 ] . it has been shown that cytokines can induce expression of soluble tf , which on the other hand has been shown to accumulate in developing thrombi . however , the clinical significance and individual contributions of microparticle - derived and soluble tf remain a matter of debate . several studies have demonstrated increased levels of circulating tf in patients with unstable angina pectoris ( uap ) and acute myocardial infarction ( ami ) [ 32 , 3845 ] . therefore , it has long been speculated that , in cases with no plaque rupture or only fractional superficial erosion , thrombus formation may mainly depend on circulating levels of tf . consistent with this idea , several studies suggest that the levels of circulating tf and other haemostatic biomarkers may correlate to adverse cardiovascular events and mortality in patients with acute coronary syndrome [ 4648 ] . stimulation of the tf - thrombin pathway does not only occur at the site of the plaque but also within the ischemic myocardium where activated coagulation factors may enhance inflammatory responses and increase infarct size . furthermore , tf expression has been reported in a number of cancers , such as glioma , pancreatic cancer , non - small - cell lung cancer , colorectal cancer , ovarian cancer , prostate cancer , hepatocellular cancer , and breast cancer . tf expression in tumors not only correlates with the incidence of thrombosis but also promotes metastasis , tumor progression , and tumor angiogenesis . on one hand tf allows docking and activation of fvii and , therefore , promotes the generation of downstream coagulation factors and activation of protease - activated receptors ( pars ) which themselves possibly induce intracellular signal transduction . on the other hand there is evidence for direct signalling through the cytoplasmic domain of tf following tf - fviia complex formation [ 55 , 56 ] . the endogenous kunitz - type inhibitor tissue factor pathway inhibitor-1 ( tfpi ) inhibits initiation of tf - induced blood coagulation and is mainly expressed on vascular endothelial cells . increased levels of the tfpi - fxa complex may reflect both increased fxa generation and increased tfpi concentrations . in addition to the full length tfpi most of the plasma tfpi circulates in truncated forms that are bound to plasma lipoproteins . these truncated forms lack their c - terminal domains and exhibit reduced affinity for vascular wall proteolysis . additionally , it has been shown that endogenous proteases and elastase released by neutrophils degrade tfpi , resulting in enhanced local coagulation that contributes to prevent pathogen dissemination during infection . conversely , infusion of a mutant tfpi protein resistant to proteolysis by elastase strongly impaired host defence against systemic infection . important players in the interaction between coagulation and inflammation are protease - activated receptors ( pars ) . pars are g - protein coupled receptors that mediate various cellular reactions as cytokine release , expression of adhesion molecules , cell migration , or proliferation . unlike other receptors , pars are not activated by a soluble , external ligand . proteases , such as activated coagulation factors , detach a defined part of the nh2-terminal chain of the receptor , thereby inducing a conformational change of the receptor . pars can also be activated by synthetic peptides consisting of the sequence of amino acids representing the tethered ligand . in contrast to other receptors , the activation of pars by enzymatic cleavage is irreversible . pars are mainly expressed in vascular cells , but also in many different other cell types such as gastrointestinal and bronchial epithelial cells . four different pars are known : par-1 , -3 , and -4 show responsible for thrombin signaling whereas par-2 is activated by trypsin - like serine proteases , fviia , and matriptase but not by thrombin . par-1 and par-2 are expressed on smooth muscle cells and endothelial cells , whereas mainly par-1 is expressed on monocytes . par-1 agonists or thrombin induce il-8 and il-6 in smc , ec , and mononuclear cells ( mncs ) therefore emphasizing the role of par-1 in inflammatory processes in vascular cells and confirming data about par-1-mediated cytokine release in ec and monocytes . in smooth muscle cells par-1 and par-2 agonists induce cytokine release to a similar extent which underlines the relevance of both pars . in addition to coagulation factors other serine proteases , for example , matriptase secreted by monocytes stimulate proinflammatory cytokine release in endothelial cells via par-2 activation . increased par-2 expression in atherosclerotic lesions suggests a role for this proinflammatory pathway ( figure 1 ) . in addition , pars can also be cleaved downstream of the tethered ligand , resulting in receptor inactivation by preventing further proteolytic activation . par-1 signalling not only induces inflammatory responses but also causes antiapoptotic and vasculoprotective reactions . since the anticoagulant protease - activated protein c can activate par1 when in complex with the endothelial cell protein c receptor ( epcr ) , which may account for much of the protective effects conferred by activated protein c ( apc ) in severe sepsis first , apc acts via par-1 when attached to epcr , resulting in cellular responses distinct from thrombin signalling by a mechanism dependent in trans - activation of the sphingosine 1 phosphate receptor 1 . in mouse models with strongly reduced epcr expression or par-1 deficiency , the loss of epcr / apc signalling via par-1 resulted in increased endotoxemia - induced lethality . concordantly , apc mutants have been shown to contribute to protective effects during sepsis by pathways independent from anticoagulant properties [ 67 , 68 ] . the second pathway described is independent from epcr . in this case , the availability of the integrin cd11b / cd18 has been shown to be crucial for par-1 mediated apc signaling on macrophages , thereby exhibiting anti - inflammatory effects and reducing endotoxin - induced lethality . thus the strength of par1 and par2 activation by thrombin , factor xa , and activated protein c can either promote or protect against changes in vascular permeability depending on the status of the endothelium . platelet activation with subsequent thrombus generation plays a major role in the development of acute coronary syndromes . at low concentrations thrombin activates par-1 on platelets through a hirudin - like site and at high concentrations additional par-4 . this induces shape change , p - selectin , and cd40l mobilization to the platelet membrane and promotes the release of platelet agonists adp , thromboxane a2 , chemokines , and growth factors and , thereby , enhances proinflammatory changes . thus , inhibition of pars by thrombin of fxa inhibitors may prove beneficial in reducing not only thrombotic but also proinflammatory responses . binding of the serine protease fvii to tf results in generation of the coagulation protease fxa ( fxa ) and subsequently thrombin both known to induce cell signaling . fxa shows dose - dependent induction of intracellular calcium transients in endothelial cells that is active - site - dependent , and independent of thrombin . potential pathophysiological responses to fxa include stimulation of proliferation , production of proinflammatory cytokines , and prothrombotic tf . elevated tfpi - fxa and prothrombin fragments f1 + 2 plasma levels indicate activation of the coagulation cascade in acute coronary syndromes . under physiological conditions an inverse relationship between tfpi - fxa and f1 + 2 suggests that tfpi - fxa regulates prothrombinase activity in vivo . under conditions associated with activation of the coagulation cascade , however , increased tfpi - xa plasma levels occur [ 57 , 74 ] . activation of coagulation as measured by tfpi - fxa but not by f1 + 2 is associated with plasma concentrations of the proinflammatory cytokine il-8 in acute coronary syndromes . furthermore , subsequent elevated il-6 levels in the course of acute coronary syndromes are associated with initial tfpi - fxa concentrations . these results argue for a proinflammatory role of fxa in acute coronary syndromes that is independent of thrombin . although thrombin provokes similar proinflammatory effects as fxa in vitro the effects of thrombin may be diminished after heparin treatment in vivo . several trials of unfractionated heparin ( ufh ) , direct thrombin inhibitors , and enoxaparin have thus far failed to demonstrate mortality reductions in acute coronary syndromes . yet , the oasis-6 trial suggests a reduction in reinfarction and mortality without excess bleeding in patients not undergoing pci . therapeutic inhibition of the proinflammatory effects of factor xa may , therefore , prove additional benefits as compared to thrombin inhibition in the clinical course in acute coronary syndromes . this is currently investigated in the atlas - acs 2 timi 51 trial that is testing the hypothesis that anticoagulation with the oral factor xa inhibitor rivaroxaban reduces cardiovascular death , mi , and stroke among patients with acs treated with guideline - based therapies for acs . in vitro experiments revealed that fxa stimulates il-8 and mcp-1 transcription in endothelial cells and mononuclear leukocytes . genetic studies and receptor desensitization experiments indicate that signaling by fxa is mediated by par-1 and par-2 [ 80 , 81 ] . according to the expression of par-1 and par-2 , par-1 and par-2 agonists induce il-8 and mcp-1 release in endothelial cells , whereas , only par-1 agonists stimulated cytokine release in mononuclear cells . several studies suggest a signaling mechanism of the tf - fviia complex via par-2 . in this model , tf - bound fviia proteolytically activates par-2 and , to a lesser extent , par-1 , and thereby evokes intracellular signaling cascades . in contrast to fxa , fviia does not elicit a proinflammatory response in endothelial cells or mononuclear cells . the tf - fviia - par-2 signalling was only observed in smc since they express both tf and par-2 and both of them seem to be a prerequisite for fviia action . ec expressing par-2 but lacking tf will not permit fviia docking , whereas mnc displaying tf but only low par-2 expression , probably allow fviia binding but do not express sufficient par-2 molecules being subsequently activated . however , par-2 and tf are induced by cytokine stimulation [ 28 , 84 ] . thus , fviia may still have an important impact in atherosclerotic vessels and acute coronary syndromes . in recent studies , it has been demonstrated that fvii is synthesized by different cancer cells ( liver , ovary , prostate , lung , gastric , thyroid , and breast ) . considering that these tumor cells also synthesize tf it is conceivable that supraphysiologic concentrations of fviia after binding of fvii to tf occur . on tumor cells , the tf - fviia binary complex mediates activation of par-2 . therefore tf - fviia - par-2 interaction with subsequent cytokine release may be relevant within a tumor environment . tf / fviia / par2 signalling has been shown to promote proliferation and metastasis of tumor cells [ 87 , 88 ] . consistently , tf / fviia - specific upregulation of il-8 expression in breast cancer cells has been shown to be mediated by par-2 and to increase cell migration . whether tf - fviia - par-2 interaction may also contribute to local thrombus formation and progression of atherosclerotic disease remains to be elucidated . experimental evidence suggests that this interplay may contribute to the development of vascular remodeling or support plaque disruption of the artery . however transfer of these data to the clinical settings remains controversial since additional optimized medical and interventional treatments interfere . therefore , understanding the causes of inflammation facilitate the development of new therapeutic strategies . to analyse whether these new therapies translate to improved clinical outcome needs to be studied in appropriate clinical trials . while inhibiting proinflammatory cytokines such as tumor - necrosis factor- ( tnf ) has been shown to effectively improve survival in several animal models of sepsis [ 8991 ] , anti - tnf therapy in septic humans failed to ameliorate or even worsened clinical outcome [ 9295 ] . in chronic inflammatory diseases however , anti - inflammatory treatment has become clinical routine . for example , inhibition of il-6 by the first anti - il-6 antibody , tocilizumab , has been shown to completely block tf - dependent thrombin generation in experimental endotoxemia [ 23 , 96 ] , and tocilizumab will be of special future interest as it has been approved for rheumatoid arthritis . experimental studies that have shown that anticoagulant treatment not only diminishes activation of coagulation but also inhibits inflammation , underline the cross - talk between activation of coagulation and cytokine release in vivo [ 49 , 97 , 98 ] . in acute inflammatory disorders such as severe sepsis , administration of recombinant apc apc mutants that lack anticoagulant properties but still enable sphingosine 1 phosphate receptor 1 dependent activation of par-1 will be of special clinical interest as they have been shown to reduce sepsis - induced in mice but do not predispose to bleeding complications . so far , anti - inflammatory treatments displayed no explicit benefit in patients with acute coronary syndromes . however , there is evidence that fxa inhibitors prove to be superior to thrombin inhibitors . particularly , treatment with low molecular weight heparins , that include additional anti - fxa activity as compared to unfractionated heparin , has been shown to decrease inflammatory changes in vitro and in vivo [ 101 , 102 ] . yet the question remains if and what anticoagulant therapies will prove beneficial to alter systemic inflammatory responses .

the intimate connection between coagulation and inflammation in the pathogenesis of vascular disease has moved more and more into focus of clinical research . this paper focuses on the essential components of this interplay in the settings of cardiovascular disease and acute coronary syndrome . tissue factor , the main initiator of the extrinsic coagulation pathway , plays a central role via causing a proinflammatory response through activation of coagulation factors and thereby initiating coagulation and downstream cellular signalling pathways . regarding activated clotting factors ii , x , and vii , protease - activated receptors provide the molecular link between coagulation and inflammation . hereby , par-1 displays deleterious as well as beneficial properties . unravelling these interrelations may help developing new strategies to ameliorate the detrimental reciprocal aggravation of inflammation and coagulation .

industrial growth , technological advancements , and higher living standards in today 's society contribute to the ever increasing generation of solid wastes . industrial park is one of the most important manufacturing bases and an indicator of development in each country . however , each manufacturing process usually generates some amount of nonconsumable waste causing adverse effects on environment . geographical information system ( gis ) technologies are effectively used in the process of site selection , which is a spatial problem . despite the consensus of all countries to achieve the goal of zero waste in industries , the accomplishment of this goal is predicted to be difficult and generation of various wastes arising from manufacturing processes is currently inevitable . a focus on sources of solid waste with respect to management is justified by the fact that waste characteristics and composition differ according to source . the appearance of such problems caused some countries to move their waste to developing countries in order to be saved from the probable risks of the hazardous waste . uncontrolled expansion of industries and ignoring environmental principles in industrial development and overuse have caused much environmental problems . a number of countries have ratified state and national legislation for managing and controlling hazardous waste whose main purpose is to minimize the potential risks of industrial hazardous waste for humans and the environment . in iran , despite the excess growth of industries , urbanization , and industrial centralization around major cities particularly tehran , no fundamental effort has been yet made in the field of industrial waste disposal , and subsequently , there are few rules for controlling and disposal of such materials . evidence shows that solid , semisolid , and liquid wastes of the factories are disposed regardless of health issues and effluent of these industries enters the absorbing wells or spread in surrounding land . solid waste is piled up around the factories for a period , and then a part of the waste is incinerated outdoor and some part is transferred out of the factories along with household waste by municipal general service . large scale land use transportation from agricultural to industrial has made tehran a city for a large center for industries centralization and their peripheral activities . because of the importance of industrial hazardous waste minimization in prevention of pollutant emissions in the environment , it is emphasized by current laws in many countries . moreover , establishment and development of minimization programs have been adopted more than ever through legislation of laws and more restrictions in controlling hazardous waste and prevention of leaving waste in the environment . the application of waste minimization techniques does not necessarily mean the use of complicated technologies or investments on expensive machines . many methods require a simple change in the process of production or in transferring the materials . generally , hazardous waste minimization methods can be used for any production process and the mutual factor in all techniques is the reduction of production costs . in this study , basic and technical information required for the waste management were collected and analyzed due to the increasing growth of industries and the need for developing efficient methods in proportion to the conditions of the studied region . in fact , the main objective of this study is to examine the quantity of industrial solid waste regarding the production line of cb province , iran . the population of this study included all operational industries in 19 industrial zones of cb province , iran , which were studied for 8 months from march to october , 2009 . data were collected partly through questionnaires and partly through field investigation and literature review on the subject on matter , which was carried out using various books and articles . industrial zones of the province were identified in order to specify the production resources . in this regard , detailed information on the industrial zones was taken from the iran small industries and industrial parks organization , in cb province . metal industries with 95 units and electricity and electronics industries with 10 units had maximum and minimum numbers of units in the province , respectively . the first stage of the study including separation and classification of the industrial units was done based on their activities and locations according to the available statistics in the province . the questionnaire contained type of industrial group , the number of personnel , the kind of product and raw materials , the amount and type of generated waste , waste physical state , frequency of generation , method of storage and maintenance , recycling methods , final disposal , and the responsible organizations for waste collection in order to identify industrial waste in cb province . various questionnaires presented by different organizations such as recycle organization , environmental protection organization , and ministry of industries were analyzed and finally a questionnaire containing two forms was prepared . according to the investigations , 19 industrial parks with the total area of 2356.10 hectares were under the coverage of industrial parks organization . among these 19 industrial parks , shahrekord , lordegan , saman , faradnbeh , junghan , farokhshahr , ben , and dastgerd industrial parks had 154 , 22 , 16 , 1 , 2 , 3 , 4 , and 3 units , respectively . therefore , shahrekord with 154 units ( or 49.8% ) and faradnbeh with 1 unit ( or 0.3 percent ) had the most and the least units , respectively . a number of 309 questionnaires were completed by the industrial units . generally , these industries performed various activities , so the amount of industrial waste was influenced by the kind of activities . in this regard , these industries were divided according to the classification of ministry of industries . food , metal , chemical , nonmetallic minerals , cellulose , textile , and electrical and electronics industries comprised 21 , 30.4 , 22 , 12.6 , 7.1 , 3.8 , and 2.9 percent of the evaluated industries in this study . the workers of these 14 industrial parks were 6033 people . based on table 1 , the total industrial waste was 1246 tons per month , and semihousehold waste per capita ( for food consumption of the workers ) was 350 gram per day . table 2 shows that the amount of wastes generated in cb province was approximately 32 ton per month . according to table 3 , the total food waste generated in studied industries was an average of 65516 kg per month which depended on the number of workers . herbal waste was 384 tons a month of which 99.4% and 0.6% were generated in food and chemical industries , respectively . herbal waste of food industries encompassed wheat , straw , bran , fruit , vegetable , and dried fruit waste . herbal waste of chemical industries was related to yarn and fabric dyeing industries . in these industries , flour and doughy waste was generated in industries like macaroni manufacturing , noodle making , and industrial bread . the amount of flour and doughy waste was 22.3 tons per month in the province which was totally generated in food industries . sweets , chocolate , and candy waste was 6.5 tons per month which was totally generated and recycled in food industries . meat and bone waste encompassed chicken and fish packaging waste and sausage production waste which was totally generated in food industries . according to investigations , wood waste was generated in metal , chemical , cellulose , and electricity and electronics industries in a form of wooden pallets which entered the factories along with purchase of tools like the engines of various machines and variety of metal ingots . the amount of wood waste was 17 tons per month , of which the maximum amount of 55.2 percent was related to metal industries . the amounts of plastic waste generated in food , metal , nonmetallic minerals , cellulose , textiles , and electrical and electronics industries were 40.8 , 1.9 , 0.8 , 0.3 , 0.3 , and 2.2 percent , respectively . these types of waste were recycled in chemical industries ; however , no action has been yet done for recycling these wastes by other industries . the amount of glass waste in the entire study was 8.2 tons per month of which 91.8 , 6 , and 2.2 percent were generated in nonmetallic minerals , cellulose , and food industries , respectively . the amount of paper and cardboard waste was 84.5 tons per month of which 83.1 percent was generated in cellulose industries and 12.2 percent was generated in food and other industries . ferrous metals waste included iron chips , iron scraps , and snipping which was 160 tons per month of which 94.8 , 6 , and 0.3 percent were generated in metal and cellulose industries , respectively . nonferrous metal waste included aluminum , copper , and cast iron of which the maximum amount was related to electricity and electronics industries for the use of aluminum and copper in wire and cable industry . the amount of nonferrous metal waste in studied samples was 5.7 tons per month of which 52.2 , 31.4 , 8.7 , 1.2 , and 3.5 percent were generated in electricity and electronics , metal , cellulose , nonmetal , and food industries , respectively . gunny waste consisting of sugar and flour sacks generated 4003 kg per month of which 81.9 , 15.6 , and 2.5 percent were generated from food , chemical , and metal industries . it should be noted that 100 percent of these wastes were reused and exploited in the same industries . trash waste included concrete , mosaics , ceramics , and stone body waste which were totally generated in nonmetal industries . the amount of generated pvc waste in the studied zones was 2.5 tons per month of which chemical , and electricity and electronics industries comprised 2300 kg ( 91.6 percent ) and 70 kg ( 2.8 percent ) , respectively . the amount of generated pet waste was 18 tons ( 93.7 percent ) per month of which 200 kg ( 1.1 percent ) and 935 kg ( 5.2 percent ) were generated in metal and food industries , respectively . the amount of polystyrene waste was 5.8 tons per month which was totally generated in chemical industries . the amount of polypropylene waste was 6.2 tons per month of which 96.8 percent and 3.2 percent were generated in chemical and textile industries , respectively . soil waste consisting of wheat soil and bentonite clay was used for vinegar treatment in industrial vinegar - making and stabilization of edible oils . furthermore , burnt sand waste ( co2 silica sand co2 waste ) is contained in soil waste . the amount of soil waste generated in the province was approximately 130 tons per month of which 96.6% and 3.4% were generated in food and metal industries , respectively . as noted above , burnt sand waste was classified in this category and was generated in cast iron foundry industry . the amount of yarn and fabric waste in all studied zones was about 7.4 tons a month . these wastes included yarn and fabrics , wool , fiber , and polymer of which 93.9 percent and 6.1 percent were generated in textile and chemical industries . the amount of chemical and special waste in all studied zones was about 21 tons per month of which 3 , 41 , 36.9 , 16.6 , 2 , and 4 percent were generated in metal , chemical , nonmetal , cellulose , textile , and electricity and electronics industries . the initial chemical waste produced from industries which produced detergents and disinfectants comprised 1 percent of the raw chemical materials . these wastes consisted of sulfuric acid , diethanolamine , coconut fatty acid , betaine , essence , resin , sulfate , salt , and so forth . the amount of these wastes was 1775 kg per month which comprised 8.1 percent of the total chemical waste . the amount of color waste was 581 kg per month which comprised 2.6 percent of the total special waste . it should be noted that this type of waste was found in metal , chemical , cellulose , and textile industries . the amount of adhesives waste was 172 kg per month which was related to chemical and cellulose industries and was most generated in card board industry . a study on the industries of industrial parks in other region of iran showed that 250 kg of waste containing degradable materials and scrap iron was transferred out of the city and incinerated outdoor by municipal service . overestablishment of industries and conversion of agricultural land to industrial land in tehran , iran , have changed the city to a large center for industries centralization and their peripheral activities . in this study , the maximum amount of generated sludge waste was 8 tons per month which was related to nonmetal industries that was generated in a mosaic - building industrial unit in boroujen industrial zone . the tar waste generated in the province was 220 kg per month which was related to the waterproof membrane industries . the zinc oxide waste was 35 kg per month which comprised 0.1 percent of total special waste in the studied industrial parks . moreover , zinc clay waste was generated by a zinc ingot production cooperative in shahrekord . it was 600 kg per month which comprised 2.7 percent of total chemical and special waste . an ideal waste disposal site is that one which is located fairly close to the source of the waste , has easy transportation access , is not located in a low - lying area or floodplain , and is underlain by geologically stable , strong , and competent rock material . the site selection depends on several factors like land use , environmental , hydrology , socioeconomic , and so forth . according to the literature , the following criteria can be applied for site selection : site must be close to at least a street with a buffer of 30 m;site must not be too far from a transfer station;site must be 3 km from residential areas , with the exception of areas with barriers;there should be a minimum distance of 100 m between site and roads;site must be on a suitable soil;site should be constructed in areas which do not have an important economic or ecological value.such kinds of criteria could be combined in a hierarchal structure shown in figure 1 . site must be close to at least a street with a buffer of 30 m ; site must not be too far from a transfer station ; site must be 3 km from residential areas , with the exception of areas with barriers ; there should be a minimum distance of 100 m between site and roads ; site must be on a suitable soil ; site should be constructed in areas which do not have an important economic or ecological value . the combination of gis and multicriteria decision models ( mcdm ) can be useful in this regard . such approach provides flexible methods for exploring relationships among geographic data and assisting experts from diverse fields in pooling their knowledge to solve complex problems . a list of potential sites which generally satisfy the minimum requirements can be identified for the purpose of effective sanitary landfill - type waste disposal . among these areas , the integration of gis in multicriteria decision analysis ( mcda ) is a powerful tool in solving disposal site selection problem , because it provides efficient spatial data manipulation and presentation . one of the most important issues in industrial waste management is its special waste which distinguishes the industrial waste from the general waste . the waste of this category includes chemical raw materials , color , adhesives , tar , zinc oxide , zinc clay , and sludge . the total generated waste in the studied industrial zones was 1246 tons per month , of which 48.2 , 14.9 , 6.7 , 22 , 0.9 , 0.6 , and 6.5 percent were generated in food , metal , chemical , nonmetallic minerals , textile , electrical and electronics , and cellulose industries , respectively . the results achieved in this study are considered the first step toward proper industrial waste management especially in recycling and disposal of waste . according to the results of this study , environmental general department recommended to use rewards and punishments for reduction of hazardous waste . coordination with financial institutions for giving loans with lower interests in order to take environmental actions or imposing large fines for each kilogram of generated hazardous waste gives instances of reward and punishment . collection , delivery , and disposal of hazardous waste should be done by private section and the officials of environmental general department supervise the performance of the private section . costs of monitoring water , soil , and weather analyses in hazardous waste disposal site should be adopted from pollutant industries and the environmental report on the landfill should be submitted to environmental department every three months . regarding fragile ecosystem of cb province , layout and extension of industrial parks in future finally , an approach to the integration of gis and mcdm was introduced as a powerful tool in solving disposal site selection problem considering several criteria .

the aim of this study is the comprehensive planning for integrated management of solid waste at the industrial parks . the share of each industrial group including food , metal , chemical , non - metallic minerals , textile , electrical and electronical , and cellulose industries were 48.2 , 14.9 , 6.7 , 22 , 0.9 , 0.6 , and 6.5 percent , respectively . the results showed that nearly half of total industrial waste produced from the range of biological materials are biodegradable and discharging them without observing environmental regulations leads to short - term pollution and nuisance in the acceptor environment . also some parts of case study waste were recyclable which is considerable from viewpoint of economical and environmental pollution . long - term impacts will appear due to improper site selection of disposal from the spatial standpoint . in this way , an approach for site selection using several socioeconomic , physical , and environmental criteria based on multicriteria decision making model ( mcdm ) is introduced . health risks and environment pollution such as soil and surface water may be done . it is essential to revise the studied industries layout , particularly those units which produce special waste which should be more cautious . also stricter enforcement is required as an effective step in reducing the harmful impacts of it .

differences in intestinal microbial composition in dizygotic as well as monozygotic twins suggest that the environment is in fact an important factor shaping the microbiome . obesity is associated with phylum - level changes in the microbiota , less bacterial diversity , and different expression of bacterial genes and metabolic pathways . for instance , the microbial inhabitants from populations with similar cultural factors such as hygiene , exposure to chemicals and/or antibiotics , and especially diet share more similarities in the microbiome structure when compared with populations in other countries . the observed associations between gut microbes and nutrient absorption indicate that human gut microbiota regulates nutrient harvest . a 20% increase in firmicutes and a corresponding decrease in bacteroidetes were associated with an increased energy harvest of 150 kcal . furthermore , the efficiency of extracting energy from various dietary ingredients favors the growth and/or colonization of certain bacterial strains , thereby contributing to this complex and competitive environment . conventionally raised mice have a 40% higher body fat content and 47% higher epididymal fat content than germ - free mice even though they consumed less food than their germ - free counterparts . fecal transplantation to gnotobiotic mice resulted in a 60% increase in body fat within 2 weeks without any increase in food consumption or obvious differences in energy expenditure . alcohol causes intestinal bacterial overgrowth in humans20 , 21 and in animal models of chronic alcohol administration.12 , 14 alcohol also results in alcohol - associated qualitative changes of the microbiota in humans20 , 21 , 22 and experimental animal models.12 , 14 , 23 , 24 , 25 in particular , beneficial commensal bacteria including lactobacillus are relatively lower after chronic alcohol consumption.12 , 20 using metagenomics and metabolomics , we have recently demonstrated that chronic alcohol administration results in a decreased capacity of the intestinal microbiome to produce long - chain fatty acids ( lcfa ) in both alcoholics and mouse models of alcoholic liver disease . this leads to reduced intestinal levels of saturated lcfa and commensal lactobacilli , which are able to use saturated lcfa as an energy source . a decrease in beneficial commensal bacteria contributes to a tight junction barrier disruption . supplementing saturated lcfa maintains eubiosis , stabilizes the intestinal gut barrier , and reduces ethanol - induced liver disease in mice . ethanol exerts a direct effect on the saturated fatty acid biosynthetic gene abundance in intestinal bacteria independently of the host . antibiotics are important drugs to treat infectious diseases and other diseases that depend on translocated microbial products . however , antibiotics also can promote dysbiosis , which might not only favor colonization but also affect body physiology by inducing weight gain . subtherapeutic administration alters the population structure of the gut microbiome and its metabolic capabilities , and increases adiposity in young mice . thus , dysbiosis induced by antibiotics is an important pathogenic factor in the onset or progression of systemic diseases . this concept has been shown in ibd.29 , 30 , 31 other environmental factors that shape microbiota are delivery mode , smoking,33 , 34 , 35 and parasitic infections . the microbiota from family members has more similarities than that from unrelated individuals,37 , 38 which raises the possibility that genetic factors affect the microbiome composition . this is supported by genetic loci in mice that have been linked to the abundance of gut bacteria . it is important to consider that studies that have failed to find significant genotype effects on microbiome diversity3 , 17 were not accounting for environmental conditions and might have been underpowered . newer , sufficiently powered studies have demonstrated that the christensenellaceae family is a heritable taxon which forms a co - occurrence network with other heritable taxa and is enriched in individuals with a low body mass index ( bmi ) . adding christensenellaceae to an obese - associated microbiome resulted in reduced weight gain in the recipient mice . similarly , there is a significant association between the nucleotide - binding oligomerization domain 2 ( nod2 ) risk allele for intestinal bowel disease and an increased relative abundance of enterobacteriaceae . concerning liver disease , nod2 variants increase the risk for culture - positive spontaneous bacterial peritonitis and bacterascites in cirrhosis and may affect survival.42 , 43 genetic polymorphisms of nod2 are associated with increased mortality in nonalcoholic liver transplant patients . inflammasomes recognize pathogen - associated molecular patterns ( pamps ) or damage - associated molecular patterns ( damps ) that cleavage proinflammatory cytokines such as pro - interleukin ( il)-1 and pro - il-18 . mice deficient for nucleotide - binding domain and leucine - rich repeat - containing protein 6 ( nlrp-6 ) , and hence less intestinal il-18 , showed altered fecal microbiota characterized by an increase of the bacterial phyla bacteroidetes ( prevotellaceae ) and tm7 . intestinal dysbiosis causes inflammation of the colon , which is mediated by chemokine ( c - c motif ) ligand 5 ( ccl5 ) . intestinal inflammation results in an increase in intestinal permeability , which leads to translocation of microbial products to the liver . binding of these microbial products to toll - like receptors ( tlrs ) in the liver is associated with exacerbated hepatic steatosis driving nash progression . furthermore , cohousing of inflammasome - deficient mice with wild - type mice results in exacerbation of hepatic steatosis . nlrp3 and nlrp6 inflammasomes and the effector il-18 ameliorate nafld / nash progression , thus highlighting the importance of genetic factors for intestinal dysbiosis and systemic diseases . the fact that healthy siblings of patients with crohn s disease manifest crohn 's disease associated immune and microbiologic features supports the relevance of genetic factors in the composition of microbiota . separating the influence of genetics and environment on microbiota composition requires carefully designed studies to identify a microbiota that is associated with liver disease . variables such as antibiotics usage , diet preferences , and environmental exposures need to be controlled for when the influence of a genetic or environmental factor is evaluated . whether known genetic risk alleles for liver disease affect the microbiome composition or the mucosal immune system deserves future studies . the immune system is very important in maintaining the symbiotic relationship between the host and the intestinal microbiome . intestinal bacteria develop and regulate the host immune system , and the immune system affects the composition of the intestinal microbiome . in particular , the mucosal immune system ensures a beneficial microbiota composition by restricting the growth of pathogens , controlling bacterial overgrowth , and reacting to pathogens and bacteria that reach the intestinal barriers , chemical barriers ( iga and antimicrobial peptides and proteins [ amp ] ) , and physical barriers ( the mucus layer and the tightly interconnected intestinal epithelial lining).49 , 50 , 51 host iga response is primarily directed toward specific bacteria within the mucosa , and this bias is disrupted in animals deficient for myd88 in t cells ( t - myd88 ) . t - myd88 animals showed increased dissimilarity in both their iga - bound and mucosa - associated communities and increased total bacterial load at the mucosa . thus , innate signaling by t cells dictates iga specificity to constrain the composition of the microbial community , while also limiting mucosal association of commensal microbes . myd88 signaling in t cells directs iga - mediated control of the microbiota to promote health . in addition , gut microbiota - derived flagellin is recognized by dendritic cells in lamina propria via tlr-5 , inducing the differentiation of b cells into iga - producing plasma cells . iga - deficient mice produce gut microbiota - specific serum igg antibodies due to mucosal barrier disruption and activation of the systemic immune system . furthermore , the host protein programmed cell death 1 ( pd1 ) partially regulates the microbial modulation of iga homeostasis ; pd1-deficient mice had an altered iga repertoire and changes in microbial communities of the gut . the importance of iga for microbiota composition and mucosal defense remains to be studied in chronic liver diseases . the gut microbiota also regulates the production of amp such as defensins , c - type lectins ( eg , regenerating islet - derived 3b [ reg3b ] and reg3 g ) , ribonucleases ( eg , angiopoietin 4 ) , and s100 proteins ( eg , psoriasin ) in intestinal epithelial cells and paneth cells , which rapidly kill or inactivate microorganisms . altered amp production is evident in mice deficient for myd88 , nod2 , or metalloproteinase-7 ( mmp7 ) , a protease involved in the regulation of defensin activity , as well as in mice that are transgenic for -defensin 5.55 , 56 , 57 we have shown that chronic alcohol administration results in suppression of intestinal reg3b and reg3 g expression in mice.12 , 14 the reg3 g level can be restored using prebiotics , which are associated with suppression of intestinal bacterial overgrowth . cirrhotic rats with ascites and translocation of viable bacteria to mesenteric lymph nodes produce lower levels of defensins and reg3 molecules compared with cirrhotic rats without bacterial translocation . further studies are required to determine whether and to what extent lower levels of amp contribute to liver disease progression by either modulating the composition of the intestinal microbiota or facilitating bacterial translocation . furthermore , goblet cells are secreting mucins , which are highly glycosylated and responsible for the formation of the inner and outer mucus layer . mucin-2 is the major secreted mucin in the small and large intestines of mice and humans . bacteria express glycosidases and metabolic enzymes , which allows them to bind to or even use the mucus layer as a source of energy.60 , 61 although the absence of mucin-2 in mice increases the susceptibility to enteric infections , the innate immune system is on high alert and activated to maintain intestinal homeostasis . in fact , mucin-2deficient mice showed much higher expression of antimicrobial proteins and were protected from intestinal bacterial overgrowth and dysbiosis in response to alcohol feeding . subsequently , lower amounts of bacterial products such as endotoxin translocated into the systemic circulation , thereby decreasing ald . this is an example of the interconnection between the different intestinal defense layers and the microbiota . interestingly , in patients with liver cirrhosis most taxonomically assigned species are of buccal origin , suggesting an invasion of bacteria from the mouth to the intestine . liver cirrhosis is known to be accompanied by decreased innate immune system surveillance , including lower gastric acid secretion , reduced bile flow , and impaired amp production . compromised host defense might contribute to the migration of the oral to the intestinal microbiome . however , to which degree each individual factor contributes to compositional changes in the microbiota requires further analysis . we have summarized the bacterial taxonomic changes in ald , nafld / nash , and cirrhosis in recent reviews.67 , 68 interestingly , most of the precirrhotic liver diseases and cirrhosis are associated with intestinal bacterial overgrowth ( described in detail in our aforementioned reviews ) . no single bacterial species has been mechanistically linked to the onset or progression of liver disease . dietary habits may induce dysbiosis , characterized by an increased percentage of intestinal gram - negative bacteria . indeed , gram - negative proteobacteria are a transplantable phylum that accelerates cholestatic liver fibrosis . as shown by us and described in more detail previously , the reversal of metabolic intestinal changes improves ald . future studies should establish causative correlations between functional rather than taxonomic microbiota changes and the pathogenesis of liver disease . for example , a high - fat diet ( hfd ) increases intestinal permeability in mice and humans . patients with nafld have significantly increased intestinal permeability and more disrupted tight junctions than healthy individuals . chronic alcohol abuse results in a disruption of the intestinal barrier . how is the gut barrier being disrupted , and is this dependent on the microbiome and dysbiosis ? although there is a possibility that liver disease itself increases intestinal permeability , intestinal inflammation might directly cause a gut barrier dysfunction and the translocation of microbial products . hfd increases the activity of the transcription factor nuclear factor-b ( nfb ) and expression of tumor necrosis factor- ( tnf ) in the small intestine of mice . hfd - induced intestinal inflammation depends on the enteric microbiota , because germ - free mice are protected from inflammatory changes . as discussed earlier , nlrp3 and nlrp6 deficiency promotes intestinal dysbiosis , triggers colonic inflammation via chemokine ( c - c motif ) ligand 5 ( ccl5 ) , and increases intestinal permeability . microbial products translocate to the liver , enhance hepatic inflammation , and cause nafld to progress to steatohepatitis . as a marker of a disrupted mucosal barrier , the systemic levels of lps are elevated in hfd - fed mice and humans after a high - fat meal.76 , 77 endotoxemia is observed in children with nafld and in patients with nash . similarly , chronic alcohol feeding results in subclinical intestinal inflammation in mice . the number of tnf-producing monocytes and macrophages in the small intestine increases in mice chronically administered alcohol . intestinal decontamination with nonabsorbable antibiotics reduced intestinal bacterial overgrowth , decreased intestinal inflammation and permeability , and reduced ald in mice . these findings suggest that dysbiosis causes intestinal inflammation and gut barrier dysfunction , although the triggering microbial metabolite or product is currently not known . in tissue - specific genetically manipulated mice , the tnf - receptor-1 ( tnfr-1 ) expressed on intestinal epithelial cells was identified as a target for tnf secreted from inflammatory cells in the lamina propria . our results suggest that dysbiosis - induced intestinal inflammation and tnfr-1 signaling on enterocytes are mediating a disruption of the intestinal barrier and most importantly ald . both , experimental cirrhosis and human cirrhotics , show features of intestinal inflammation.80 , 81 , 82 whether intestinal inflammation associated with cirrhosis depends on the gut microbiome is not known . microbial products or pamps translocate through disrupted tight junctions using the paracellular route from the intestinal lumen to extraintestinal tissues and organs , but living bacteria appear to translocate via the transcellular route ( transcytosis ) . during decompensated cirrhosis , a further increase in intestinal permeability could be triggered by intestinal inflammation and may contribute to enhanced translocation of viable bacteria . pamps reach the liver via the portal system and activate specific pattern recognition receptors including tlrs . mice that express nonfunctional molecules in the tlr signaling pathway are often protected from liver disease.85 , 86 , 87 , 88 signaling via the lps receptor tlr4 in hematopoietic - derived cells is required for the development of liver steatosis but not for the development of obesity in mice . mice deficient in sensing pamps or downstream signaling are resistant to nash.90 , 91 bacterial overgrowth correlates with an increase in the luminal amount of pamps . thus , in the presence of an intestinal barrier dysfunction , the luminal bacterial load determines the amount of pamps that translocate to the systemic circulation and hence the degree of liver damage . antibiotics seem to have a beneficial effect by reducing the intestinal bacterial burden , which in turn reduces the amount of translocating pamps . intestinal decontamination improves several diseases that are dependent on increased intestinal permeability and elevated systemic levels of pamps . treatment of obese mice with antibiotics improves glucose metabolism and reduces adiposity and adipose inflammation . a clinical trial in patients with ald using the nonabsorbable antibiotic paromomycin did not show an improvement in liver damage compared with placebo - treated patients . because systemic endotoxemia was not reduced after 4 weeks of treatment , antibiotic therapy might not have been effective , or it might have induced dysbiosis . cirrhotics suffering from hepatic encephalopathy are commonly treated with nonabsorbable antibiotics , which decreases the number of ammonia - producing intestinal bacteria and improves the mental status of patients with end - stage liver disease . in addition , antibiotics reduce the translocation of viable bacteria from the intestinal lumen to extraintestinal space such as mesenteric lymph nodes in experimental cirrhosis . all the above evidence strongly argues for an important role of microbial products that translocate from the intestine to the liver . alcoholics could be divided into low and high gut permeability groups , with the low permeability group having a similar permeability as healthy controls . alcoholics with high intestinal permeability also had an altered composition and activity of the gut microbiota such as lower amounts of bifidobacterium spp . , clostridiales family xiv incertae sedis , and ruminococcaceae when compared with healthy controls . levels of these bacteria were not changed in alcoholics with a low intestinal permeability . importantly , higher intestinal permeability was associated with higher scores of depression , anxiety , and alcohol craving after 3 weeks of abstinence . whether liver disease correlates with the degree of intestinal permeability in humans requires further study . to confirm the importance of the intestinal microbiota for chronic liver disease , we induced liver fibrosis in germ - free and conventional mice by administration of thioacetamide via the drinking water . to our surprise , germ - free mice showed more liver fibrosis than conventional mice . to confirm our results we used a second model of liver fibrosis by repeated intraperitoneal injections of carbon tetrachloride , switched the source of germ - free mice , and used a different gnotobiotic facility , but we still found exacerbated liver fibrosis in the absence of the microbiota . using an additional genetic model with mice lacking myd88/trif and hence downstream innate immunity signaling , we identified hepatocytes to be more susceptible to toxin - induced cell death . thus , although microbial products are related to inflammation and progressive liver disease , the complete absence of pamps also has a deleterious effect on the liver . it is feasible that metabolic products from the microbiota might provide hepatoprotection and might ameliorate chronic liver diseases . for example , indole-3 propionic acid is a deamination product of dietary tryptophan , and its production is completely dependent on the presence of the gut microbiota . indole-3 propionic acid was recently shown to protect the intestinal barrier through the xenobiotic sensor pregnane x receptor ( pxr ) . we would like to highlight and present a few examples of other ways the microbiota contributes to liver disease . both nafld and nash are strongly linked to obesity , type 2 diabetes mellitus , and the metabolic syndrome . a western diet rich in fats and carbohydrates contributes to qualitative microbiota changes . the microbiota may contribute to obesity and nafld in a number of ways , some of which are quite unexpected . alterations in the intestinal microbiota increase energy extraction and fermentation of dietary fibers into oligosaccharides , monosaccharides , and short - chain fatty acids , respectively . for instance . there may be complex mechanisms responsible for increased energy harvest from the diet by bacteria . transplantation of fecal microbiota from adult female twin pairs discordant for obesity into germ - free mice fed low - fat mouse chow , as well as diets representing different levels of saturated fat and fruit and vegetable consumption typical of the u.s . diet , increased total body and fat mass as well as obesity - associated metabolic phenotypes . moreover , despite gaining the same body weight after hfd feeding , one group of mice developed hyperglycemia and had a high plasma concentration of proinflammatory cytokines ( called the responders ) , while the second cohort remained normoglycemic and had lower levels of systemic inflammation ( nonresponders ) . germ - free mice colonized with intestinal microbiota from the nonresponder group maintained normoglycemia whereas germ - free mice transplanted with responder microbiota showed hyperglycemia , hepatic macrovesicular steatosis , more hepatic triglycerides , and an increased expression of genes involved in de novo lipogenesis after hfd feeding . these results suggest that gut microbiota contributes to the development of nafld independently of obesity . for instance , microbiota produce endogenous ethanol through the fermentation of carbohydrates , a physiologic pathway that is strongly enhanced in the presence of gut dysmotility ( eg , associated with obesity , diabetes , or chronic alcohol abuse ) or an excess of carbohydrates in the diet.106 , 107 obese animals have higher blood ethanol levels . once ethanol reaches the liver via the portal vein , it can promote steatosis , oxidative stress , and liver inflammation . pediatric patients with nash have higher blood ethanol concentrations than healthy individuals or pediatric patients with nafld , suggesting that endogenous ethanol production by the microbiota might be important in the progression from simple hepatic steatosis to nash . ethanol can be metabolized into acetaldehyde by microbial fermentation or by the host either in the liver or the intestine.110 , 111 ethanol can reach the microbiome and intestinal epithelia cells from either the luminal side or via diffusion from the systemic blood circulation . ethanol and in particular its metabolic derivative acetaldehyde can disrupt intestinal tight junctions and increase intestinal permeability . conventionalization of adult germ - free c57bl/6 mice with a normal microbiota harvested from the distal intestine ( cecum ) of conventionally raised animals produces a 60% increase in body fat content and insulin resistance . fasting - induced adipocyte factor ( fiaf ) , a member of the angiopoietin - like family of proteins , is selectively suppressed in the intestinal epithelium of normal mice by conventionalization . analysis of germ - free and conventionalized , wild - type , and fiaf - deficient mice established that fiaf is a circulating lipoprotein lipase inhibitor and that its suppression is essential for the microbiota - induced deposition of triglycerides in adipocytes . a dysbiotic microbiome associated with hfd metabolizes and converts dietary choline into methylamines . lower hepatic choline levels reduce secretion of very - low - density lipoprotein in the liver causing nash.113 , 114 choline deficiency is linked to nafld and nash and is a common model of experimental nash in rodents . healthy subjects develop a fatty liver on a choline - deficient diet only if there is a single - nucleotide polymorphism in the promoter region of phosphatidylethanolamine n - methyltransferase , which affects de novo synthesis of phosphatidylcholine . this is an excellent example how environmental and genetic factors synergize to cause liver disease . causes for changes in the intestinal microbiota composition and consequences of intestinal dysbiosis that are relevant for the pathogenesis of chronic liver diseases are illustrated and summarized in figure 1 . this complex community influences both normal physiology and disease susceptibilities through its collective metabolic activities and host interactions . we are just starting to understand the mechanisms that result in dysbiosis and its functional consequences for liver diseases . we need to further dissect the mechanisms of how dysbiosis affects intestinal homeostasis and causes the progression of liver disease beyond triggering an increase in intestinal permeability . genetic factors ( single - nucleotide polymorphisms , gene copy numbers , etc . ) are involved in shaping the composition of the gut microbiota and modulating disease susceptibility or resistance.40 , 43 , 117 given that germ - free mice are more susceptible to experimental liver fibrosis , there might be hepatoprotective microbial metabolites . our ultimate goal should be to design therapies that restore intestinal eubiosis and homeostasis . this could be achieved by either targeting the microbiota or the intestine of the host . this goal is formidable because of the immense diversity among the microbiota , interpersonal variations , and temporal fluctuations in composition , which are especially apparent in disease .

the leaky gut hypothesis links translocating microbial products with the onset and progression of liver disease , and for a long time they were considered one of its major contributors . however , a more detailed picture of the intestinal microbiota contributing to liver disease started to evolve . the gut is colonized by trillions of microbes that aid in digestion , modulate immune response , and generate a variety of products that result from microbial metabolic activities . these products together with host - bacteria interactions influence both normal physiology and disease susceptibility . a disruption of the symbiosis between microbiota and host is known as dysbiosis and can have profound effects on health . qualitative changes such as increased proportions of harmful bacteria and reduced levels of beneficial bacteria , and also quantitative changes in the total amount of bacteria ( overgrowth ) have been associated with liver disease . understanding the link between the pathophysiology of liver diseases and compositional and functional changes of the microbiota will help in the design of innovative therapies . in this review , we focus on factors resulting in dysbiosis , and discuss how dysbiosis can disrupt intestinal homeostasis and contribute to liver disease .

heavy metals pollution in water , soils , and sediment is increasingly serious in china along with the rapid industrialization and urbanization [ 1 , 2 ] . heavy metals as a type of persistent toxic pollutants are unbiodegradable in the environment . thus , the residual heavy metals in environment are threatening human health and ecosecurity [ 4 , 5 ] . the main sources of heavy metals in the water are atmospheric precipitation , discharge of industrial wastewater and urban sewage , mineral mining , and infusion of surface runoff [ 6 , 7 ] . heavy metals are insoluble in the receiving water , and the majority of them are transformed from the aqueous phase to the solid phase and finally deposit in the sediment . due to this process , the contents of heavy metals in sediments were higher than those in aqueous phase ; hence , that can be regarded as the accumulation library of heavy metals . however , the heavy metals in the sediments can be released into the environment again , causing secondary pollution of the water and chronically damaging the ecoenvironment [ 13 , 14 ] . the heavy metals as nondegradable toxic substances in the water can be enriched via food chains from low- to high - level organisms . such enrichment leads to direct or indirect accumulation of heavy metals in human body , causing chronic poisoning and threatening human health or even life [ 5 , 17 , 18 ] . the rapid economic development in northeast china has led to the quick increase of urban water demand and the expanding discharge of industrial wastewater and sewage , polluting the water system there severely . this paper aims to investigate the heavy metal pollution in groundwater and the sediments , and the spatial distribution characterization of the heavy metals in water system . the risks of heavy metal pollution in groundwater and the degrees and potential ecological risks of heavy metal pollution in sediments were assessed . the migration scope of as in water system was predicted by using the groundwater modeling system ( gms ) . the study area was dominated by croplands along the river , and the elevation ( mean 4550 m ) generally declined from northeast to southwest . totally 33 groundwater sampling sites were distributed along the water flow direction of the river . totally 12 sampling sites of sediment were distributed from 12310 to 12328e , with reference to the sampling sites of groundwater . cr , as , cd , and pb were selected as monitoring factors based on the historical research data of environment pollution in the study area as well as the tasks of this study . samples of sediments were dried in an oven at 105c for 24 h and the samples were microwave digested with nitric acid , hydrochloric acid , hydrofluoric acid , and hydrogen peroxide before determination , as described in a previous study . the contents of heavy metals ( cr(vi ) , as , cd , and pb ) in groundwater samples and in sediment samples digested were measured using an inductively coupled plasma mass spectrometer ( icp - ms ) . analytical quality control included analysis of reagent blank , sample blank , reference material , and three parallel samples . the content distributions of cr(vi ) , as , cd , and pb in groundwater were plotted in figure 2 using arcgis according to the test results . cr(vi ) , as , cd , and pb pollution in groundwater was assessed on basis of chinese national quality standard for groundwater ( gb / t 14848 - 93 ) . clearly , the as , cr(vi ) , and cd contents in groundwater did not exceed the thresholds in sanitary standard for drinking water ( gb 5749 - 2006 ) , and the contents of heavy metals in most sites met class i in gb / t 14848 - 93 ( figure 2 ) . however , the pb contents in some sampling sites exceeded the thresholds in the above two standards . two kinds of risk assessment models of heavy metal pollution degree were used to evaluate the heavy metal pollution in the groundwater in the study area . single - factor pollution index ( i ) is used to assess how a single heavy metal pollutes groundwater at a sampling site:(1)ii = cisi , where c i is the measured content of pollutant i in surface water ( ml ) and s i is the evaluation standard of pollutant i in surface water ( ml ) . when i i is > 1 , the content of that heavy metal exceeds the standard . the results of single - factor pollution index of heavy metals in groundwater in the study areas are showed in figure 3 using the class ii standards in gb / t 14848 - 93 . the is of cd at sites 10 and 17 were > 1 , indicating cd contents exceeded the standard . the is of cr(vi ) and as did not exceed 1 , indicating the contents of these two heavy metals met the class ii standards in gb / t 14848 - 93 . nemerow pollution index ( ni ) is used to assess how several heavy metals pollute groundwater at a sampling site . this index considers the mean and maximum values of single - factor pollution index and highlights the pollutants with high pollution degrees . it is expressed as ( 2)ni=1/nci / si2+maxci / si221/2,where n is the number of indices ; c i is the measured content of heavy metal i ; s i is the standard value . the heavy metal pollution in groundwater the pollution degree and pollution level of heavy metals in groundwater were assessed with ni . the heavy metal pollution was evaluated using class ii standard in gb / t 14848 - 93 . the nis of heavy metals in groundwater in the study area are showed in figure 4 . the ni at site 28 exceeded the standard ; the nis at sites 10 , 17 , and 18 were at warming level . the solute transport of as was simulated by using gms with the above models and the results , under the natural state without adding any external factors such as anthropogenic impact . the forms and distribution of pollution halo were characterized and the contents of specific pollutants in groundwater were predicted . the solute transport was simulated by using the measured as contents as initial conditions without considering other pollution sources . the simulated as transport at different time periods in the study area is illustrated in figure 5 . the distribution area of as at different concentration ranges in different years was calculated by using the arcgis software . the simulated results showed that the as polluted area was 286.28 km in the west of the study area and 98.36 km in the east of the study area at the moment , which indicated the main polluted area was in the downstream of the hun river . g / l in the west polluted area with 1.02 km ; the as content in the second district was 3.5 g / l with 2.28 km , followed by the as content being 2.25 g / l with 23.41 km , 1.5 g / l with 38.91 km , and 1.0 g / l with 286.28 km , respectively . the contents and the areas after as transfer in different years were showed in table 2 . the results showed that the polluted areas were decreased gradually for the 10-year transfer in low content districts and decreased gradually for first 5-year transfer and increased gradually for last 5-year transfer in high content districts . after 10 years of transfer , the as polluted areas were 2.25 km with 4.25 g / l , 12.63 km with 3.5 g / l , 24.22 km with 2.25 g / l , 36.13 km with 1.5 g / l , and 119.30 km with 1.0 the reason why the as polluted areas were increased in high content districts because these were some pump wells downstream , which pumped about 2000 m / d each well and made the solute transfer upstream because of the funnels formed . into the severely polluted areas , a continuous pollution source with constant pollution concentration was added and the solute transfer was simulated . the simulated results were showed in figure 6 . with the addition of a pollution source , the simulated results showed that as transfer areas did not change largely from those under the initial conditions ; the as polluted area was 286.28 km in the west of the study area at the moment . the highest as content in the center was 4.25 g / l in the west polluted area with 3.82 km , 3.5 g / l with 4.63 km , 2.25 g / l with 24.88 km , 1.5 g / l with 42.04 km , and 1.0 g / l with 295.09 , respectively . the contents and the areas after as transfer in different years were showed in table 3 . the results showed that the changing trend of polluted areas was almost the same with those as initial conditions . however , the highest as content area was not changed almost because of the addition of a pollution source . after 10 years of transfer , the as polluted areas were 3.23 km with 4.25 g / l , 14.24 km with 3.5 g / l , 27.31 km with 2.25 g / l , 35.94 km with 1.5 g / l , and 135.80 km with 1.0 the contents of heavy metals in sediment reflect the degree of heavy metal pollution in the water . to clarify the sources of heavy metals in groundwater , we also collected sediment at varying depth . the contents of cr(vi ) , as , cd , and pb were measured using icp - ms . the results are showed in table 4 . clearly , cr pollution was the most severe in sediment of hun river , and the cr contents in most samples exceeded the background value , followed by pb . cd content only at site n21 - 2 exceeded the background value , and no as content exceeded the background value . single - factor pollution index c f is expressed as follows:(3)cfi = cdicri , where c d is the measured heavy metal content in sediment and c r is the background content of a heavy metal ( table 4 ) . c f > 1 indicates pollution of a heavy metal , and c f < 1 indicates a clean compound pollution index ( cpi ) was used to assess the heavy metal pollution in sediment , which is expressed as follows : ( 4)cpi=i=1mcfim , where c f is a single - factor index of a heavy metal and m is the number of heavy metal types . cpi < 1 indicates no heavy metal pollution in sediment ; cpi 1 indicates heavy metal pollution . the pollution levels of the heavy metal were clarified in table 6 . based on these results , we can see that cr pollution was the most severe in the river sediment , and the cr contents in most sampling sites exceeded the background value , followed by pb ( tables 5 - 6 ) . cd content only at site n21 - 2 exceeded the background value , and no as content exceeded the background value . n21 - 2 , and s20 - 1 were at comprehensive pollution , and in particular , the cpi of n21 - 2 was 2.1716 . the potential ecological risk index of a heavy metal , e r , is expressed as follows : ( 5)eri = tricfi , where t r is the toxicity response coefficient of that heavy metal and reflects the toxicity of the heavy metal and the water body 's sensitivity to the heavy metal pollution ; c f is the coefficient of pollution . the potential ecological risk index of multiple heavy metal ( ri ) is expressed as follows : ( 6)ri=i=1meri . the ecological risk of the study area was assessed using the classification of all potential ecological risk indices ( table 7 ) . clearly , e r of four heavy metals was < 40 , indicating the four heavy metals were at low potential ecological risks . the major states of heavy metals include weak acid - extractable form , reducible form , oxidizable form , and residual form . we analyzed the forms of heavy metals at the severely polluted sites ( n19 - 2 , s20 - 1 , n21 - 1 , and n21 - 2 ) according to soil and sediment sequential extraction procedure of speciation of 13 trace elements ( gb / t25282 - 2010 ) . the results are showed in table 8 . clearly , the proportions of bioavailability forms of cd were all the highest among the four metals at four sites , especially the weak acid - extractable form . the forms indicate that cd is most soluble in rivers and its migration features facilitate its migration via the water into groundwater , which is basically consistent with the heavy metal pollution situations in groundwater described in section 3.1 . then pb was mainly in the reducible form , indicating that pb can be easily released into the environment to pollute the water . therefore , we deduce that the release of heavy metals from sediment is a major pollution source of heavy metals in groundwater . ( 1 ) the comprehensive pollution at groundwater site 28 reached the pollution level , sites 10 , 17 , and 18 reached warning level , and other sites were all clean . the changes and migration scope of as content indicate that as migrated downstream to hun river in recent 10 years ; the polluted areas were decreased gradually in low content districts and decreased gradually first and increased gradually later in high content districts because these were some pump wells downstream to form groundwater depression cone , which made the solute transfer upstream . ( 2 ) both the cr(vi ) and pb contents in most sediment samples exceeded the national background values . the potential ecological risk of cd was very high at site n21 - 2 , but not at other sites . ( 3 ) through the morphological analysis of heavy metals at four sites where the contents were the highest , the proportions of biodegradable form of cd were all the highest among the four metals at four sites , secondly was the weak acid - extractable , which all indicated cd could be easily released into the water and polluted the groundwater due to the migration . pb was mainly in the reduction form , indicating that pb also can be released into the environment and pollute the water . therefore , we deduce that the release from sediment is a major source of heavy metal pollution in groundwater .

the areas with typical municipal sewage discharge river and irrigation water function were selected as study sites in northeast china . the samples from groundwater and river sediment in this area were collected for the concentrations and forms of heavy metals ( cr(vi ) , cd , as , and pb ) analysis . the risk assessment of heavy metal pollution was conducted based on single - factor pollution index ( i ) and nemerow pollution index ( ni ) . the results showed that only one groundwater sampling site reached a polluted level of heavy metals . there was a high potential ecological risk of cd on the n21 - 2 sampling site in river sediment . the morphological analysis results of heavy metals in sediment showed that the release of heavy metals can be inferred as one of the main pollution sources of groundwater . in addition , the changes in the concentration and migration scope of as were predicted by using the groundwater modeling system ( gms ) . the predicted results showed that as will migrate downstream in the next decade , and the changing trend of as polluted areas was changed with as content districts because of some pump wells downstream to form groundwater depression cone , which made the solute transfer upstream .

glaucoma , an optic neuropathy affecting over 60 million people , is the second cause of global blindness and draws intensive attention because of the irreversibility of glaucomatous optic nerve damage . previous studies have found that collagen tissues create changes in glaucoma patients , such as biomechanics changes of trabecular meshwork ( tm ) , thinner sclera , and laminar cribrosa [ 35 ] . however , are these changes the result of elevated intraocular pressure ( iop ) or are they the primary original factors causing glaucoma ? elevated iop is generally considered as the main risk factor in glaucomatous pathogenesis , which is primarily caused by increased aqueous humor outflow resistance . previous studies hypothesized that one of the main causes of this is biomechanical and molecular changes of the extracellular matrix ( ecm ) in the tm . this study assumes that changes in collagen , as the main component of ecm in the tm , may be involved in increased aqueous humor outflow resistance and elevation of iop . we can find that patients have variable susceptibility to glaucomatous optic neuropathy , which presents with high - tension glaucoma , normal - tension glaucoma ( ntg ) , or ocular hypertension . sclera and lamina cribrosa , known as the load - bearing tissues , may be responsible for that . collagen plays structural roles and contributes to mechanical properties , organization , and shape of tissues . collagen changes can result in a weaker structure , including changes in elasticity and compliance of collagen tissues , and decreased density and thickness of these tissues , which may be more sensitive to elevated iop . based on the above , this study hypothesized that primary change of collagen is an original factor in the pathogenesis of glaucoma . ecm in tm is believed to be essential for maintenance of the normal outflow system . changes in biomechanics of ecm , caused by dysfunction and structural alteration of collagen , affect the tm function and the aqueous humor outflow . comparing the stiffness of tm in normal tissue and glaucomatous tissue , last et al . found that the stiffness of glaucomatous tm significantly increased , which resulted from dysregulation of ecm . studies also found that glaucomatous eyes have different forms of ecm deposited within the cribriform layer to increase the outflow resistance . hence , changes in tm elasticity and mechanical load may have a significant role in glaucoma . several alterations in collagen expression and transcription have been characterized in the tm of primary open - angle glaucoma ( poag ) . type i collagen is the major component of structures within the tm collagen beans and uveoscleral aqueous humor outflow pathways . targeted type i collagen mutation and induced an elevation of iop in mice , and their results suggested an association between iop regulation and collagen turnover . the turnover of collagen causes difficulty in hydrolyzation by matrix metalloproteinases ( mmps ) , leading to accumulation of type i collagen . another collagen , type vi , also increases , which is associated with sheath - derived plaques in the cribriform meshwork . thus , collagen abnormality in the outflow pathway appears to play an important role in the elevation of iop and may be one of the significant original factors of glaucoma . a variety of cellular and molecular changes can occur that modify the connective tissues of the optic nerve and surrounding sclera in aging , ocular development , and glaucomatous disease . these changes remodel the microenvironment of the optic nerve , and presumably change the susceptibility to axonal injury in this region . many experimental and clinical studies have found that the biomechanical properties of sclera shift to being less elastic and stiffer in glaucoma [ 1517 ] . sclera has a collagen - rich ecm , and collagen constitutes 90% of scleral dry weight . collagen fibers in sclera are organized into irregularly arranged and somewhat interwoven lamellae ; the lamellae varying in thickness . the variations of collagen may lead to inter - individual differences in scleral material properties . previous detailed modeling studies found that scleral material properties were varied over physiologic ranges , suggesting that there could be significant inter - individual differences . inter - individual variations in sclera , particularly peripapillary scleral thickness , can result in vastly different biomechanical responses to iop . thinner sclera is more sensitive to developing glaucoma . for example , decreased density of collagen in the peripapillary sclera was found in glaucoma ; and in high myopia eyes , sclera is elongated and thinned , which increases risk of developing poag . the region of laminar cribrosa in the optic nerve head ( onh ) is the principal site of retinal ganglion cells ( rgcs ) axonal insult in glaucoma . the lamina cribrosa provides structural and functional support to the rgcs axons while passing from the relatively high - pressure environment in the eye to a low - pressure region in the retro bulbar cerebrospinal space . however , because of discontinuity in the corneal - scleral shell , lamina cribrosa is often considered a weak spot in mechanically loaded systems , and is the site of substantial stress concentration . collagen types i , iii , iv , v , and vi constitute the main composition in the lamina cribrosa , and these macromolecules change with age . as this tissue ages , individual differences , leading to more or less of a particular macromolecule of the extracellular matrix , may alter the support function of the lamina cribrosa and influence the degeneration of the optic nerve associated with glaucoma . the inter - individual variations in lamina cribrosa thickness are responsible for ability to resist damage . recently , using spectral domain optical coherence tomography ( sd - oct ) , park et al . on the other hand , thin lamina cribrosa leads to a decreased distance between the intraocular space and the space of the retrobulbar cerebrospinal fluid compartment at a given trans - lamina cribrosa pressure difference between both compartments , the pressure gradient gets steeper due to the reduced distance between both compartments . hence , one may assume that even at the same iop level , individuals with thinner lamina cribrosa will have increased susceptibility to glaucomatous optic neuropathy . a recent study strongly claimed that the lower and upper quadrants of onh are indeed lower density , and that if exposed in increased iop , this region is an area of vulnerability and may be the first site to become damaged . this may help understand the selective visual field loss observed in the initial stages of glaucoma . besides thickness and density , ocular developmental differences and mechanical properties changes with aging form the inter - individual variations of susceptibility to developing glaucoma . laboratory evidence has also demonstrated the potential role for collagens in glaucomatous optic neuropathy . in glaucomatous monkey eyes , alterations in the three - dimensional organization of collagen fibrils were observed in the optic nerve head , suggesting that these architectural changes may affect the flexibility and resilience required of the lamina cribrosa in supporting optic nerve fibers . in glaucoma and suspected glaucoma , the content and/or the composition of the collagen molecules in the lamina cribrosa is significantly changed , and differs from that of normal eyes . one can therefore speculate that a primary collagen disturbance might be involved in the pathogenesis of glaucoma . evidence , as mentioned above , indicates that the inter - individual differences of sclera and lamina cribrosa will affect susceptibility to glaucoma . this may explain the individual variation to elevated iop seen clinically . during rises in iop , if these tissues are strong enough to resist elevated iop and to prevent the optic nerve from being damaged , ocular hypertension occurs . in contrast , if these tissues are weak , normal - tension glaucoma is presented . based on this evidence , this study hypothesizes primary changes in collagen molecules results in a weaker structure , which increases susceptibility to glaucoma . evidence from a meta - analysis strongly indicates that individuals with myopia have an increased risk of developing poag . people with moderate and especially high myopia have a two - fold to three - fold increased risk of glaucoma compared with that of non - myopic subjects , and this risk is independent of other glaucoma risk factors and iop . myopia is a complex pathology of ocular structure with elongated axial length . increasing axial length results in many structural changes such as thinner sclera and lamina cribrosa and weakness of the fibroglial matrix of the nerve fibers at the optic disc . the explanation is thought to be that variation of collagen structure and amounts leads to weakness of these tissues , which could contribute to the high susceptibility of the optic disc to iop fluctuations and to the increasing risk of glaucomatous neuropathy . collagen undergoes significant changes during the development of myopia . in regard to the development of myopia , previous studies found a significant loss of scleral tissue weight and subsequent scleral thinning associated with a narrowing and disconnection of collagen fiber bundles and a reduction in the number of bundles . reduction of collagen amounts in sclera is a result of both decreased collagen synthesis and increased collagen degradation . these ultrastructural variations suggest a derangement of the organization and growth of the collagen fibers , which result in the thinning of sclera . moreover , the thinning of the peripapillary sclera is an additional biomechanical factor that increases tension in the lamina cribrosa beams , leading to increased glaucoma susceptibility . many experimental and clinical studies have found that the biomechanical properties of sclera shift to being less elastic and stiffer in glaucoma [ 1517 ] . some scientists indicate that these changes may be related to the development of glaucoma . sclera has a collagen - rich ecm , and collagen constitutes 90% of scleral dry weight . collagen fibers in sclera are organized into irregularly arranged and somewhat interwoven lamellae ; the lamellae varying in thickness . the variations of collagen may lead to inter - individual differences in scleral material properties . previous detailed modeling studies found that scleral material properties were varied over physiologic ranges , suggesting that there could be significant inter - individual differences . inter - individual variations in sclera , particularly peripapillary scleral thickness , can result in vastly different biomechanical responses to iop . thinner sclera is more sensitive to developing glaucoma . for example , decreased density of collagen in the peripapillary sclera was found in glaucoma ; and in high myopia eyes , sclera is elongated and thinned , which increases risk of developing poag . the region of laminar cribrosa in the optic nerve head ( onh ) is the principal site of retinal ganglion cells ( rgcs ) axonal insult in glaucoma . the lamina cribrosa provides structural and functional support to the rgcs axons while passing from the relatively high - pressure environment in the eye to a low - pressure region in the retro bulbar cerebrospinal space . however , because of discontinuity in the corneal - scleral shell , lamina cribrosa is often considered a weak spot in mechanically loaded systems , and is the site of substantial stress concentration . collagen types i , iii , iv , v , and vi constitute the main composition in the lamina cribrosa , and these macromolecules change with age . as this tissue ages , individual differences , leading to more or less of a particular macromolecule of the extracellular matrix , may alter the support function of the lamina cribrosa and influence the degeneration of the optic nerve associated with glaucoma . the inter - individual variations in lamina cribrosa thickness are responsible for ability to resist damage . recently , using spectral domain optical coherence tomography ( sd - oct ) , park et al . found that lamina cribrosa thickness was significantly thinner in ntg than in poag . thin lamina cribrosa provide less biomechanical support for the optic nerve . on the other hand , thin lamina cribrosa leads to a decreased distance between the intraocular space and the space of the retrobulbar cerebrospinal fluid compartment at a given trans - lamina cribrosa pressure difference between both compartments , the pressure gradient gets steeper due to the reduced distance between both compartments . hence , one may assume that even at the same iop level , individuals with thinner lamina cribrosa will have increased susceptibility to glaucomatous optic neuropathy . a recent study strongly claimed that the lower and upper quadrants of onh are indeed lower density , and that if exposed in increased iop , this region is an area of vulnerability and may be the first site to become damaged . this may help understand the selective visual field loss observed in the initial stages of glaucoma . besides thickness and density , ocular developmental differences and mechanical properties changes with aging form the inter - individual variations of susceptibility to developing glaucoma . laboratory evidence has also demonstrated the potential role for collagens in glaucomatous optic neuropathy . in glaucomatous monkey eyes , alterations in the three - dimensional organization of collagen fibrils were observed in the optic nerve head , suggesting that these architectural changes may affect the flexibility and resilience required of the lamina cribrosa in supporting optic nerve fibers . in glaucoma and suspected glaucoma , the content and/or the composition of the collagen molecules in the lamina cribrosa is significantly changed , and differs from that of normal eyes . one can therefore speculate that a primary collagen disturbance might be involved in the pathogenesis of glaucoma . evidence , as mentioned above , indicates that the inter - individual differences of sclera and lamina cribrosa will affect susceptibility to glaucoma . this may explain the individual variation to elevated iop seen clinically . during rises in iop , if these tissues are strong enough to resist elevated iop and to prevent the optic nerve from being damaged , ocular hypertension occurs . in contrast , if these tissues are weak , normal - tension glaucoma is presented . based on this evidence , this study hypothesizes primary changes in collagen molecules results in a weaker structure , which increases susceptibility to glaucoma . evidence from a meta - analysis strongly indicates that individuals with myopia have an increased risk of developing poag . people with moderate and especially high myopia have a two - fold to three - fold increased risk of glaucoma compared with that of non - myopic subjects , and this risk is independent of other glaucoma risk factors and iop . myopia is a complex pathology of ocular structure with elongated axial length . increasing axial length results in many structural changes such as thinner sclera and lamina cribrosa and weakness of the fibroglial matrix of the nerve fibers at the optic disc . the explanation is thought to be that variation of collagen structure and amounts leads to weakness of these tissues , which could contribute to the high susceptibility of the optic disc to iop fluctuations and to the increasing risk of glaucomatous neuropathy . collagen undergoes significant changes during the development of myopia . in regard to the development of myopia , previous studies found a significant loss of scleral tissue weight and subsequent scleral thinning associated with a narrowing and disconnection of collagen fiber bundles and a reduction in the number of bundles . reduction of collagen amounts in sclera is a result of both decreased collagen synthesis and increased collagen degradation . these ultrastructural variations suggest a derangement of the organization and growth of the collagen fibers , which result in the thinning of sclera . moreover , the thinning of the peripapillary sclera is an additional biomechanical factor that increases tension in the lamina cribrosa beams , leading to increased glaucoma susceptibility . in addition , recent theories about glaucomatous optic neuropathy can not effectively explain the different susceptibility of individuals to elevated iop . this article hypothesizes that glaucoma is a disorder disease with a series of characteristic pathological alterations of collagen in its content , distribution , ultrastructure , and metabolism . inter - individual differences in scleral and lamina cribrosa caused by variations of collagen contribute to inter - individual variation in susceptibility to elevated iop . collagen , as an original cause of glaucoma , plays an important role in glaucoma pathogenesis . some changes in eye collagen may occur before the development of glaucoma or glaucomatous optic neuropathy .

numerous studies have been completed on glaucoma pathogenesis . however , the potential and controversial interaction between ocular biomechanical properties and the glaucomatous diseases process has received much more attention recently . previous studies have found that collagen tissues gain mutation change in glaucoma patients . this study was conducted to determine the role of collagen in the biomechanics of glaucoma in humans . its changes may be the result of mechanical modifications brought on by intraocular pressure ( iop ) fluctuations . more importantly , biomechanics and genetic evidence indicate that the mutation of collagen may play a role in the process of glaucoma . alteration of collagen in the outflow pathway may alter mechanical tissue characteristics and a concomitant increase of aqueous humor outflow resistance and elevation of iop . the variations of collagen , leading to inter - individual differences in scleral and lamina cribrosa properties , result in different susceptibility of individuals to elevated iop . therefore , this study hypothesized that collagen mutations may be an original cause of glaucoma .

according to the 2014 report from the world health organization , 39% of the world 's adult population is overweight , and 13% is obese . in the near future an important feature of obesity and aging is dysregulation of fat in relation to morbidity and mortality . adipokines , proteins secreted by the adipose tissue ( at ) , can trigger metabolic syndromes such as obesity and type 2 diabetes . metabolic diseases are mostly caused by excessive energy storage in the lipid droplets of adipocytes , which results in at expansion . it is therefore of interest to determine the causes and the molecular mechanisms of at expansion in order to find opportunities to control it . over - nutrition leads to at expansion , which is regulated by two events : excessive energy storage into the lipid droplets of adipocytes , a process leading to hypertrophy ( increase in adipocyte size ) , and increased adipogenesis , also known as adipocyte hyperplasia . adipogenesis is a process of differentiation of multipotent mesenchymal stem cells ( msc ) into adipocytes . several transcription factors have been identified as master regulators for preadipocyte determination , such as zinc finger protein 423 ( zfp423 ) and early b cell factor 1 ( ebf1 ) . whereas zfp423 induces early commitment , terminal differentiation is tightly controlled by a transcriptional cascade , whereby peroxisome proliferator - activated receptor ( ppar ) is the essential transcription factor . further key transcriptional factors are the ccaat / enhancer - binding protein ( c / ebp ) family members ( i.e. , c / ebp , c / ebp , and c / ebp ) , kruppel - like factors ( klfs ) , camp responsive element binding protein ( creb ) and early growth response 20 ( krox20 ) . recently , it has been shown that the activator protein-1 ( ap-1 ) family is involved in the adipocyte differentiation process . the ap-1 family is formed by a dimeric protein complex , composed of fos , jun and/or activating transcription factor ( atf ) members . fos - related antigen 1 and 2 ( fra-1 and fra-2 ) are able to regulate adipocyte differentiation . fra-1 impairs adipocyte differentiation by inhibiting c / ebp , whereas fra-2 controls adipocyte turnover . fra-2 thereby not only decreases the adipocyte number by repressing ppar2 expression during adipocyte differentiation , but also decreases adipocyte apoptosis through direct repression of hypoxia - inducible factors ( hifs ) expression . the hif family is a heterodimeric transcription factor complex , composed of hif-1 , hif-2 and hif-1. the heterodimers consist of an oxygen - sensitive hif- protein ( hif-1 or hif-2 ) and the oxygen - insensitive hif-1 subunit . during normoxia , hif- proteins are poly - ubiquitinylated and are finally degraded by proteasomes . under hypoxic conditions , occurring in at during expansion they therefore become stabilized and form dimers with the constitutively expressed hif-1. transcriptional activation of genes controlled by the hif response elements is involved in the regulation of angiogenesis , metabolism , and inflammation . indeed , hif-1 promotes at dysfunction by inducing glucose tolerance , inhibiting energy expenditure and peripheral use of lipid , as well as by increasing leptin level and hfd - induced hepatic steatosis . the present protocol describes methods for studying at status to unravel the molecular characteristics of adipocyte homeostasis in adult mice . it shows how apoptosis , proliferation and differentiation of adipocytes in vivo and in vitro can be regulated by hypoxia . to do so , we use mice with adipocyte specific deletion of fra-2 generated by crossing mice carrying the fra-2 floxed alleles with fabp4-creert mice . by using fabp4-cre ert mice , the deletion is adipocyte specific and inducible by tamoxifen injection . for the adult model , intra peritoneal injections of tamoxifen are performed over 5 consecutive days starting at the age of 6 weeks . thus , the mice are subjected to a normal diet or high - fat diet for 6 weeks before the analysis is done . the mice used in this study were male based on a c57bl6 background to avoid female hormones , such as estrogens , shown to regulate the body fat distribution . using another genetic background might also alter the metabolic phenotype , due to strain - related differences in lipid management . this protocol demonstrates how to analyze at under hypoxia using histology and how to quantify adipocyte apoptosis , proliferation and differentiation in vivo using immunohistochemistry and gene profiling analyses . the study is completed by in vitro experiments , showing how to analyze primary adipocyte differentiation and apoptosis altered by exposure to hypoxia . ethics statement : animals are housed in standardized conditions following the guidelines of the german animal welfare act . animals are fed a standard diet and water ad libitum and kept with a 12 hr day / night cycle . all experiments with animals are authorized by the local ethics committee . to quantify hypoxia in vivo , first determine the body weight of the mice , then inject 60 mg / kg body weight of solid pimonidazole hydrochloride intra - peritoneally ( for example : pimonidazole is an effective hypoxic marker , which forms adducts with thiol groups in proteins , peptides and amino acids and is detected by a specific antibody . 45 min after injection , sacrifice mice by co2 asphyxiation and subsequent cervical dislocation.pin down the limbs of mice ( as illustrated in figure 1 ) and open the peritoneal cavity . remove the left and the right perigonadal ( epididymal ) fat pad inside the peritoneal cavity . note : fat pad are bound to the epididymis by the peritoneal leaflets as shown in figure 1 ( perigonadal fat pads are indicated by arrows).take care to remove the gonadal tissues from the fat pad . determine fat pad weights to calculate the ratio : fat pad weight ( g ) per body weight ( g ) . pin down the limbs of mice ( as illustrated in figure 1 ) and open the peritoneal cavity . remove the left and the right perigonadal ( epididymal ) fat pad inside the peritoneal cavity . note : fat pad are bound to the epididymis by the peritoneal leaflets as shown in figure 1 ( perigonadal fat pads are indicated by arrows ) . determine fat pad weights to calculate the ratio : fat pad weight ( g ) per body weight ( g ) . please click here to view a larger version of this figure . to compile a quantitative gene expression profile , use one perigonadal fat pad to isolate rna . note : until the tissue is processed , store tissue samples in rna stabilization solution at -80 c or in liquid nitrogen . to homogenize the fat pad , add the fat pad to 1 ml single - phase solution of guanidine isothiocyanate and phenol . use tubes containing ceramic beads ( 1.4 mm ) to crush the tissue in a homogenizer at 6,500 rpm ( 2 times 20 sec , with 30 sec pause).isolate the rna as follows ( single - step method by chomczynski and sacchi ) . to separate the phases , transfer homogenized fat pad to microcentrifuge tube , add 0.2 ml volume chloroform , shake for 15 sec , incubate for 5 min at room temperature and centrifuge 12,000 x g for 5 min . transfer the upper aqueous phase ( around 400 l ) , which contains the rna , into a new microcentrifuge tube . do not include the dna - containing interphase or the protein - containing phenol phase.to precipitate the rna , add 1 volume of isopropanol , mix and incubate for 15 min at 4 c ( it is also possible to incubate at -20 c overnight ) . remove isopropanol supernatant carefully . wash twice with 75% ethanol with centrifuge steps of 12,000 x g for 10 min.after drying the precipitated rna for around 10 min at room temperature , dissolve it in 50 l h2o ( rnase - free ) . to facilitate this , incubate for 2 min at 65 c . note : keep rna in 75% ethanol at -80 c or in liquid nitrogen for long - term storage.quantify the rna preparations by a260/280 ( optimal quotient is between 1.8 and 2.2 ) and calculate the concentration by a260 : to avoid dna contamination , digest 1 g of the rna preparation with 1 u dnase i for 30 min at 37 c in a volume of 10 l . note : this step is optional.use 10 l of rna preparation containing 1 g rna for the reverse transcriptase reaction to generate single - stranded cdna , suitable for quantitative pcr application . the components and their amounts are listed in table 1.use a pcr master mix for a quantitative real - time pcr reaction . to determine metabolic changes in the whole fat pad , use specific primers for genes involved in at homeostasis ( table 2 ) and the pcr conditions listed in table 3.real-time pcr data analysis . define the baseline ( figure 2 ) , normally cycle 1 to 15 , where there is no change in fluorescence signals . the real - time pcr software normalizes specific fluorescence signals to the baseline fluorescence and to the internal reference dye , rox , resulting in the magnitude of the specific signals by the primers , delta rn ( rn).set the threshold within the exponential phase of the amplification curve . based on the ct value , calculate the relative expression ( ct ) and the fold change ( ct ) : to homogenize the fat pad , add the fat pad to 1 ml single - phase solution of guanidine isothiocyanate and phenol . use tubes containing ceramic beads ( 1.4 mm ) to crush the tissue in a homogenizer at 6,500 rpm ( 2 times 20 sec , with 30 sec pause ) . isolate the rna as follows ( single - step method by chomczynski and sacchi ) . to separate the phases , transfer homogenized fat pad to microcentrifuge tube , add 0.2 ml volume chloroform , shake for 15 sec , incubate for 5 min at room temperature and centrifuge 12,000 x g for 5 min . transfer the upper aqueous phase ( around 400 l ) , which contains the rna , into a new microcentrifuge tube . do not include the dna - containing interphase or the protein - containing phenol phase.to precipitate the rna , add 1 volume of isopropanol , mix and incubate for 15 min at 4 c ( it is also possible to incubate at -20 c overnight ) . wash twice with 75% ethanol with centrifuge steps of 12,000 x g for 10 min.after drying the precipitated rna for around 10 min at room temperature , dissolve it in 50 l h2o ( rnase - free ) . to facilitate this , incubate for 2 min at 65 c . note : keep rna in 75% ethanol at -80 c or in liquid nitrogen for long - term storage.quantify the rna preparations by a260/280 ( optimal quotient is between 1.8 and 2.2 ) and calculate the concentration by a260 : to separate the phases , transfer homogenized fat pad to microcentrifuge tube , add 0.2 ml volume chloroform , shake for 15 sec , incubate for 5 min at room temperature and centrifuge 12,000 x g for 5 min . transfer the upper aqueous phase ( around 400 l ) , which contains the rna , into a new microcentrifuge tube . do not include the dna - containing interphase or the protein - containing phenol phase . to precipitate the rna , add 1 volume of isopropanol , mix and incubate for 15 min at 4 c ( it is also possible to incubate at -20 c overnight ) . wash twice with 75% ethanol with centrifuge steps of 12,000 x g for 10 min . after drying the precipitated rna for around 10 min at room temperature , dissolve it in 50 l h2o ( rnase - free ) . to facilitate this , incubate for 2 min at 65 c . note : keep rna in 75% ethanol at -80 c or in liquid nitrogen for long - term storage . quantify the rna preparations by a260/280 ( optimal quotient is between 1.8 and 2.2 ) and calculate the concentration by a260 : to avoid dna contamination , digest 1 g of the rna preparation with 1 u dnase i for 30 min at 37 c in a volume of 10 l . use 10 l of rna preparation containing 1 g rna for the reverse transcriptase reaction to generate single - stranded cdna , suitable for quantitative pcr application . use a pcr master mix for a quantitative real - time pcr reaction . to determine metabolic changes in the whole fat pad , use specific primers for genes involved in at homeostasis ( table 2 ) and the pcr conditions listed in table 3 . real - time pcr data analysis . define the baseline ( figure 2 ) , normally cycle 1 to 15 , where there is no change in fluorescence signals . the real - time pcr software normalizes specific fluorescence signals to the baseline fluorescence and to the internal reference dye , rox , resulting in the magnitude of the specific signals by the primers , delta rn ( rn).set the threshold within the exponential phase of the amplification curve . based on the ct value , calculate the relative expression ( ct ) and the fold change ( ct ) : define the baseline ( figure 2 ) , normally cycle 1 to 15 , where there is no change in fluorescence signals . the real - time pcr software normalizes specific fluorescence signals to the baseline fluorescence and to the internal reference dye , rox , resulting in the magnitude of the specific signals by the primers , delta rn ( rn ) . based on the ct value , calculate the relative expression ( ct ) and the fold change ( ct ) : table 1 : components with respective volume for the reverse transcriptase reaction to generate single - stranded cdna . table 2 : list of genes with sequence of the respective primers used for analyzing adipocyte homeostasis . to perform histological analysis of the adipocyte homeostasis , use the second perigonadal fat pad . do not desiccate the tissue ! fix the fat pad in 3.7% pbs - buffered formaldehyde overnight , embed in paraffin ( follow the instructions as described elsewhere ) and cut the embedded tissue into 2 - 5 m thick sections ( maximum 5 m).determine the number of adipocytes per field and adipocyte size in a bright - field microscope after hematoxylin and eosin ( h&e ) staining : deparaffinize sections by washing 3 times for 5 min in xylene and rehydrate the section 2 times for 2 min in 100% ethanol and 2 times for 2 min in 96% ethanol . finally , wash the section in distilled h2o for 5 min.stain with hematoxylin , diluted 1:5 with distilled h2o , for 10 min at room temperature and wash in h2o for 5 min . stain with eosin solution ( 20 ml 5% eosin y/210 ml distilled h2o/25 l glacial acetic acid ) for 30 sec and wash again with h2o for 5 min.dehydrate sections using 96% ethanol and 100% ethanol 2 times each for 2 min , and xylene 3 times for 5 min . mount the sections with anhydrous mounting agent.evaluate the sections under a bright - field microscope . a representative example of how to analyze adipocytes with imagej 1.48v is shown in fig . 3 : number of cells per area ( m ) , cell area ( x 10 m ) , cell size ( m ) . open the picture of the section with imagej 1.48v . if the parameters of the image are indicated in pixels instead of a unit of length , adjust the scale.select * straight line * in the toolbar and adjust the line to a known distance by reference to the scale bar . go to analyze - > set scale . the distance of the line is shown in pixels ; add the known distance and the unit of length , e.g. , m . the size of the picture and the analysis of the parameters are indicated in the unit of length given.to determine the parameters of the adipocytes , adjust the threshold . select the following settings : thresholding method : default ; threshold color : b&w ; color space : hsb . the picture is now shown in black and white . adjust the brightness to clear white adipocytes and to close black intercellular spaces , as in fig . 3b.count the number of adipocyte per m ( figure 3e ) with the * multi - point * selection in the toolbar and mark each cell for counting.to determine the adipocyte size , select * straight line * again in the toolbar and draw the diameter of an adipocyte ( figure 3c ) . go to analyze - > measure and a new window will appear , with the length of the diameter in m.to determine the adipocyte area , select * wand ( tracing ) tool * and click inside the adipocyte . go to analyze - > measure and a new window will appear , with the area of this adipocyte in m . for immunohistochemistry , prepare the section for antibody and tdt - mediated dutp - biotin nick end labeling ( tunel ) staining as follows : deparaffinize sections by washing 3 times for 5 min in xylene and rehydrate the section 2 times for 2 min in 100% ethanol and 2 times for 2 min in 96% ethanol . finally , wash the section in distilled h2o.for antigen retrieval , digest the tissue section for 30 min at 37 c with proteinase k working solution ( 20 g / ml in 10 mm tris / hcl , ph 7.4 - 8 ) and rinse with pbs . perform antibody staining in wet chambers : to block the endogenous peroxidase , use 3% hydrogen peroxide in pbs for 10 min , with subsequently washing 2 times for 5 min in pbs . to block the unspecific binding of antibodies , use 10% serum in pbs . use the serum of the host of the secondary antibody , in this case goat.for the staining , use the antibodies for apoptosis , proliferation and hypoxia detection ( table 4 ) . dilute antibodies in pbs/10% goat serum . wash the sections 3 times for 5 min in pbs.to enhance the signal use a biotinylated secondary antibody , with the dilution as listed in table 5 . for hypoxia detection , use hrp conjugated rabbit anti note : for the hypoxia staining with fitc - mab1 , skip step 1.4.4.4 ) and continue with step 1.4.4.5).for each slide , pre - incubate 50 l avidin solution with 50 l biotinylated peroxidase h for 30 min at room temperature ( this method is also referred to as avidin / biotin abc complex formulation ) and then add to the sections for another 45 min . wash 2 times for 5 min with pbs.incubate the sections in peroxidase substrate solution until the staining become more intense ( between 5 to 10 min ) . wash for 5 min with distilled h2o.for counterstaining , stain with hematoxylin diluted 1:5 with distilled h2o for 10 min at room temperature and wash in h2o for 5 min.dehydrate sections in 96% ethanol and 100% ethanol 2 times each for 2 min , and xylene 3 times for 5 min . determine apoptosis by tunel assay and follow manufacturer 's instructions : add 50 l enzyme solution into 450 l label solution . then apply 50 l tunel reaction mixture on histologic sections and incubate for 60 min at + 37 c in a humidified atmosphere in the dark . wash the sections 3 times with pbs for 5 min and mount with fluorescence mounting medium including dapi ( 4',6-diamidino-2-phenylindole ) for counterstaining.evaluate the section under a fluorescence microscope with 100 fold magnification . for measurement of fluorescein use an excitation wavelength of 488 nm and detect between 515 - 565 nm ( green laser ) ; dapi excites at about 360 nm and emits at about 460 nm when bound to dna ( blue laser).quantify the overlays of dapi and fluorescein : fix the fat pad in 3.7% pbs - buffered formaldehyde overnight , embed in paraffin ( follow the instructions as described elsewhere ) and cut the embedded tissue into 2 - 5 m thick sections ( maximum 5 m ) . determine the number of adipocytes per field and adipocyte size in a bright - field microscope after hematoxylin and eosin ( h&e ) staining : deparaffinize sections by washing 3 times for 5 min in xylene and rehydrate the section 2 times for 2 min in 100% ethanol and 2 times for 2 min in 96% ethanol . finally , wash the section in distilled h2o for 5 min.stain with hematoxylin , diluted 1:5 with distilled h2o , for 10 min at room temperature and wash in h2o for 5 min . stain with eosin solution ( 20 ml 5% eosin y/210 ml distilled h2o/25 l glacial acetic acid ) for 30 sec and wash again with h2o for 5 min.dehydrate sections using 96% ethanol and 100% ethanol 2 times each for 2 min , and xylene 3 times for 5 min . mount the sections with anhydrous mounting agent.evaluate the sections under a bright - field microscope . a representative example of how to analyze adipocytes with imagej 1.48v is shown in fig . 3 : number of cells per area ( m ) , cell area ( x 10 m ) , cell size ( m ) . open the picture of the section with imagej 1.48v . if the parameters of the image are indicated in pixels instead of a unit of length , adjust the scale.select * straight line * in the toolbar and adjust the line to a known distance by reference to the scale bar . go to analyze - > set scale . the distance of the line is shown in pixels ; add the known distance and the unit of length , e.g. , m . the size of the picture and the analysis of the parameters are indicated in the unit of length given.to determine the parameters of the adipocytes , adjust the threshold . select the following settings : thresholding method : default ; threshold color : b&w ; color space : hsb . the picture is now shown in black and white . adjust the brightness to clear white adipocytes and to close black intercellular spaces , as in fig . 3b.count the number of adipocyte per m ( figure 3e ) with the * multi - point * selection in the toolbar and mark each cell for counting.to determine the adipocyte size , select * straight line * again in the toolbar and draw the diameter of an adipocyte ( figure 3c ) . go to analyze - > measure and a new window will appear , with the length of the diameter in m.to determine the adipocyte area , select * wand ( tracing ) tool * and click inside the adipocyte . go to analyze - > measure and a new window will appear , with the area of this adipocyte in m . deparaffinize sections by washing 3 times for 5 min in xylene and rehydrate the section 2 times for 2 min in 100% ethanol and 2 times for 2 min in 96% ethanol . finally , wash the section in distilled h2o for 5 min . stain with hematoxylin , diluted 1:5 with distilled h2o , for 10 min at room temperature and wash in h2o for 5 min . stain with eosin solution ( 20 ml 5% eosin y/210 ml distilled h2o/25 l glacial acetic acid ) for 30 sec and wash again with h2o for 5 min . dehydrate sections using 96% ethanol and 100% ethanol 2 times each for 2 min , and xylene 3 times for 5 min . a representative example of how to analyze adipocytes with imagej 1.48v is shown in fig . 3 : number of cells per area ( m ) , cell area ( x 10 m ) , cell size ( m ) . open the picture of the section with imagej 1.48v . if the parameters of the image are indicated in pixels instead of a unit of length , adjust the scale.select * straight line * in the toolbar and adjust the line to a known distance by reference to the scale bar . the distance of the line is shown in pixels ; add the known distance and the unit of length , e.g. , m . the size of the picture and the analysis of the parameters are indicated in the unit of length given.to determine the parameters of the adipocytes , adjust the threshold . select the following settings : thresholding method : default ; threshold color : b&w ; color space : hsb . the picture is now shown in black and white . adjust the brightness to clear white adipocytes and to close black intercellular spaces , as in fig . 3b.count the number of adipocyte per m ( figure 3e ) with the * multi - point * selection in the toolbar and mark each cell for counting.to determine the adipocyte size , select * straight line * again in the toolbar and draw the diameter of an adipocyte ( figure 3c ) . go to analyze - > measure and a new window will appear , with the length of the diameter in m.to determine the adipocyte area , select * wand ( tracing ) tool * and click inside the adipocyte . go to analyze - > measure and a new window will appear , with the area of this adipocyte in m . open the picture of the section with imagej 1.48v . if the parameters of the image are indicated in pixels instead of a unit of length , adjust the scale . select * straight line * in the toolbar and adjust the line to a known distance by reference to the scale bar . the distance of the line is shown in pixels ; add the known distance and the unit of length , e.g. , m . confirm with ok . the size of the picture and the analysis of the parameters are indicated in the unit of length given . to determine the parameters of the adipocytes , adjust the threshold . select the following settings : thresholding method : default ; threshold color : b&w ; color space : hsb . the picture is now shown in black and white . adjust the brightness to clear white adipocytes and to close black intercellular spaces , as in fig . 3b . count the number of adipocyte per m ( figure 3e ) with the * multi - point * selection in the toolbar and mark each cell for counting . to determine the adipocyte size , select * straight line * again in the toolbar and draw the diameter of an adipocyte ( figure 3c ) . go to analyze - > measure and a new window will appear , with the length of the diameter in m . to determine the adipocyte area , select * wand ( tracing ) tool * and click inside the adipocyte . go to analyze - > measure and a new window will appear , with the area of this adipocyte in m . for immunohistochemistry , prepare the section for antibody and tdt - mediated dutp - biotin nick end labeling ( tunel ) staining as follows : deparaffinize sections by washing 3 times for 5 min in xylene and rehydrate the section 2 times for 2 min in 100% ethanol and 2 times for 2 min in 96% ethanol . finally , wash the section in distilled h2o.for antigen retrieval , digest the tissue section for 30 min at 37 c with proteinase k working solution ( 20 g / ml in 10 mm tris / hcl , ph 7.4 - 8 ) and rinse with pbs . deparaffinize sections by washing 3 times for 5 min in xylene and rehydrate the section 2 times for 2 min in 100% ethanol and 2 times for 2 min in 96% ethanol . finally , wash the section in distilled h2o . for antigen retrieval , digest the tissue section for 30 min at 37 c with proteinase k working solution ( 20 g / ml in 10 mm tris / hcl , ph 7.4 - 8 ) and rinse with pbs . perform antibody staining in wet chambers : to block the endogenous peroxidase , use 3% hydrogen peroxide in pbs for 10 min , with subsequently washing 2 times for 5 min in pbs . to block the unspecific binding of antibodies , use 10% serum in pbs . use the serum of the host of the secondary antibody , in this case goat.for the staining , use the antibodies for apoptosis , proliferation and hypoxia detection ( table 4 ) . dilute antibodies in pbs/10% goat serum . wash the sections 3 times for 5 min in pbs.to enhance the signal use a biotinylated secondary antibody , with the dilution as listed in table 5 . for hypoxia detection , use hrp conjugated rabbit anti note : for the hypoxia staining with fitc - mab1 , skip step 1.4.4.4 ) and continue with step 1.4.4.5).for each slide , pre - incubate 50 l avidin solution with 50 l biotinylated peroxidase h for 30 min at room temperature ( this method is also referred to as avidin / biotin abc complex formulation ) and then add to the sections for another 45 min . wash 2 times for 5 min with pbs.incubate the sections in peroxidase substrate solution until the staining become more intense ( between 5 to 10 min ) . wash for 5 min with distilled h2o.for counterstaining , stain with hematoxylin diluted 1:5 with distilled h2o for 10 min at room temperature and wash in h2o for 5 min.dehydrate sections in 96% ethanol and 100% ethanol 2 times each for 2 min , and xylene 3 times for 5 min . evaluate the sections under a bright - field microscope . to block the endogenous peroxidase , use 3% hydrogen peroxide in pbs for 10 min , with subsequently washing 2 times for 5 min in pbs . to block the unspecific binding of antibodies , use 10% serum in pbs . use the serum of the host of the secondary antibody , in this case goat . for the staining , use the antibodies for apoptosis , proliferation and hypoxia detection ( table 4 ) . dilute antibodies in pbs/10% goat serum . wash the sections 3 times for 5 min in pbs . to enhance the signal use a biotinylated secondary antibody , with the dilution as listed in table 5 . for hypoxia detection , use hrp conjugated rabbit anti note : for the hypoxia staining with fitc - mab1 , skip step 1.4.4.4 ) and continue with step 1.4.4.5 ) . for each slide , pre - incubate 50 l avidin solution with 50 l biotinylated peroxidase h for 30 min at room temperature ( this method is also referred to as avidin / biotin abc complex formulation ) and then add to the sections for another 45 min . incubate the sections in peroxidase substrate solution until the staining become more intense ( between 5 to 10 min ) . for counterstaining , stain with hematoxylin diluted 1:5 with distilled h2o for 10 min at room temperature and wash in h2o for 5 min . dehydrate sections in 96% ethanol and 100% ethanol 2 times each for 2 min , and xylene 3 times for 5 min . determine apoptosis by tunel assay and follow manufacturer 's instructions : add 50 l enzyme solution into 450 l label solution . then apply 50 l tunel reaction mixture on histologic sections and incubate for 60 min at + 37 c in a humidified atmosphere in the dark . wash the sections 3 times with pbs for 5 min and mount with fluorescence mounting medium including dapi ( 4',6-diamidino-2-phenylindole ) for counterstaining.evaluate the section under a fluorescence microscope with 100 fold magnification . for measurement of fluorescein use an excitation wavelength of 488 nm and detect between 515 - 565 nm ( green laser ) ; dapi excites at about 360 nm and emits at about 460 nm when bound to dna ( blue laser).quantify the overlays of dapi and fluorescein : add 50 l enzyme solution into 450 l label solution . then apply 50 l tunel reaction mixture on histologic sections and incubate for 60 min at + 37 c in a humidified atmosphere in the dark . wash the sections 3 times with pbs for 5 min and mount with fluorescence mounting medium including dapi ( 4',6-diamidino-2-phenylindole ) for counterstaining . evaluate the section under a fluorescence microscope with 100 fold magnification . for measurement of fluorescein use an excitation wavelength of 488 nm and detect between 515 - 565 nm ( green laser ) ; dapi excites at about 360 nm and emits at about 460 nm when bound to dna ( blue laser ) . quantify the overlays of dapi and fluorescein : figure 3 : analyzing adipocyte characteristics in fat pad sections . section pictures of the perigonadal fat pad of male mice treated with a high - fat diet ( hfd ) or normal diet ( nd ) with a bright - field microscope ( a ) ; the threshold is adjusted into black and white ( b ) and the adipocyte size ( length ; c ) , area ( d ) and adipocyte cell number per mm ( e ) are quantified with imagej 1.48v . table 4 : antibodies with respective dilution used for the immunohistological staining of at sections . table 5 : secondary antibodies with dilution used for immunohistological staining . sacrifice mice and remove the subcutaneous adipose tissue . sacrifice the mice by co2 asphyxiation and subsequent cervical dislocation.pin down the limbs of mice as illustrated in figure 4 . detach the skin from the upper leg , loin and flank and pin it down with needles as in figure 4 . then remove the subcutaneous adipose tissue , which is located posterior at the base of the hind legs , surrounding the inguinal lymph nodes ( as shown in figure 4 , left : subcutaneous fat pads indicated by the arrows ; right : inguinal lymph node indicated by the arrow ) . detach the skin from the upper leg , loin and flank and pin it down with needles as in figure 4 . then remove the subcutaneous adipose tissue , which is located posterior at the base of the hind legs , surrounding the inguinal lymph nodes ( as shown in figure 4 , left : subcutaneous fat pads indicated by the arrows ; picture of subcutaneous fat pad ; the left arrows indicate the subcutaneous fat pad and the right arrow indicates the inguinal lymph node . seed adsc 4,000 cells / cm in dulbecco 's modified eagle 's medium - ham 's f-12 supplemented with 10% normal calf serum , 1% penicillin / streptomycin , 0.5% amphotericin b , 16 m biotin , 18 m pantothenic acid and 100 m ascorbic acid and grow culture to confluence around 70 to 80% , which is reached after 4 to 6 days of culture . induce adipogenic differentiation . remove the adherent adsc from the surface by trypsin treatment . remove medium , wash with pbs and add 0.025% trypsin solution ( preheated to 37 c ) for 2 min ( until cells detach from the surface ) . count the cells using a neubauer chamber . put the glass cover on the central area of the neubauer chamber . dilute the cell suspension 1:10 and load the chamber with 10 l diluted cell suspension . count the cells in 4 squares located at the corners , each composed of 16 smaller squares . calculate the cell number per ml : seed adsc ( as described in point 2.2 ) in 12-well culture plates and grow culture to confluence around 70 to 80% ( reached after 4 to 6 days).induce adipogenic differentiation by adding 5 g / ml insulin , 1 m dexamethasone and 5 m 3-isobutyl-1-methylxanthine ( ibmx ) to the cultures . remove the adherent adsc from the surface by trypsin treatment . remove medium , wash with pbs and add 0.025% trypsin solution ( preheated to 37 c ) for 2 min ( until cells detach from the surface ) . immediately add medium and wash the cells . remove medium , wash with pbs and add 0.025% trypsin solution ( preheated to 37 c ) for 2 min ( until cells detach from the surface ) . immediately add medium and wash the cells . count the cells using a neubauer chamber . put the glass cover on the central area of the neubauer chamber . dilute the cell suspension 1:10 and load the chamber with 10 l diluted cell suspension . count the cells in 4 squares located at the corners , each composed of 16 smaller squares . calculate the cell number per ml : put the glass cover on the central area of the neubauer chamber . dilute the cell suspension 1:10 and load the chamber with 10 l diluted cell suspension . count the cells in 4 squares located at the corners , each composed of 16 smaller squares . calculate the cell number per ml : seed adsc ( as described in point 2.2 ) in 12-well culture plates and grow culture to confluence around 70 to 80% ( reached after 4 to 6 days ) . induce adipogenic differentiation by adding 5 g / ml insulin , 1 m dexamethasone and 5 m 3-isobutyl-1-methylxanthine ( ibmx ) to the cultures . cells will be fully differentiated after 7 days . to analyze the adipogenic differentiation , stain with oil red o , which stains triglycerides of mature adipocytes . note : work must be performed under a fume hood ! remove the medium , wash the adipocytes gently with pbs and fix the cells for 60 min with 2 ml 10% formalin.to prepare oil red o staining solution , mix 3 parts of the red oil o stock solution ( 300 mg red oil o powder dissolved in 100 ml 99% isopropanol ) with 2 parts distilled h2o and incubate for 10 min at room temperature . note : the working solution is stable for 2 hr.for the oil red o staining , remove the formalin , wash adipocytes with h2o , incubate with 2 ml 60% isopropanol for 5 min , remove the isopropanol and add 2 ml oil red o working solution for 5 min . counterstain with hematoxylin as in point 1.4.4.5).evaluate the plates under a phase contrast microscope with 100 fold magnification . the lipids of the adipocytes will appear red and the nuclei will appear blue . remove the medium , wash the adipocytes gently with pbs and fix the cells for 60 min with 2 ml 10% formalin . to prepare oil red o staining solution , mix 3 parts of the red oil o stock solution ( 300 mg red oil o powder dissolved in 100 ml 99% isopropanol ) with 2 parts distilled h2o and incubate for 10 min at room temperature . o staining , remove the formalin , wash adipocytes with h2o , incubate with 2 ml 60% isopropanol for 5 min , remove the isopropanol and add 2 ml oil red o working solution for 5 min . rinse the cells with tap water until the water is clear . optional step : silence the gene of interest by transfection with shrna . change the medium and add serum - free medium.for the transfection of adipocytes , use lipofection . follow the manufacturer 's instructions and use 1 g shrna per 12-well tissue plates . after addition of the lipid - dna - complex , incubate adipocytes for 48 hr at 37 c . change the medium and add serum - free medium . for the transfection of adipocytes , use lipofection . follow the manufacturer 's instructions and use 1 g shrna per 12-well tissue plates . after addition of the lipid - dna - complex , incubate adipocytes for 48 hr at 37 c . to analyze adipocytes subjected to hypoxia , use a hypoxic work station or hypoxic incubator to maintain the cells under hypoxic conditions . gather adipocytes with an extra soft cell scraper , wash with pbs and add 1 x 10 cells to 100 l annexin v - binding buffer ( 10 mm hepes / naoh , ph 7.4 ; 140 mm nacl ; 2.5 mm cacl2 ) . add the amount of annexin v - fitc recommended by the manufacturer and incubate at room temperature for 15 min.for flow cytometry measurement , add 200 l annexin v - binding buffer and 1 m of nuclear counterstain . annexin v / nuclear counterstain cells are defined as secondary necrotic and annexin v / nuclear counterstain cells are defined as apoptotic cells ( figure 5 ) . add 1 ml single - phase solution of guanidine isothiocyanate and phenol to each well and isolate the rna [ using single - step method by chomczynski and sacchi ( step 1.3.2 ) ] and proceed as in steps 1.3.2.1 ) to 1.3.5.2 ) . analyze apoptosis by fitc - labeled annexin v and subsequent flow cytometry analyses . gather adipocytes with an extra soft cell scraper , wash with pbs and add 1 x 10 cells to 100 l annexin v - binding buffer ( 10 mm hepes / naoh , ph 7.4 ; 140 mm nacl ; 2.5 mm cacl2 ) . add the amount of annexin v - fitc recommended by the manufacturer and incubate at room temperature for 15 min.for flow cytometry measurement , add 200 l annexin v - binding buffer and 1 m of nuclear counterstain . annexin v / nuclear counterstain cells are defined as secondary necrotic and annexin v / nuclear counterstain cells are defined as apoptotic cells ( figure 5 ) . gather adipocytes with an extra soft cell scraper , wash with pbs and add 1 x 10 cells to 100 l annexin v - binding buffer ( 10 mm hepes / naoh , ph 7.4 ; 140 mm nacl ; 2.5 mm cacl2 ) . add the amount of annexin v - fitc recommended by the manufacturer and incubate at room temperature for 15 min . for flow cytometry measurement , add 200 l annexin v - binding buffer and 1 m of nuclear counterstain . annexin v / nuclear counterstain cells are defined as secondary necrotic and annexin v / nuclear counterstain cells are defined as apoptotic cells ( figure 5 ) . add 1 ml single - phase solution of guanidine isothiocyanate and phenol to each well and isolate the rna [ using single - step method by chomczynski and sacchi ( step 1.3.2 ) ] and proceed as in steps 1.3.2.1 ) to 1.3.5.2 ) . add 1 ml single - phase solution of guanidine isothiocyanate and phenol to each well and isolate the rna [ using single - step method by chomczynski and sacchi ( step 1.3.2 ) ] and proceed as in steps 1.3.2.1 ) to 1.3.5.2 ) . dot plot presentations of the facs for annexin v - fitc and to - pro-3 staining of adipocytes . to quantify hypoxia in vivo , first determine the body weight of the mice , then inject 60 mg / kg body weight of solid pimonidazole hydrochloride intra - peritoneally ( for example : inject 1.5 mg into a 25 g mouse ) . pimonidazole is an effective hypoxic marker , which forms adducts with thiol groups in proteins , peptides and amino acids and is detected by a specific antibody . 45 min after injection , sacrifice mice by co2 asphyxiation and subsequent cervical dislocation.pin down the limbs of mice ( as illustrated in figure 1 ) and open the peritoneal cavity . remove the left and the right perigonadal ( epididymal ) fat pad inside the peritoneal cavity . note : fat pad are bound to the epididymis by the peritoneal leaflets as shown in figure 1 ( perigonadal fat pads are indicated by arrows).take care to remove the gonadal tissues from the fat pad . determine fat pad weights to calculate the ratio : fat pad weight ( g ) per body weight ( g ) . pin down the limbs of mice ( as illustrated in figure 1 ) and open the peritoneal cavity . remove the left and the right perigonadal ( epididymal ) fat pad inside the peritoneal cavity . note : fat pad are bound to the epididymis by the peritoneal leaflets as shown in figure 1 ( perigonadal fat pads are indicated by arrows ) . take care to remove the gonadal tissues from the fat pad . determine fat pad weights to calculate the ratio : fat pad weight ( g ) per body weight ( g ) . please click here to view a larger version of this figure . to compile a quantitative gene expression profile , use one perigonadal fat pad to isolate rna . note : until the tissue is processed , store tissue samples in rna stabilization solution at -80 c or in liquid nitrogen . to homogenize the fat pad , add the fat pad to 1 ml single - phase solution of guanidine isothiocyanate and phenol . use tubes containing ceramic beads ( 1.4 mm ) to crush the tissue in a homogenizer at 6,500 rpm ( 2 times 20 sec , with 30 sec pause).isolate the rna as follows ( single - step method by chomczynski and sacchi ) . to separate the phases , transfer homogenized fat pad to microcentrifuge tube , add 0.2 ml volume chloroform , shake for 15 sec , incubate for 5 min at room temperature and centrifuge 12,000 x g for 5 min . transfer the upper aqueous phase ( around 400 l ) , which contains the rna , into a new microcentrifuge tube . do not include the dna - containing interphase or the protein - containing phenol phase.to precipitate the rna , add 1 volume of isopropanol , mix and incubate for 15 min at 4 c ( it is also possible to incubate at -20 c overnight ) . wash twice with 75% ethanol with centrifuge steps of 12,000 x g for 10 min.after drying the precipitated rna for around 10 min at room temperature , dissolve it in 50 l h2o ( rnase - free ) . to facilitate this , incubate for 2 min at 65 c . note : keep rna in 75% ethanol at -80 c or in liquid nitrogen for long - term storage.quantify the rna preparations by a260/280 ( optimal quotient is between 1.8 and 2.2 ) and calculate the concentration by a260 : to avoid dna contamination , digest 1 g of the rna preparation with 1 u dnase i for 30 min at 37 c in a volume of 10 l . note : this step is optional.use 10 l of rna preparation containing 1 g rna for the reverse transcriptase reaction to generate single - stranded cdna , suitable for quantitative pcr application . the components and their amounts are listed in table 1.use a pcr master mix for a quantitative real - time pcr reaction . to determine metabolic changes in the whole fat pad , use specific primers for genes involved in at homeostasis ( table 2 ) and the pcr conditions listed in table 3.real-time pcr data analysis . define the baseline ( figure 2 ) , normally cycle 1 to 15 , where there is no change in fluorescence signals . the real - time pcr software normalizes specific fluorescence signals to the baseline fluorescence and to the internal reference dye , rox , resulting in the magnitude of the specific signals by the primers , delta rn ( rn).set the threshold within the exponential phase of the amplification curve . based on the ct value , calculate the relative expression ( ct ) and the fold change ( ct ) : to homogenize the fat pad , add the fat pad to 1 ml single - phase solution of guanidine isothiocyanate and phenol . use tubes containing ceramic beads ( 1.4 mm ) to crush the tissue in a homogenizer at 6,500 rpm ( 2 times 20 sec , with 30 sec pause ) . isolate the rna as follows ( single - step method by chomczynski and sacchi ) . to separate the phases , transfer homogenized fat pad to microcentrifuge tube , add 0.2 ml volume chloroform , shake for 15 sec , incubate for 5 min at room temperature and centrifuge 12,000 x g for 5 min . transfer the upper aqueous phase ( around 400 l ) , which contains the rna , into a new microcentrifuge tube . do not include the dna - containing interphase or the protein - containing phenol phase.to precipitate the rna , add 1 volume of isopropanol , mix and incubate for 15 min at 4 c ( it is also possible to incubate at -20 c overnight ) . wash twice with 75% ethanol with centrifuge steps of 12,000 x g for 10 min.after drying the precipitated rna for around 10 min at room temperature , dissolve it in 50 l h2o ( rnase - free ) . to facilitate this , incubate for 2 min at 65 c . note : keep rna in 75% ethanol at -80 c or in liquid nitrogen for long - term storage.quantify the rna preparations by a260/280 ( optimal quotient is between 1.8 and 2.2 ) and calculate the concentration by a260 : to separate the phases , transfer homogenized fat pad to microcentrifuge tube , add 0.2 ml volume chloroform , shake for 15 sec , incubate for 5 min at room temperature and centrifuge 12,000 x g for 5 min . transfer the upper aqueous phase ( around 400 l ) , which contains the rna , into a new microcentrifuge tube . do not include the dna - containing interphase or the protein - containing phenol phase . to precipitate the rna , add 1 volume of isopropanol , mix and incubate for 15 min at 4 c ( it is also possible to incubate at -20 c overnight ) . wash twice with 75% ethanol with centrifuge steps of 12,000 x g for 10 min . after drying the precipitated rna for around 10 min at room temperature , dissolve it in 50 l h2o ( rnase - free ) . to facilitate this , incubate for 2 min at 65 c . note : keep rna in 75% ethanol at -80 c or in liquid nitrogen for long - term storage . quantify the rna preparations by a260/280 ( optimal quotient is between 1.8 and 2.2 ) and calculate the concentration by a260 : to avoid dna contamination , digest 1 g of the rna preparation with 1 u dnase i for 30 min at 37 c in a volume of 10 l . use 10 l of rna preparation containing 1 g rna for the reverse transcriptase reaction to generate single - stranded cdna , suitable for quantitative pcr application . use a pcr master mix for a quantitative real - time pcr reaction . to determine metabolic changes in the whole fat pad , use specific primers for genes involved in at homeostasis ( table 2 ) and the pcr conditions listed in table 3 . define the baseline ( figure 2 ) , normally cycle 1 to 15 , where there is no change in fluorescence signals . the real - time pcr software normalizes specific fluorescence signals to the baseline fluorescence and to the internal reference dye , rox , resulting in the magnitude of the specific signals by the primers , delta rn ( rn).set the threshold within the exponential phase of the amplification curve . based on the ct value , calculate the relative expression ( ct ) and the fold change ( ct ) : define the baseline ( figure 2 ) , normally cycle 1 to 15 , where there is no change in fluorescence signals . the real - time pcr software normalizes specific fluorescence signals to the baseline fluorescence and to the internal reference dye , rox , resulting in the magnitude of the specific signals by the primers , delta rn ( rn ) . based on the ct value , calculate the relative expression ( ct ) and the fold change ( ct ) : table 1 : components with respective volume for the reverse transcriptase reaction to generate single - stranded cdna . table 2 : list of genes with sequence of the respective primers used for analyzing adipocyte homeostasis . please click here to view a larger version of this figure . to perform histological analysis of the adipocyte homeostasis , use the second perigonadal fat pad . fix the fat pad in 3.7% pbs - buffered formaldehyde overnight , embed in paraffin ( follow the instructions as described elsewhere ) and cut the embedded tissue into 2 - 5 m thick sections ( maximum 5 m).determine the number of adipocytes per field and adipocyte size in a bright - field microscope after hematoxylin and eosin ( h&e ) staining : deparaffinize sections by washing 3 times for 5 min in xylene and rehydrate the section 2 times for 2 min in 100% ethanol and 2 times for 2 min in 96% ethanol . finally , wash the section in distilled h2o for 5 min.stain with hematoxylin , diluted 1:5 with distilled h2o , for 10 min at room temperature and wash in h2o for 5 min . stain with eosin solution ( 20 ml 5% eosin y/210 ml distilled h2o/25 l glacial acetic acid ) for 30 sec and wash again with h2o for 5 min.dehydrate sections using 96% ethanol and 100% ethanol 2 times each for 2 min , and xylene 3 times for 5 min . mount the sections with anhydrous mounting agent.evaluate the sections under a bright - field microscope . a representative example of how to analyze adipocytes with imagej 1.48v is shown in fig . 3 : number of cells per area ( m ) , cell area ( x 10 m ) , cell size ( m ) . open the picture of the section with imagej 1.48v . if the parameters of the image are indicated in pixels instead of a unit of length , adjust the scale.select * straight line * in the toolbar and adjust the line to a known distance by reference to the scale bar . go to analyze - > set scale . the distance of the line is shown in pixels ; add the known distance and the unit of length , e.g. , m . confirm with ok . the size of the picture and the analysis of the parameters are indicated in the unit of length given.to determine the parameters of the adipocytes , adjust the threshold . select the following settings : thresholding method : default ; threshold color : b&w ; color space : hsb . the picture is now shown in black and white . adjust the brightness to clear white adipocytes and to close black intercellular spaces , as in fig . 3b.count the number of adipocyte per m ( figure 3e ) with the * multi - point * selection in the toolbar and mark each cell for counting.to determine the adipocyte size , select * straight line * again in the toolbar and draw the diameter of an adipocyte ( figure 3c ) . go to analyze - > measure and a new window will appear , with the length of the diameter in m.to determine the adipocyte area , select * wand ( tracing ) tool * and click inside the adipocyte . go to analyze - > measure and a new window will appear , with the area of this adipocyte in m . for immunohistochemistry , prepare the section for antibody and tdt - mediated dutp - biotin nick end labeling ( tunel ) staining as follows : deparaffinize sections by washing 3 times for 5 min in xylene and rehydrate the section 2 times for 2 min in 100% ethanol and 2 times for 2 min in 96% ethanol . finally , wash the section in distilled h2o.for antigen retrieval , digest the tissue section for 30 min at 37 c with proteinase k working solution ( 20 g / ml in 10 mm tris / hcl , ph 7.4 - 8 ) and rinse with pbs . perform antibody staining in wet chambers : to block the endogenous peroxidase , use 3% hydrogen peroxide in pbs for 10 min , with subsequently washing 2 times for 5 min in pbs . to block the unspecific binding of antibodies , use 10% serum in pbs . use the serum of the host of the secondary antibody , in this case goat.for the staining , use the antibodies for apoptosis , proliferation and hypoxia detection ( table 4 ) . dilute antibodies in pbs/10% goat serum . wash the sections 3 times for 5 min in pbs.to enhance the signal use a biotinylated secondary antibody , with the dilution as listed in table 5 . for hypoxia detection , use hrp conjugated rabbit anti note : for the hypoxia staining with fitc - mab1 , skip step 1.4.4.4 ) and continue with step 1.4.4.5).for each slide , pre - incubate 50 l avidin solution with 50 l biotinylated peroxidase h for 30 min at room temperature ( this method is also referred to as avidin / biotin abc complex formulation ) and then add to the sections for another 45 min . wash 2 times for 5 min with pbs.incubate the sections in peroxidase substrate solution until the staining become more intense ( between 5 to 10 min ) . wash for 5 min with distilled h2o.for counterstaining , stain with hematoxylin diluted 1:5 with distilled h2o for 10 min at room temperature and wash in h2o for 5 min.dehydrate sections in 96% ethanol and 100% ethanol 2 times each for 2 min , and xylene 3 times for 5 min . determine apoptosis by tunel assay and follow manufacturer 's instructions : add 50 l enzyme solution into 450 l label solution . then apply 50 l tunel reaction mixture on histologic sections and incubate for 60 min at + 37 c in a humidified atmosphere in the dark . wash the sections 3 times with pbs for 5 min and mount with fluorescence mounting medium including dapi ( 4',6-diamidino-2-phenylindole ) for counterstaining.evaluate the section under a fluorescence microscope with 100 fold magnification . for measurement of fluorescein use an excitation wavelength of 488 nm and detect between 515 - 565 nm ( green laser ) ; dapi excites at about 360 nm and emits at about 460 nm when bound to dna ( blue laser).quantify the overlays of dapi and fluorescein : fix the fat pad in 3.7% pbs - buffered formaldehyde overnight , embed in paraffin ( follow the instructions as described elsewhere ) and cut the embedded tissue into 2 - 5 m thick sections ( maximum 5 m ) . determine the number of adipocytes per field and adipocyte size in a bright - field microscope after hematoxylin and eosin ( h&e ) staining : deparaffinize sections by washing 3 times for 5 min in xylene and rehydrate the section 2 times for 2 min in 100% ethanol and 2 times for 2 min in 96% ethanol . finally , wash the section in distilled h2o for 5 min.stain with hematoxylin , diluted 1:5 with distilled h2o , for 10 min at room temperature and wash in h2o for 5 min . stain with eosin solution ( 20 ml 5% eosin y/210 ml distilled h2o/25 l glacial acetic acid ) for 30 sec and wash again with h2o for 5 min.dehydrate sections using 96% ethanol and 100% ethanol 2 times each for 2 min , and xylene 3 times for 5 min . mount the sections with anhydrous mounting agent.evaluate the sections under a bright - field microscope . a representative example of how to analyze adipocytes with imagej 1.48v is shown in fig . 3 : number of cells per area ( m ) , cell area ( x 10 m ) , cell size ( m ) . open the picture of the section with imagej 1.48v . if the parameters of the image are indicated in pixels instead of a unit of length , adjust the scale.select * straight line * in the toolbar and adjust the line to a known distance by reference to the scale bar . go to analyze - > set scale . the distance of the line is shown in pixels ; add the known distance and the unit of length , e.g. , m . confirm with ok . the size of the picture and the analysis of the parameters are indicated in the unit of length given.to determine the parameters of the adipocytes , adjust the threshold . select the following settings : thresholding method : default ; threshold color : b&w ; color space : hsb . the picture is now shown in black and white . adjust the brightness to clear white adipocytes and to close black intercellular spaces , as in fig . 3b.count the number of adipocyte per m ( figure 3e ) with the * multi - point * selection in the toolbar and mark each cell for counting.to determine the adipocyte size , select * straight line * again in the toolbar and draw the diameter of an adipocyte ( figure 3c ) . go to analyze - > measure and a new window will appear , with the length of the diameter in m.to determine the adipocyte area , select * wand ( tracing ) tool * and click inside the adipocyte . go to analyze - > measure and a new window will appear , with the area of this adipocyte in m . deparaffinize sections by washing 3 times for 5 min in xylene and rehydrate the section 2 times for 2 min in 100% ethanol and 2 times for 2 min in 96% ethanol . finally , wash the section in distilled h2o for 5 min . stain with hematoxylin , diluted 1:5 with distilled h2o , for 10 min at room temperature and wash in h2o for 5 min . stain with eosin solution ( 20 ml 5% eosin y/210 ml distilled h2o/25 l glacial acetic acid ) for 30 sec and wash again with h2o for 5 min . dehydrate sections using 96% ethanol and 100% ethanol 2 times each for 2 min , and xylene 3 times for 5 min . a representative example of how to analyze adipocytes with imagej 1.48v is shown in fig . 3 : number of cells per area ( m ) , cell area ( x 10 m ) , cell size ( m ) . open the picture of the section with imagej 1.48v . if the parameters of the image are indicated in pixels instead of a unit of length , adjust the scale.select * straight line * in the toolbar and adjust the line to a known distance by reference to the scale bar . go to analyze - > set scale . the distance of the line is shown in pixels ; add the known distance and the unit of length , e.g. , m . confirm with ok . the size of the picture and the analysis of the parameters are indicated in the unit of length given.to determine the parameters of the adipocytes , adjust the threshold . select the following settings : thresholding method : default ; threshold color : b&w ; color space : hsb . the picture is now shown in black and white . adjust the brightness to clear white adipocytes and to close black intercellular spaces , as in fig . 3b.count the number of adipocyte per m ( figure 3e ) with the * multi - point * selection in the toolbar and mark each cell for counting.to determine the adipocyte size , select * straight line * again in the toolbar and draw the diameter of an adipocyte ( figure 3c ) . go to analyze - > measure and a new window will appear , with the length of the diameter in m.to determine the adipocyte area , select * wand ( tracing ) tool * and click inside the adipocyte . go to analyze - > measure and a new window will appear , with the area of this adipocyte in m . open the picture of the section with imagej 1.48v . if the parameters of the image are indicated in pixels instead of a unit of length , adjust the scale . select * straight line * in the toolbar and adjust the line to a known distance by reference to the scale bar . go to analyze - > set scale . the distance of the line is shown in pixels ; add the known distance and the unit of length , e.g. , m . the size of the picture and the analysis of the parameters are indicated in the unit of length given . to determine the parameters of the adipocytes , adjust the threshold . select the following settings : thresholding method : default ; threshold color : b&w ; color space : hsb . the picture is now shown in black and white . adjust the brightness to clear white adipocytes and to close black intercellular spaces , as in fig . 3b . count the number of adipocyte per m ( figure 3e ) with the * multi - point * selection in the toolbar and mark each cell for counting . to determine the adipocyte size , select * straight line * again in the toolbar and draw the diameter of an adipocyte ( figure 3c ) . go to analyze - > measure and a new window will appear , with the length of the diameter in m . to determine the adipocyte area , go to analyze - > measure and a new window will appear , with the area of this adipocyte in m . for immunohistochemistry , prepare the section for antibody and tdt - mediated dutp - biotin nick end labeling ( tunel ) staining as follows : deparaffinize sections by washing 3 times for 5 min in xylene and rehydrate the section 2 times for 2 min in 100% ethanol and 2 times for 2 min in 96% ethanol . finally , wash the section in distilled h2o.for antigen retrieval , digest the tissue section for 30 min at 37 c with proteinase k working solution ( 20 g / ml in 10 mm tris / hcl , ph 7.4 - 8 ) and rinse with pbs . deparaffinize sections by washing 3 times for 5 min in xylene and rehydrate the section 2 times for 2 min in 100% ethanol and 2 times for 2 min in 96% ethanol . finally , wash the section in distilled h2o . for antigen retrieval , digest the tissue section for 30 min at 37 c with proteinase k working solution ( 20 g / ml in 10 mm tris / hcl , ph 7.4 - 8 ) and rinse with pbs . perform antibody staining in wet chambers : to block the endogenous peroxidase , use 3% hydrogen peroxide in pbs for 10 min , with subsequently washing 2 times for 5 min in pbs . to block the unspecific binding of antibodies , use 10% serum in pbs . use the serum of the host of the secondary antibody , in this case goat.for the staining , use the antibodies for apoptosis , proliferation and hypoxia detection ( table 4 ) . dilute antibodies in pbs/10% goat serum . wash the sections 3 times for 5 min in pbs.to enhance the signal use a biotinylated secondary antibody , with the dilution as listed in table 5 . for hypoxia detection , note : for the hypoxia staining with fitc - mab1 , skip step 1.4.4.4 ) and continue with step 1.4.4.5).for each slide , pre - incubate 50 l avidin solution with 50 l biotinylated peroxidase h for 30 min at room temperature ( this method is also referred to as avidin / biotin abc complex formulation ) and then add to the sections for another 45 min . wash 2 times for 5 min with pbs.incubate the sections in peroxidase substrate solution until the staining become more intense ( between 5 to 10 min ) . wash for 5 min with distilled h2o.for counterstaining , stain with hematoxylin diluted 1:5 with distilled h2o for 10 min at room temperature and wash in h2o for 5 min.dehydrate sections in 96% ethanol and 100% ethanol 2 times each for 2 min , and xylene 3 times for 5 min . evaluate the sections under a bright - field microscope . to block the endogenous peroxidase , use 3% hydrogen peroxide in pbs for 10 min , with subsequently washing 2 times for 5 min in pbs . to block the unspecific binding of antibodies , use 10% serum in pbs . use the serum of the host of the secondary antibody , in this case goat . for the staining , use the antibodies for apoptosis , proliferation and hypoxia detection ( table 4 ) . dilute antibodies in pbs/10% goat serum . wash the sections 3 times for 5 min in pbs . to enhance the signal use a biotinylated secondary antibody , with the dilution as listed in table 5 . for hypoxia detection , use hrp conjugated rabbit anti - fitc as the secondary antibody . note : for the hypoxia staining with fitc - mab1 , skip step 1.4.4.4 ) and continue with step 1.4.4.5 ) . for each slide , pre - incubate 50 l avidin solution with 50 l biotinylated peroxidase h for 30 min at room temperature ( this method is also referred to as avidin / biotin abc complex formulation ) and then add to the sections for another 45 min . incubate the sections in peroxidase substrate solution until the staining become more intense ( between 5 to 10 min ) . for counterstaining , stain with hematoxylin diluted 1:5 with distilled h2o for 10 min at room temperature and wash in h2o for 5 min . dehydrate sections in 96% ethanol and 100% ethanol 2 times each for 2 min , and xylene 3 times for 5 min . determine apoptosis by tunel assay and follow manufacturer 's instructions : add 50 l enzyme solution into 450 l label solution . then apply 50 l tunel reaction mixture on histologic sections and incubate for 60 min at + 37 c in a humidified atmosphere in the dark . wash the sections 3 times with pbs for 5 min and mount with fluorescence mounting medium including dapi ( 4',6-diamidino-2-phenylindole ) for counterstaining.evaluate the section under a fluorescence microscope with 100 fold magnification . for measurement of fluorescein use an excitation wavelength of 488 nm and detect between 515 - 565 nm ( green laser ) ; dapi excites at about 360 nm and emits at about 460 nm when bound to dna ( blue laser).quantify the overlays of dapi and fluorescein : add 50 l enzyme solution into 450 l label solution . then apply 50 l tunel reaction mixture on histologic sections and incubate for 60 min at + 37 c in a humidified atmosphere in the dark . wash the sections 3 times with pbs for 5 min and mount with fluorescence mounting medium including dapi ( 4',6-diamidino-2-phenylindole ) for counterstaining . evaluate the section under a fluorescence microscope with 100 fold magnification . for measurement of fluorescein use an excitation wavelength of 488 nm and detect between 515 - 565 nm ( green laser ) ; dapi excites at about 360 nm and emits at about 460 nm when bound to dna ( blue laser ) . quantify the overlays of dapi and fluorescein : figure 3 : analyzing adipocyte characteristics in fat pad sections . section pictures of the perigonadal fat pad of male mice treated with a high - fat diet ( hfd ) or normal diet ( nd ) with a bright - field microscope ( a ) ; the threshold is adjusted into black and white ( b ) and the adipocyte size ( length ; c ) , area ( d ) and adipocyte cell number per mm ( e ) are quantified with imagej 1.48v . table 4 : antibodies with respective dilution used for the immunohistological staining of at sections . sacrifice mice and remove the subcutaneous adipose tissue . sacrifice the mice by co2 asphyxiation and subsequent cervical dislocation.pin down the limbs of mice as illustrated in figure 4 . detach the skin from the upper leg , loin and flank and pin it down with needles as in figure 4 . then remove the subcutaneous adipose tissue , which is located posterior at the base of the hind legs , surrounding the inguinal lymph nodes ( as shown in figure 4 , left : subcutaneous fat pads indicated by the arrows ; right : inguinal lymph node indicated by the arrow ) . detach the skin from the upper leg , loin and flank and pin it down with needles as in figure 4 . then remove the subcutaneous adipose tissue , which is located posterior at the base of the hind legs , surrounding the inguinal lymph nodes ( as shown in figure 4 , left : subcutaneous fat pads indicated by the arrows ; picture of subcutaneous fat pad ; the left arrows indicate the subcutaneous fat pad and the right arrow indicates the inguinal lymph node . seed adsc 4,000 cells / cm in dulbecco 's modified eagle 's medium - ham 's f-12 supplemented with 10% normal calf serum , 1% penicillin / streptomycin , 0.5% amphotericin b , 16 m biotin , 18 m pantothenic acid and 100 m ascorbic acid and grow culture to confluence around 70 to 80% , which is reached after 4 to 6 days of culture . induce adipogenic differentiation . remove the adherent adsc from the surface by trypsin treatment . remove medium , wash with pbs and add 0.025% trypsin solution ( preheated to 37 c ) for 2 min ( until cells detach from the surface ) . count the cells using a neubauer chamber . put the glass cover on the central area of the neubauer chamber . dilute the cell suspension 1:10 and load the chamber with 10 l diluted cell suspension . count the cells in 4 squares located at the corners , each composed of 16 smaller squares . calculate the cell number per ml : seed adsc ( as described in point 2.2 ) in 12-well culture plates and grow culture to confluence around 70 to 80% ( reached after 4 to 6 days).induce adipogenic differentiation by adding 5 g / ml insulin , 1 m dexamethasone and 5 m 3-isobutyl-1-methylxanthine ( ibmx ) to the cultures . remove the adherent adsc from the surface by trypsin treatment . remove medium , wash with pbs and add 0.025% trypsin solution ( preheated to 37 c ) for 2 min ( until cells detach from the surface ) . immediately add medium and wash the cells . remove medium , wash with pbs and add 0.025% trypsin solution ( preheated to 37 c ) for 2 min ( until cells detach from the surface ) . immediately add medium and wash the cells . count the cells using a neubauer chamber . put the glass cover on the central area of the neubauer chamber . dilute the cell suspension 1:10 and load the chamber with 10 l diluted cell suspension . count the cells in 4 squares located at the corners , each composed of 16 smaller squares . calculate the cell number per ml : put the glass cover on the central area of the neubauer chamber . dilute the cell suspension 1:10 and load the chamber with 10 l diluted cell suspension . count the cells in 4 squares located at the corners , each composed of 16 smaller squares . calculate the cell number per ml : seed adsc ( as described in point 2.2 ) in 12-well culture plates and grow culture to confluence around 70 to 80% ( reached after 4 to 6 days ) . induce adipogenic differentiation by adding 5 g / ml insulin , 1 m dexamethasone and 5 m 3-isobutyl-1-methylxanthine ( ibmx ) to the cultures . cells will be fully differentiated after 7 days . to analyze the adipogenic differentiation , stain with oil red o , which stains triglycerides of mature adipocytes . note : work must be performed under a fume hood ! remove the medium , wash the adipocytes gently with pbs and fix the cells for 60 min with 2 ml 10% formalin.to prepare oil red o staining solution , mix 3 parts of the red oil o stock solution ( 300 mg red oil o powder dissolved in 100 ml 99% isopropanol ) with 2 parts distilled h2o and incubate for 10 min at room temperature . note : the working solution is stable for 2 hr.for the oil red o staining , remove the formalin , wash adipocytes with h2o , incubate with 2 ml 60% isopropanol for 5 min , remove the isopropanol and add 2 ml oil red o working solution for 5 min . counterstain with hematoxylin as in point 1.4.4.5).evaluate the plates under a phase contrast microscope with 100 fold magnification . the lipids of the adipocytes will appear red and the nuclei will appear blue . remove the medium , wash the adipocytes gently with pbs and fix the cells for 60 min with 2 ml 10% formalin . to prepare oil red o staining solution , mix 3 parts of the red oil o stock solution ( 300 mg red oil o powder dissolved in 100 ml 99% isopropanol ) with 2 parts distilled h2o and incubate for 10 min at room temperature . note : the working solution is stable for 2 hr . for the oil red o staining , remove the formalin , wash adipocytes with h2o , incubate with 2 ml 60% isopropanol for 5 min , remove the isopropanol and add 2 ml oil red o working solution for 5 min . optional step : silence the gene of interest by transfection with shrna . change the medium and add serum - free medium.for the transfection of adipocytes , use lipofection . follow the manufacturer 's instructions and use 1 g shrna per 12-well tissue plates . after addition of the lipid - dna - complex , incubate adipocytes for 48 hr at 37 c . change the medium and add serum - free medium . for the transfection of adipocytes , use lipofection . after addition of the lipid - dna - complex , incubate adipocytes for 48 hr at 37 c . to analyze adipocytes subjected to hypoxia , use a hypoxic work station or hypoxic incubator to maintain the cells under hypoxic conditions . analyze apoptosis by fitc - labeled annexin v and subsequent flow cytometry analyses . gather adipocytes with an extra soft cell scraper , wash with pbs and add 1 x 10 cells to 100 l annexin v - binding buffer ( 10 mm hepes / naoh , ph 7.4 ; 140 mm nacl ; 2.5 mm cacl2 ) . add the amount of annexin v - fitc recommended by the manufacturer and incubate at room temperature for 15 min.for flow cytometry measurement , add 200 l annexin v - binding buffer and 1 m of nuclear counterstain . annexin v / nuclear counterstain cells are defined as secondary necrotic and annexin v / nuclear counterstain cells are defined as apoptotic cells ( figure 5 ) . add 1 ml single - phase solution of guanidine isothiocyanate and phenol to each well and isolate the rna [ using single - step method by chomczynski and sacchi ( step 1.3.2 ) ] and proceed as in steps 1.3.2.1 ) to 1.3.5.2 ) . analyze apoptosis by fitc - labeled annexin v and subsequent flow cytometry analyses . gather adipocytes with an extra soft cell scraper , wash with pbs and add 1 x 10 cells to 100 l annexin v - binding buffer ( 10 mm hepes / naoh , ph 7.4 add the amount of annexin v - fitc recommended by the manufacturer and incubate at room temperature for 15 min.for flow cytometry measurement , add 200 l annexin v - binding buffer and 1 m of nuclear counterstain . annexin v / nuclear counterstain cells are defined as secondary necrotic and annexin v / nuclear counterstain cells are defined as apoptotic cells ( figure 5 ) . gather adipocytes with an extra soft cell scraper , wash with pbs and add 1 x 10 cells to 100 l annexin v - binding buffer ( 10 mm hepes / naoh , ph 7.4 ; 140 mm nacl ; 2.5 mm cacl2 ) . add the amount of annexin v - fitc recommended by the manufacturer and incubate at room temperature for 15 min . for flow cytometry measurement , add 200 l annexin v - binding buffer and 1 m of nuclear counterstain . annexin v / nuclear counterstain cells are defined as secondary necrotic and annexin v / nuclear counterstain cells are defined as apoptotic cells ( figure 5 ) . add 1 ml single - phase solution of guanidine isothiocyanate and phenol to each well and isolate the rna [ using single - step method by chomczynski and sacchi ( step 1.3.2 ) ] and proceed as in steps 1.3.2.1 ) to 1.3.5.2 ) . add 1 ml single - phase solution of guanidine isothiocyanate and phenol to each well and isolate the rna [ using single - step method by chomczynski and sacchi ( step 1.3.2 ) ] and proceed as in steps 1.3.2.1 ) to 1.3.5.2 ) . dot plot presentations of the facs for annexin v - fitc and to - pro-3 staining of adipocytes . we show how to determine adipocyte homeostasis in vivo and in vitro using the example of fra-2 fabp4-creert mice compared to wild - type littermates . our protocol defines how increased hif expression by hypoxia is correlated with adipocyte dysfunction as indicated by increased adipocyte apoptosis . increased adipocyte size and area in high - fat diet ( hfd ) treated mice over - nutrition , among other factors , results in adipocyte hypertrophy , caused by excessive energy storage in the lipid droplets . sections of the fat pad from normal ( nd ; figure 6a ) and high - fat diet ( hfd ; figure 6b ) mice as well as quantifications of the adipocyte size and area clearly show adipocyte hypertrophy after 6 weeks of hfd , which is indicated by increased adipocyte size in the hfd treated mice ( figure 6c and d ) . increased hypoxia in the adipose tissue ( at ) of adult fra-2 fabp4-creert mice leads to increased hif-1 level and adipocyte apoptosis to determine the in vivo status of hypoxia in at , fra-2 fabp4-creert mice are analyzed 6 weeks after fra-2 deletion at the age of 12 weeks and compared with wild - type littermates . pimonidazole is administered to the mice intraperitoneally as an effective hypoxia marker ; it is nontoxic and able to distribute into the at . hypoxic adipocytes in the at in vivo are defined by immunohistochemical antibody staining ( figure 7a ) . furthermore , the increased hypoxic status of the at in mice is accompanied by increased hif-1 positive adipocytes as indicated by the immunohistochemical staining figure 7b ) , which is confirmed by quantification of hif-1 expression levels and its targets genes . additionally , tunel staining of at sections from fra-2 fabp4-creert mice and control littermates ( figure 7c ) shows that increased adipocyte apoptosis is correlated with the presence of hypoxia and hif-1 expression . increased hif-1 expression in primary adipocytes through hypoxia induced adipocyte apoptosis to analyze adipocyte apoptosis in vitro , we use adipocytes generated from subcutaneous fat pads as described elsewhere . as expected , the hif-1 expression in adipocytes is increased after 24 hr of hypoxia ( figure 8a ) . to further analyze hif-1 activities , the rna levels of hif target genes such as inos ( inducible nitric oxide synthase ) are quantified by qpcr . figure 8b shows that the increased expression of hif-1 under hypoxic conditions leads to increased inos mrna level . since we have already shown in vivo ( figure 8) that increased hif-1 expression in adipocytes correlates with increased adipocyte apoptosis , apoptosis is also quantified in in vitro cultures by annexin v staining under hypoxic conditions . consistent with the in vivo data ( figure 7 ) , an increased hif-1 level is accompanied by increased adipocyte apoptosis induced by hypoxic conditions ( figure 8b ) . moreover , to prove that hypoxic - induced apoptosis is hif - dependent ; hif-1 or hif-2 is silenced by rna interference in adipocytes derived from wild - type or fra-2 deficient mice . the increased adipocyte apoptosis is restored by silencing hif-1 or hif-2 as shown by annexin v staining in figure 8b , proving that the hypoxia sensor hif- regulates the adipocyte apoptosis . figure 6 : increased adipocyte size and area in high - fat diet ( hfd ) mice . ( a , b ) h&e staining of sections from the perigonadal fat pad of male wild - type mice fed with normal diet ( nd ) ( a ) or high - fat diet ( hfd ) ( b ) for 6 weeks . ( c , d ) quantifications of adipocyte size ( c ) and area ( d ) from the perigonadal fat pad of wild - type mice fed with nd ( a ) or hfd ( b ) for 6 weeks . statistical analysis was performed using students t - test . * * * p < 0.0001.please click here to view a larger version of this figure . figure 7 : increased hif-1 level and apoptosis in adipocytes of adult fra-2 fabp4-creert mice . hypoxia ( a ) and hif-1 ( b ) staining in the at of male fra-2 fabp4-creert mice and male control littermates 6 weeks after tamoxifen injection . ( c ) tunel staining in fra-2 fabp4-creert mice and control littermates at 6 weeks after tamoxifen injection . this figure has been modified from luther et al .. please click here to view a larger version of this figure . ( a ) real - time pcr analysis of hif-1 and hifs target inos mrna levels in primary adipocytes placed in hypoxic chambers analyzed at the indicated time points . ( b ) quantification of apoptosis by annexin v facs staining in primary adipocytes isolated from fra-2 fabp4-creert mice or wild - type controls transfected with sh control or sh plasmid against hif-1 or hif-2 and placed under hypoxia ( 1% o2 ) for 24 hr . statistical analysis was performed using student 's t - test . * p < 0.05 and * * p < 0.01 were accepted as significant adipocytes are characterized phenotypically by their size , numbers and area , revealing adipocyte hyperplasia and hypertrophy , induced by excessive energy storage due to over - nutrition . these events leading to fatty acid dysregulation and subsequent metabolic syndromes are also states of increased fat mass with preserved metabolisms , which is also referred to as " healthy " fat expansion . for example , kusminski et al . showed that mice with massive fat expansion remain metabolically healthy , suggesting that fat expansion is not necessarily linked to metabolic syndromes and needs to be carefully determined to evaluate the characteristics of the adipocytes . the adipose tissue ( at ) plays a pivotal role in the regulation of body metabolism . at is the biggest endocrine organ that could influence dyslipidemia , atherosclerosis , hyperinsulinemia and hyperglycemia . evaluating at homeostasis and the molecular mechanisms regulating it could allow a better understanding of metabolic system disorders . therefore , unravelling the mechanisms regulating adipocyte differentiation , adipocyte size and fat pad mass would help to develop new therapeutic treatment for obesity disorders . using in vivo and in vitro methods , it is possible to determine the role of food and gene expression impacts on adipocyte differentiation and activity . to determine the at homeostasis , determining the balance between adipocyte differentiation , proliferation and apoptosis as suggested by our protocol is as important as analyzing the glucose and insulin metabolic response . expression profiling analyses of genes involved in adipogenesis , lipogenesis , lipolysis , fatty acid uptake , hypoxia , apoptosis and proliferation in primary adipocytes and visceral at is a high throughput method for obtaining an overview on adipocyte homeostasis and their possible dysfunction . interesting candidates should be further analyzed at the protein level by western blot or immunohistological staining . to obtain optimal results through real - time pcr system using unsymmetrical cyanine dyes , the concentrations of cdna ranging from 1 to 10 ng and the optimal primer concentrations ranging from 50 to 900 nm the critical components are the primers ; for each run , the melting curves need to be strictly controlled to ensure the specificity and to exclude the formation of primer dimers . furthermore , commercial available unsymmetrical cyanine dyes are provided as master mixes that contain a passive reference dye ( such as rox ) to provide an internal reference signal . the cdna signal is normalized during data analysis to the rox signals to correct well - to - well signal fluctuations . another point to be considered in order to establish a good qpcr system is the choice of the housekeeping gene . for each condition , several housekeeping genes are used , e.g. , hprt , -actin , gapdh , -2-mg or hsp90 . hif proteins stabilized by hypoxic conditions are master regulators determining not only adipocytes survival , but also metabolic changes , such as glucose , insulin tolerance and lipid metabolism . to ascertain hypoxic areas in the fat pad , hif-1 is determined in at sections by immunohistochemistry . since hif proteins are rapidly degraded within 5 to 10 min under normoxic conditions , the procedure and the fixation of the fat pad for the histological analysis should be tightly controlled to avoid latency time . therefore , to ensure hypoxia not only through hif staining , pimonidazole is used to determine hypoxic areas in the at . pimonidazole is able to distribute into tissues , as it was already shown in bones , and effectively mark hypoxic areas by binding to thiol - containing proteins specifically in hypoxic cells , which is further detected in histological sections by specific antibody binding . for example , the involvement of the prolyl hydroxylase ( phd ) enzyme , which induce hydroxylation of proline residues under normoxia , as well as the von hippel - lindau ( vhl ) protein , which recognize hydroxylated prolines and induce the poly - ubiquitination to mediate proteasomal hif degradation , need to be analyzed for a full overview of the pathway . moreover , ubiquitous detection of hifs would also determine the protein stability and degradation that can be alter . furthermore , proliferation by ki67 and apoptosis by tunel staining are determined in vivo through staining of at histological sections . quantification of proliferation by ki67 and apoptosis by tunel or annexin v staining through flow cytometry analysis is also carried out . proliferation could of course be measured by other techniques such as the analyses of the adipocyte cell cycle , which is not addressed by the measurement of ki67 positive cells . moreover , apoptosis study by tunel can be completed by facs analyses of annexin v and top - pr-3 which will determine the levels of necrosis versus the apoptosis cell death process . apoptosis is a fundamental process for the program of cell death , which is important for at homeostasis . indeed , dysregulation of adipocyte apoptosis has been implicated previously in processes contributing to obesity and lipodystrophy . moreover , in 2011 , keuper et al . linked adipose tissue inflammation to adipocyte apoptosis . they showed that macrophages induced apoptosis in preadipocytes and adipocytes , which in turn attract macrophages . the recruitment of macrophages accelerates inflammation , which contributes to metabolic syndromes such as glucose and insulin tolerance . however , adipocyte apoptosis is still a poorly studied phenomenon , despite the hypothesis that induced adipocyte apoptosis could lead to decreased weight . the present protocol uses immunohistochemical approaches to study different phenomenon such as proliferation , apoptosis and hypoxia in vivo . therefore , the tissue was fixed with 4% formaldehyde , which is a critical step . an extended tissue fixation time leads to change of the epitopes , which become non - accessible for the antibody . moreover , the thickness of the sections also influences the antibodies binding to their epitopes ; optimal thickness is between 2 and 5 m . sections thicker than 5 m will give false positive results due to increased binding sites . in contrast , sections thinner than 2 m contain less binding sites and positive areas are not well defined . further critical factors are the antibody itself , incubation time , concentration and even temperature , which influence the quality of the specific binding to the epitopes . therefore , validating antibody concentration and incubation time is necessary for each condition . to complete the study , we provide an in vitro adipocyte differentiation protocol , which could be extended by different treatments , stimulation or co - cultures . by using in vitro adipocyte cultures , it is feasible to determine defects in adipocyte differentiation and functions . to obtain reliable results , as for all primary cells , the healthy behavior and appearance of the adscs and adipocytes is quite important . the granularity , cytoplasmic vacuolations and/or detachment are signs of deterioration , indicating inadequate medium , microbial contamination or senescence of the primary cells . this protocol is using isolated adipocytes from the fat pad tissue , whereas it is as well possible to use mesenchymal stem cell isolated from bone marrow as described by other protocols . the latest includes stromal progenitor cells , which might reflect additional differentiation problems occurring at the very early step of adipocyte differentiation , this might be missed in our current protocol . moreover , adscs can be expanded rapidly ( more than 10 times within one week ) , and long - term cultured adscs after some passages still retain their mesenchymal pluripotency . another advantage using adscs is that one can easily switch to human , since adscs can be harvested from patients by liposuction which is a simple and minimally invasive method . as at influences several other organs in an endocrine manner , the protocol should be extended to adipokines . adipokines , such as leptin , adiponectin , tumor necrosis factor- ( tnf ) and resistin , secreted by adipocytes are known to affect metabolic diseases by controlling fat metabolism , energy homeostasis and insulin sensitivity . therefore , serum and adipocyte secretome analyses should be performed . in the case of at dysfunction , adipokines and pro - inflammatory cytokines , such as il-6 , can lead to dysregulation of organs such as the liver and pancreas , and of muscle function . in order to exclude systemic organ dysfunction , animal models or cell cultures we provide a protocol for analyzing the basic state of the at and adipocytes in vivo and in vitro to reveal molecular mechanisms of adipocyte homeostasis and functionality .

considering that adipose tissue ( at ) is an endocrine organ , it can influence whole body metabolism . excessive energy storage leads to the dysregulation of adipocytes , which in turn induces abnormal secretion of adipokines , triggering metabolic syndromes such as obesity , dyslipidemia , hyperglycemia , hyperinsulinemia , insulin resistance and type 2 diabetes . therefore , investigating the molecular mechanisms behind adipocyte dysregulation could help to develop novel therapeutic strategies . our protocol describes methods for evaluating the molecular mechanism affected by hypoxic conditions of the at , which correlates with adipocyte apoptosis in adult mice . this protocol describes how to analyze at in vivo through gene expression profiling as well as histological analysis of adipocyte differentiation , proliferation and apoptosis during hypoxia exposure , ascertained through staining of hypoxic cells or hif-1 protein . furthermore , in vitro analysis of adipocyte differentiation and its responses to various stimuli completes the characterization of the molecular pathways behind possible adipocyte dysfunction leading to metabolic syndromes .

hypoxic - ischemic encephalopathy due to birth asphyxia is the major factor that induces neuronal cell injury before , during , and after birth . although there has been marked development in the fields of obstetrics and neonatology , the incidence of and mortality caused by hypoxic - ischemic encephalopathy remain elevated ( 1 , 2 ) . low birth weight infants and infants with intrauterine growth retardation , who have already experienced intrauterine repetitive hypoxic distress , seem to have milder hypoxic - ischemic brain damage than full - term newborns who are exposed to a lethal ischemic insult in the perinatal period . therefore , some have suggested the possibility that sublethal hypoxic episodes afford a protective effect against further hypoxic exposure ( 3 , 4 ) . hypoxic preconditioning has been described in the brain , heart , retina , and other tissues . ( 5 ) first showed that exposure of neonatal rat pups to hypoxia alone ( 8% oxygen for 3 hr ) protected these animals 1 day later from a stroke induced by combined hypoxia / ischemia . although the mechanisms of preconditioning are still undetermined , it appears to require synthesis of new rna and proteins during the reoxygenation period before permanent ligation ( 6 , 7 ) . ( 8) explained that the difference in the degree of hypoxia and the delay between preconditioning and ischemia determines the efficacy of preconditioning . this study was conducted to evaluate the protective effect of hypoxic preconditioning on hypoxic - ischemic injury in the neonatal rat and the persistence of a protective window after hypoxic preconditioning . furthermore , this study investigated the relationship between the downregulation of apoptosis and its possible role in the protective effect of hypoxic preconditioning . the survival rates , as well as ratios of lipid to n - acetyl aspartate ( naa ) and lipid to creatine ( cr ) , were investigated in positive cells , and morphologic changes were investigated with proton ( h ) magnetic resonance spectroscopy ( mrs ) and terminal deoxynucleotidyl transferase - mediated dutp - biotin nick end - labeling ( tunel ) staining . a total of 231 seven - day - old sprague - dawley newborn rats weighing 12 - 18 grams were divided into six groups : preconditioned 6 hr before hypoxic - ischemic injury ( pre-6 hr , n = 30 ) , 12 hr before ( pre-12 hr , n = 32 ) , 1 day before ( pre-1 day , n = 34 ) , 3 days before ( pre-3 day , n = 41 ) , 6 days before ( pre-6 day , n = 43 ) , and control without preconditioning ( n = 51 ) . all experimental animal protocols were approved by the university of ulsan college of medicine iacuc committee ( 2008 - 12 - 011 ) . the rats were preconditioned by hypoxic exposure ( 8% oxygen/92% nitrogen ) for three hours at 37. exhaled carbon dioxide was removed through a one - way valve in the lower part of a hypoxic chamber . rats were returned to their home cages at room temperature immediately after the hypoxic preconditioning . control group rats were isolated during preconditioning , and then they were also returned to their home cages . using ligation of the right common carotid artery , 6 hr , 12 hr , 1 , 3 , and 6 days after receiving three hours of hypoxic preconditioning , all rats , including the control group , 5 ) under isoflurane anesthesia , and the rats were submitted to hypoxia ( 8% oxygen , 92% nitrogen ) for 3 hr under the same conditions as in the preconditioning procedure . after the hypoxic - ischemic insult , the specimens were returned to their home cages at room temperature . h mrs was performed 24 hr after hypoxic - ischemic insult , and the results were examined using bruker biospec 4.7 tesla mri / mrs system ( bruker , fallanden , switzerland ) . the voxel for mrs was located in the parietotemporal area , and the volume of a single voxel was 3 3 4 l . the spin echo signal was detected by tr = 3,000 msec , te = 30 msec , and ns = 128 , and the h2o signal was inhibited by a chemical shift selective ( chess ) sequence . spectra were analyzed semi - quantitively using an aspect 3000 computer and tomikon software ( bruker , fallanden , switzerland ) . in order to measure the ratios of lipid to naa and lipid to cr as early predictors of apoptosis , the h spectra of naa , creatine , choline , and lipids were assessed at 2.02 , 3.03 , 3.24 , and 1.3 ppm , respectively . to differentiate between lipids and lactate at 1.3 ppm , the presence of the peak at te = 135 msec was analyzed . one day after the hypoxic - ischemic injury , the rats were anesthetized with an intra - abdominal ketamine ( 2 mg / kg ) injection following h mrs examination . the rats were then perfused through the left ventricle of the heart with heparinized saline ( 2 unit heparin/1 ml saline , 2.5 ml / g ) followed by buffered paraformaldehyde ( 4% in phosphate buffer , ph 7.4 ) . two rats in each group were sacrificed one day after hypoxic - ischemic injury , and these brains were examined for histologic study using tunel staining . tunel staining for the detection of dna fragmentation produced during cell apoptosis began with the digestion of deparaffinized sections using proteinase k , blocking solution , and permeabilization solution . these sections were then stained with the tunel reaction mixture ( 50 l ) of an in situ cell death detection kit ( boehringer - mannheim , germany ) . the remaining rats were morphologically evaluated on the fourteenth day after hypoxic - ischemic injury and scored according to the following scale of modified palmer 's classification ( 9 ) : 0 , no difference between the two hemispheres in shape and size ; 1 , mild reduction of the right hemisphere volume only ; 2 , volume reduction and atrophic changes with small cysts in the right hemisphere ; 3 , moderate atrophic changes of the right hemisphere ; and 4 , severe atrophic changes of the right hemisphere . the results were expressed as mean standard deviation , and the statistics were calculated using an unpaired t - test , one way anova , tukey 's test , dunn 's method , mann - whitney u test , and pearson 's correlation with the threshold of significance at 0.05 . a total of 231 seven - day - old sprague - dawley newborn rats weighing 12 - 18 grams were divided into six groups : preconditioned 6 hr before hypoxic - ischemic injury ( pre-6 hr , n = 30 ) , 12 hr before ( pre-12 hr , n = 32 ) , 1 day before ( pre-1 day , n = 34 ) , 3 days before ( pre-3 day , n = 41 ) , 6 days before ( pre-6 day , n = 43 ) , and control without preconditioning ( n = 51 ) . all experimental animal protocols were approved by the university of ulsan college of medicine iacuc committee ( 2008 - 12 - 011 ) . the rats were preconditioned by hypoxic exposure ( 8% oxygen/92% nitrogen ) for three hours at 37. exhaled carbon dioxide was removed through a one - way valve in the lower part of a hypoxic chamber . rats were returned to their home cages at room temperature immediately after the hypoxic preconditioning . control group rats were isolated during preconditioning , and then they were also returned to their home cages . using ligation of the right common carotid artery , 6 hr , 12 hr , 1 , 3 , and 6 days after receiving three hours of hypoxic preconditioning , all rats , including the control group , 5 ) under isoflurane anesthesia , and the rats were submitted to hypoxia ( 8% oxygen , 92% nitrogen ) for 3 hr under the same conditions as in the preconditioning procedure . after the hypoxic - ischemic insult , the specimens were returned to their home cages at room temperature . h mrs was performed 24 hr after hypoxic - ischemic insult , and the results were examined using bruker biospec 4.7 tesla mri / mrs system ( bruker , fallanden , switzerland ) . the voxel for mrs was located in the parietotemporal area , and the volume of a single voxel was 3 3 4 l . the spin echo signal was detected by tr = 3,000 msec , te = 30 msec , and ns = 128 , and the h2o signal was inhibited by a chemical shift selective ( chess ) sequence . spectra were analyzed semi - quantitively using an aspect 3000 computer and tomikon software ( bruker , fallanden , switzerland ) . in order to measure the ratios of lipid to naa and lipid to cr as early predictors of apoptosis , the h spectra of naa , creatine , choline , and lipids were assessed at 2.02 , 3.03 , 3.24 , and 1.3 ppm , respectively . to differentiate between lipids and lactate at 1.3 ppm , the presence of the peak at te = 135 msec was analyzed . one day after the hypoxic - ischemic injury , the rats were anesthetized with an intra - abdominal ketamine ( 2 mg / kg ) injection following h mrs examination . the rats were then perfused through the left ventricle of the heart with heparinized saline ( 2 unit heparin/1 ml saline , 2.5 ml / g ) followed by buffered paraformaldehyde ( 4% in phosphate buffer , ph 7.4 ) . two rats in each group were sacrificed one day after hypoxic - ischemic injury , and these brains were examined for histologic study using tunel staining . tunel staining for the detection of dna fragmentation produced during cell apoptosis began with the digestion of deparaffinized sections using proteinase k , blocking solution , and permeabilization solution . these sections were then stained with the tunel reaction mixture ( 50 l ) of an in situ cell death detection kit ( boehringer - mannheim , germany ) . the remaining rats were morphologically evaluated on the fourteenth day after hypoxic - ischemic injury and scored according to the following scale of modified palmer 's classification ( 9 ) : 0 , no difference between the two hemispheres in shape and size ; 1 , mild reduction of the right hemisphere volume only ; 2 , volume reduction and atrophic changes with small cysts in the right hemisphere ; 3 , moderate atrophic changes of the right hemisphere ; and 4 , severe atrophic changes of the right hemisphere . the results were expressed as mean standard deviation , and the statistics were calculated using an unpaired t - test , one way anova , tukey 's test , dunn 's method , mann - whitney u test , and pearson 's correlation with the threshold of significance at 0.05 . here we confirmed the preconditioning groups , followed by 8% oxygen hypoxia , reduced brain injury in newborn rats . the pre-6 hr , pre-12 hr , and pre-1 day hypoxic preconditioning groups had significantly higher survival rates when evaluating 152 rats among 231 : control ( 25 rats , 47% ) ; pre-6 hr ( 26 rats , 86% ) ; pre-12 hr ( 32 rats , 100% ) ; pre-1 day ( 32 rats , 94% ) ; pre-3 day ( 19 rats , 45% ) ; and pre-6 day ( 18 rats , 42% ) ( fig . we performed h mrs study after hypoxic - ischemic insult ; and then lipid / naa ( n - acetyl aspartate ) and lipid / cr ( creatine ) ratios were measured as early predictors of apoptosis . the pre-6 hr , pre-12 hr , and pre-1 day hypoxic preconditioning groups also had lower lipid / naa and lipid / cr ratios on h mrs in comparison with the pre-3 day and pre-6 day ( p < 0.05 , fig . 3 ) . the lipid / naa ratios in control , pre-6 hr , pre-12 hr , pre-1 day , pre-3 day and pre-6 day ratios were 8.05 2.95 , 4.64 2.78 , 5.25 2.58 , 5.05 1.86 , 7.09 2.87 , and 6.19 2.62 , respectively . results of the histologic evaluation demonstrated that the hypoxic preconditioning attenuated hypoxic - ischemic induced apoptosis in newborn brain . on day 1 , the groups that received hypoxic preconditioning 6 hr , 12 hr , and 1 day prior to hypoxic - ischemic injury had fewer tunel - positive apoptotic cells present than in the control group , which received no preconditioning . in addition , the h mrs characteristics are shown and lower lipid peaks are observed in the preconditioning groups in fig . we monitored the structural damage in rats of different groups on the fourteenth day after hypoxic - ischemic injury . the morphologic scores of the preconditioning groups were also significantly lower than those of the control group ( p < 0.05 , fig . 5 ) . the morphologic scores in the control , pre-6 hr , pre-12 hr , pre-1 day , pre-3 day , and pre-6 day groups were 3.28 0.84 , 1.12 1.21 , 1.59 1.46 , 1.69 1.15 , 2.95 1.08 , and 2.61 1.54 , respectively , at day 14 . the high lipid / naa and lipid / cr ratios on h mrs were obtained on the first day after hypoxic - ischemic insult and were predictive of morphologic changes ( fig . however , the h mrs and morphologic scores of the pre-3 day and pre-6 day preconditioning groups did not show a protective effect on survival rates or on lipid / naa and lipid / cr ratios . both fetal and neonatal asphyxia can cause cerebral hypoxic - ischemic injury , resulting in severe neurologic sequelae and death . survivors of perinatal asphyxia frequently have moderate to severe brain injury for which there is currently no promising therapy . reports published in the last decade have made increasing use of the term hypoxic or ischemic preconditioning to describe an adaptive response to subsequent lethal events ( 3 ) . focusing on protective strategies to prevent and reduce hypoxic - ischemic injury , sublethal stimuli , such as hypoxia ( 4 ) , hyperthermia ( 10 ) , hypothermia ( 11 ) , glutamate and seizure ( 12 ) , oxygen - glucose deprivation ( 13 ) , receptor blockade / activation ( 14 ) , and cytokine ( 15 ) , have been reported . the mechanism of the brain tolerance induced by hypoxic preconditioning has not been fully elucidated , but three major mechanisms have been proposed . first , the formation of various types of prolonged neuronal adaptive responses is associated with the activation of the cell genome and protein synthesis . these include stress proteins ( heat shock proteins [ hsp ] ) ( 10 ) ; immediate early genes ( c - fos , c - jun , krox-24 [ ngfi - a ] ) ( 16 ) ; transcription factors ( hypoxia - inducible factor-1 [ hif-1 ] ) ( 17 ) ; nfb ( nuclear factor b ) ( 18 ) ; camp response element - binding protein ( creb ) ( 19 ) ; neuromodulatory peptides for survival , differentiation , and plasticity ; growth factors ( igf-1 , brain - derived neurotrophic factor [ bdnf ] , ngf ) ( 20 ) ; and anti - apoptotic genes ( bcl-2 , bcl - xl , bax ) ( 3 ) . in particular , an important role is played by immediate early gene superfamilies whose protein products are transcription factors that regulate the expression of phenotype - specific late genes . a rather long time ( 24 hr or more ) between mild preconditioning episodes and severe hypoxia is needed for the maximal protective effect of preconditioning to appear , which suggests the involvement of neuronal gene expression and de novo protein synthesis in the mechanisms of brain tolerance ( 7 ) . for example , kitagawa et al . ( 21 ) found that sublethal ischemia at 1 and 2 days prior to an ischemic insult that is normally lethal to neurons was neuroprotective in vivo . our data , however , showed significant neuroprotective effects in the pre-6 hr hypoxic preconditioning group . this suggests that the factors mentioned above could be induced to influence the tolerance of the brain in as little as 6 hr , and the persistence of the protective effect lasted 3 days after preconditioning . the second possibility involves the roles of signaling proteins ( no , erk1/2 , akt ) ( 22 ) . no , an important proximal component of the transduction pathway of adaptive genomic changes , was shown to induce diverse paracrine actions in the central nervous system . third , the roles of molecules , including adenosine a1 receptors ( 23 ) and manganese superoxide dismutase ( mnsod ) ( 24 ) have suggested a possible mechanism . finally , activation of n - methyl - d - aspartate ( nmda ) receptors has been shown to be involved in the development of both adaptive and pathological brain reactions . paradoxically , subtoxic concentrations of nmda protect neurons against glutamate - mediated excitotoxicity ( 1 , 14 ) . the major mechanism of nmda receptor - medicated tolerance after preconditioning involves hsp induction ; bcl-2 gene expression ; activation of adenosine a1 receptors ; release and synthesis of bdnf by activation of nfb ; trkb receptor , akt activation through phosphoinositide 3-kinase , and calmodulin - dependent protein kinase ; and downregulation of the mixed - lineage kinase 3 ( mlk3)-c - jun n - terminal kinase ( jnk ) signaling pathway via akt activation ( 22 ) . according to data from the present study , the pre-6 hr , pre-12 hr , and pre-1 day hypoxic preconditioning groups had significantly higher survival rates than those of the control group . these results suggest that preconditioning was effective in myocardial cells as well as cells in the brain . mild , short ischemic events in myocytes contributed to the resistance to subsequent , lethal ischemic exposure , which reduced cardiac infarction and improved cardiac function during reperfusion after ischemic insult . it is well known that apoptosis plays an important role in hypoxic - ischemic brain injury and that the apoptotic cell counts are closely related to the degree of brain injury ( 25 ) . evidence of fragmented and condensed dna is apparent using the tunel method of in situ dna end labeling . cantagrel et al . ( 3 ) found a decrease in the number of apoptotic cells 24 and 48 hr after the insult using a newborn rat model of hypoxic preconditioning ( 8% oxygen/92% nitrogen , 3 hr , 37 ) on day 6 followed by carotid ligation and hypoxic insult on day 7 , suggesting that regulation of apoptotic cell death is one of the mechanisms involved in the tolerance to hypoxia - ischemia that is induced by hypoxic preconditioning . using the same experimental model , we investigated the preconditioning effects in the present study . mrs allows for the study of brain biochemistry and metabolism and for indirect characterization of the composition of brain tissue in vivo . mrs has become an important adjunct to diagnostic structural imaging that permits more accurate and earlier determination of prognosis after hypoxia - ischemia in the newborn ( 26 - 28 ) . furthermore , non - invasive in vivo h mrs of a hypoxic - ischemic brain might reveal apoptosis ( 29 ) . in h mrs of hypoxic - ischemic newborn rat brains , the prominent lipid peaks at 1.3 ppm appear early , and the apoptotic cell counts might be correlated with the intensity of the lipid peak and the ratios of lipid / naa and lipid / cr . therefore , the ratios of lipid / naa and lipid / cr could be used as an early indicator to diagnose hypoxic - ischemic injury by apoptosis and also to assess the effect of preconditioning on decreasing apoptosis . we have used h mrs in vivo for the purpose of early detection in the hypoxic - ischemic brain injury model ( 30 ) . additionally , the results of the current study demonstrate that the increase of the lipid peak at 24 hr following hypoxic - ischemic injury is correlated with morphologic scores . we suggest a promising future for early in vivo detection of apoptosis using h mrs , which can be used to develop new therapeutic methods to attenuate apoptosis in the hypoxic - ischemic injured brain . in conclusion , using h mrs , tunel staining , and morphologic scores , this study showes that hypoxic preconditioning 6 hr to 1 day before hypoxic - ischemic injury produces higher survival rates and the presence of neuroprotective effects against subsequent hypoxic - ischemic injury . however , hypoxic preconditioning in the pre-3 day & pre-6 day groups does not show these protective effects . the downregulation of apoptotic cell death may be a mechanism underlying the protective effects of hypoxic preconditioning in the newborn rat brain . protective effect of hypoxic preconditioning on hypoxic - ischemic injured newborn rats hyun - kyung park , in - joon seol and ki - soo kim brief episodes of hypoxia before subsequent lethal ischemic events could be helpful . the protective effect of hypoxic preconditioning on hypoxic - ischemic brain injury was investigated in the neonatal rat . our study showed that hypoxic preconditioning 6-hr to 1-day before hypoxic - ischemic injury increased survival rates and had neuroprotective effects against subsequent hypoxic - ischemic injury .

brief episodes of cerebral hypoxia - ischemia cause transient ischemic tolerance to subsequent ischemic events that are otherwise lethal . this study was conducted to evaluate the protective effect of hypoxic preconditioning on hypoxic - ischemic injury in the neonatal rat and the persistence of a protective window after hypoxic preconditioning . the rats were preconditioned with hypoxia ( 8% oxygen , 92% nitrogen ) for three hours , subjected to ischemia using ligation of the right common carotid artery , and then exposed to another three hours of hypoxia . using proton magnetic resonance spectroscopy , terminal deoxynucleotidyl transferase - mediated dutp - biotin nick end - labeling ( tunel ) staining , and morphologic scores , this study shows that hypoxic preconditioning 6-hr to 1-day before hypoxic - ischemic injury increases survival rates and has neuroprotective effects against subsequent hypoxic - ischemic injury . the mechanism of the protective effects of hypoxic preconditioning in the newborn rat brain may involve downregulation of apoptotic cell death .

a 55-year - old man in good general health had pain and intermittent locking of the left knee of two months duration . magnetic resonance imaging ( mri ) showed a displaced bucket - handle tear of the lateral meniscus and so arthroscopic lateral meniscectomy was performed . increasing pain and swelling in the left popliteal fossa developed postoperatively over the following five days . a 65-ml hematoma was aspired and repeat arthroscopy of the knee detected only remnants of blood . the same symptomatology was displayed four days later and so the patient was referred for endovascular management . selective arteriography was performed via the right femoral artery , and a 4-fr catheter was placed into the left proximal popliteal artery . after contrast injection ( 30 ml at 8 ml / sec ; iopromida , shering , madrid , spain ) , a pseudoaneurysm measuring 2.51.60.9 mm and arising from the lateral inferior genicular artery was detected ; there was early venous drainage via the hypertrophied genicular veins , forming an arteriovenous ( av ) fistula ( fig . 1 ) . a hydrophilic 3-fr catheter ( terumo , leuven , belgium ) was selectively advanced over a guide wire into the injured genicular artery . the pseudoaneurysm was embolized with two small ( 2 mm in diameter , 20 mm in length ) fibered platinum coils ( cook europe , bjaeverskov , denmark ) . follow - up angiography demonstrated complete occlusion of the av fistula , with no filling of the pseudoaneurysm ( fig . clinically , the patient became free of symptoms a few days after the embolization and further clinical follow - up at six months was uneventful . vascular injuries arising from artroscopy of the knee are very rare , and several large series have reported an incidence of less than 1% ( 1 , 2 ) . most vascular injuries involve the popliteal artery or vein , or both ( 2 ) . pseudoaneurysms of the popliteal and lateral genicular arteries after knee surgery are rare and only a few , isolated case reports have been published ( 3 - 6 ) . pseudoaneurysms are more likely to form when a vessel is incompletely divided and blood dissects into the surrounding soft tissues . the susceptibility of the popliteal artery and its branches to injury during arthroscopic meniscectomy is due to several factors , including the employed surgical technique , and the operator 's experience and knowledge of their anatomic location ( 3 , 4 ) . the popliteal artery is close to the posterior capsule of the joint and it is moved forward during knee flexion . however , visualization of this region during knee arthroscopy is limited and any injury is often not immediately recognized ( 4 ) . nevertheless , the formation of combined pseudoaneurysm and av fistula after knee arthorscopy is extremely rare . only two such cases have been previously reported on , and both of these occurred in the popliteal artery ( 5 , 6 ) . reported a case of av fistula of the lateral superior and inferior geniculate arteries , and the etiology of this was unclear ( 7 ) . in our case , the mechanism to injury was probably direct trauma by cutting all three layers of both the artery and vein with scissors , which resulted in an av fistula . alternatively , hematoma formation around the artery and vein with degradation of the enclosed vessels may have also resulted in a combined pseudoaneurysm with av fistula . when a patient has popliteal swelling , mass , bruit , thrill , recurrent hemarthosis , pain , calf edema , and/or a neurologic deficit after a knee arthroscopy , the possibility of a vascular injury should be considered early on in the diagnostic process ( 2 , 4 ) . the diagnosis can be confirmed by conventional arteriography , as in our case , by color doppler sonography , or with using three - dimensional ct or mri arteriography techniques ( 4 - 8 ) . surgical exploration with vessel ligation is the " gold standard " of treatment because it preserves distal flow if collateral circulation has formed ( 3 , 5 - 8 ) . other treatment options include correction of the vascular tear by vascular patch or direct suture ( 8) . sonographically guided compression to occlude the postcatherterization pseudoaneurysm of the femoral and brachial arteries has been reported on ( 9 ) . however , with the advances in selective endovascular techniques , percutaneous embolization has been successfully performed for the management of popliteal pseudoaneurysms and also for the delayed combined pseudoaneurysm and av fistula of the anterior tibial artery after an open tibial fracture ( 10 ) . in our case , we embolized the lesion with using microcoils to definitely occlude the pseudoaneurysm and av fistula . this case demonstrates that superselective percutaneous angiographic embolization with coils offers the advantages of a minimally invasive approach ( no surgical incision , a reduced risk of infection and a shortened hospital stay ) ; therefore , this technique may represent an effective and safe therapeutic alternative for this uncommon complication of knee arthroscopy . in summary , we report here on a unique case of a lateral inferior genicular artery pseudoaneurysm with concomitant arteriovenous fistula as a complication of knee arthroscopy .

arthroscopic meniscectomy of the knee is generally a safe and effective procedure with a low rate of vascular complications . we report here on a unique case of a 55-year - old man with a lateral inferior genicular artery pseudoaneurysm and a concomitant arteriovenous fistula that developed after arthroscopic meniscectomy ; this was successfully treated with selective angiographic embolization . this case illustrates the effectiveness of an endovascular approach as a minimally invasive treatment for this uncommon complication that occurs after an arthroscopic procedure .

promoting the components of a healthy lifestyle and improvements in many countries social and economical conditions over the past several centuries have brought about the reduction , control , and elimination of many fatal infectious diseases . on the other hand , due to changes in lifestyle and industrial advances , nowadays , there are four main groups of non - contagious illnesses : cardiovascular diseases , cancers , chronic respiratory diseases , and diabetes , all of which are known to be the most important threats to human life and abilities . their side effects can impose heavy pressure on families , societies , and governments ( 1 ) . suffering from diabetes disease , and the obligation of the patient to take special care of him or herself , can cause many challenges in diabetics life routines , as they cope with these challenges . coping strategies can play an important role in the disease control , therapy , and psychological - social adaptation of diabetes patients ( 2 ) . some researchers have claimed that problem - focused strategies can successfully increase self - caring abilities in diabetic patients , while motion - focused methods can have harmful effects . therefore , a wide range of coping behaviors , including problem - focused and motion - focused coping strategies , have been recognized for their ability to decrease stresses related to the disease . studies have indicated that diabetes disease can have a negative effect on physical function , increasing psychological and spiritual side effects , and personal , family , and social communication ( 3 ) . psychological hardiness is a very important and prominent character feature that plays an important role in recognizing humans as free and valuable beings . this feature introduces the notion of humans as efficient and powerful creatures that , even in the most critical conditions and situations under a lot of pressure , can apply logical and rational coping strategies while also protecting his body and mind ( 4 ) . hardiness is one of the beliefs in the basic role of the quality of human life . belief systems often cause improvements in behavior , increase healthiness , life satisfaction , and life quality promotion ; on the other hand , it seems that many problems and issues that people face with in their lives and experiences are as a result of these beliefs . people s system of belief have a wide effect on the establishment of equilibrium among the various dimensions of life , and as guides for patterns of behavior , they enable humans to expand the desirable consequences and avoid the unwanted consequences of their actions ( 5 ) . baron indicated in his report that people with high levels of hardiness usually behave more effectively and constructively against the pressures and difficulties that commonly occur in workplace environment , and they rarely allow such pressures to continue and lead to physiological disease . there exists strong evidence indicating that hardiness has a positive correlation with physical and mental health ( 4 ) . the quality of professional life refers to applying conscious efforts to create a high quality professional life , so that these regular efforts are organized by an organization that gives greater opportunities to its workers in order to have a positive effect on their work ( 6 , 7 ) . in recent years , programs designed to improve the quality of professional life have had a fundamental and important role in increasing the efficiency of human resources ; consequently , they have been responsible for the effectiveness and efficiency of well - respected organizations and companies . strategic programs of promoting the quality of workers professional lives within organizations is a wide approach , indicating that cultivating an efficient human workforce plays an important role in the evolution of organizations . in modern societies , the promotion of professional life quality is a comprehensive program designed to attract the attention of workers and to help them manage changes , all while preserving workers inside the organization . dissatisfaction with professional life and low professional life quality are fundamental problems that can affect all workers lives , regardless of their position and rank , and may endanger individuals mental and physical health by imposing stress and other disturbing factors upon them ( 8 , 9 ) . many studies have indicated that there is a significant relationship between professional life quality , job control , occupational stress , and the frequency and intensity of physical disease . in addition , many studies findings indicate a significant and positive association between the extent of occupational stress and physical diseases . however , a negative significant relationship is reported between professional life quality and occupational stress , and between job control and occupational stress . also , a negative significant relationship has been reported between professional life quality and physical disease , and job control and physical disease ( 10 ) . since no research to date has been conducted on the relationship between professional life quality and coping strategies , along with hardiness , in diabetes patients , this paper aims to respond this question that do coping strategies ( problem - focused and motion - focused ) and hardiness explain professional life quality of patients with type ii diabetes and healthy people ? this study employs an applied research style . the relationship between the variables of coping strategies , along with hardiness as a predictor variable and professional life quality as the dependent variable , is investigated . for analysis apart from data descriptions , the pearson correlation coefficient and multiple regression analysis are used . before executing the main regression analysis for each specific question , a complete regression analysis model was executed for the variable of professional life quality , considering hardiness and all coping components . this analysis was executed with two purposes in mind : the first purpose is to investigate the basic hypotheses of multiple regression analysis , such as the linearity of the relationship between the target variable and predictor variables , normality and homogeneity of dispersion of conditional distributions . although the f test used for r - significance is resistant to the mean deviation of some of these hypotheses in regression analysis , hypotheses such as the non - linearity of the relationship , the estimation accuracy of parameters , and resulting interpretations are effective . furthermore , to complete the first purpose , some separate regressions of professional life quality were executed individually on scales , to cause situations of data distribution to be investigated accurately . the second purpose of this analysis was to investigate the success value of the general model in predicting variables of professional life quality and determining the value of r. this study was carried out between april and june 2013 . our research sample included 40 people of both genders ( 20 men , 20 women ) suffering from diabetes and ranging in age between 24 and 60 years old ( 45.1 10.7 years ) . they were selected by a simple random sampling method from among the staff of governmental organizations in qazwin , iran . a non - diabetic group ( healthy people ) consisted of 40 people who were same age range ( mean age 44.3 11.2 years old ) and gender distribution , and from same professional organizations as members of the diabetic group . instruments of this research were walton s professional life quality questionnaire ( 11 ) , of billings and moos ( 1981 ) questionnaire of coping strategies ( 12 ) , and kobasa s hardiness questionnaire ( 13 ) . in this research , walton s ( 11 ) the questionnaire employs a 5-degree likert scale , and its variations ranges from very low to very high . the reliability of this questionnaire is reported as = 0.89 by pardakhtchi et al . ( 14 ) , and the reliability obtained in this research was based on cronbach s alpha of 0.87 . a study by khaghani and colleagues investigated the relationship between occupational stress and the professional life quality of nurses employed in selected armed forces hospitals . to evaluate the validity of their professional life quality questionnaire , they used a formal method and , in order to determine reliability , they used the retest method . the correlation coefficient calculated by these methods was equal to 0.9 , which indicates an appropriate correlation of questions ( 15 , 16 ) . primary studies were implemented to determine the validity and reliability of the professional life quality questionnaire ; cronbach s alpha was reported as 0.95 ( 17 ) . billings and moos ( 1981 ) questionnaire of coping strategies consists of 19 terms that consider two coping strategies : problem - focused coping strategies ( 11 terms ) , and emotion - focused coping strategies ( 8 terms ) . the subject should sign any term that is counted as a type of coping response based on the extent to which he or she uses that method on a four - choice likert scale ( never = 0 , sometimes = 1 , often = 2 , always = 3 ) . in iran , barahini and mousavi used a four - choice likert scale ranging from 0 to 3 , instead of a yes or no format . kobasa s 1976 hardiness questionnaire consists of 50 provisions in which subjects should determine the correctness and incorrectness levels of sentences using a four - choice scale . this test consists of components related to challenge , commitment , and control , and each has 17 , 16 , and 17 provisions , respectively . due to previous studies conducted in our country , for instance , kiamarsi and abolghasemi ( 18 ) reported validity as cronbach s alpha of hardiness scale , and subscales of commitment , control , and challenging at 0.86 , 0.83 , 0.72 , and 0.69 , respectively . in a primary investigation of a persian form of the hardiness scale , alpha coefficients ranged from 0.88 to 0.93 for the commitment subscale , from 0.85 to 0.94 for the control subscale , from 0.89 to 0.95 for the challenge subscale , and from 0.87 to 0.94 for the total hardiness score , which indicated an appropriate internal consistency of scale ( 19 , 20 ) . as the study was observational and the data were kept confidential , there seem to be no ethical conflicts associated with the study . to respond the research question is there a significant correlation between professional life quality and coping strategies ( problem - focused and emotion - focused strategies ) , and hardiness specifically ? a correlation matrix is used to shows two - by - two correlations between variables , as shown in table 1 . the relationship between the variables of problem - focused strategy and professional life quality is significant ( r = 0.345 , p < 0.001 ) . since the value of this ratio is positive , it indicates that there is a positive relationship between the variable of problem - focused strategy and variable of professional life quality . this means that the more a person uses problem - focused strategies , the greater is his or her professional life quality . correlation coefficients between professional life quality along with applying emotion strategies ( -0.009 ) and hardiness ( -0.102 ) are statistically not significant . a multiple regression model was applied to investigate the main question of the research are coping strategies ( problem - focused and emotion - focused ) , and hardiness able to explain professional life quality among type ii diabetic people and healthy people ? , and also to determine the portion of each of them in predicting professional life quality ( table 2 ) . according to the matrix of correlation of predictors with each other and with target variable , which is shown in table 1 , all correlation ratios among predictor variables were less than 0.9 , hence the probability of co - linearity is low among predictor variables . finally , these variables explain 11.2% of the variance among the target variable ( professional life quality ) in patients suffering from type ii diabetes and among healthy people ( table 3 ) . also , the results of the f test verify the significant relationship of the r ( determination ratio ) . finally , the table values verify the determinant ratios of problem - focused coping strategies with values of t = 3.468 , p = 0.001 , and = 0.38 ( table 4 ) . beta values related to problem - focused coping strategies indicate that , if one unit is added to problem - focused coping strategies , the professional life quality of both type ii diabetic people and healthy people will increase up to 38% . according to the research findings , we conclude that coping strategies ( problem - focused and emotion - focused ) and hardiness can explain the professional life quality of people suffering from type ii diabetes and for healthy people . this finding is in accordance with findings of besharat et al . and bayrami and esmaili ( 21 , 22 ) . according to the model by folkman and lazarus ( 24 ) cognition strategies are used to solve the problem . in other words , problem - focused efforts include the execution of direct activities to change stressful situations and to prevent or reduce their effects . people apply problem - focused coping strategies when they believe that they will work the face of a stressful situation . emotion - focused coping strategies are meant to eliminate the emotional effects of stressful factors . emotion - focused efforts are concentrated on changing emotional reactions of pressure - making factors . the personality trait of hardiness creates a specific internal approach that influences peoples confrontation methods when faced with various problems . hardiness is due to knowledge based on the fact that people with accesses to more resources can respond better to stressors . in other words , hardiness is a basic feeling of control that enables a tough person to draw on and have access to a list of useful strategies that he or she can afford . consequently , hardiness leads to the establishment of an optimistic view about stressors ( 4 , 23 ) . hardiness is a set of personality traits that acts as a source of resistance against life s pressures . cognition strategies are used to solve problems . accordingly , various coping strategies for problems are directly investigated , so that they include all potential abilities of person to allow positive coping with the problem and the solving of it , usually by finding suitable solutions . therefore , efficient coping methods , such as increasing people s self - confidence , improve their problem solving skills and leads to more satisfaction ( 23 ) . in the present study , we determined that none of the variables of hardiness and emotion - focused coping strategies have an effect on the professional life quality of diabetic people , since emotion - focused strategies are meant to eliminate the emotional reactions to stressful factors . emotion - focused efforts are dedicated to changing emotional reactions into pressure - making factors . a diabetic person requires both physical and mental adjustment and should have a new view of him or herself . chronic and routine stresses have important effects on physical and psychological disturbances , and type ii diabetes is very sensitive to the effects of stress ( 24 - 26 ) . both variables of hardiness and problem - focused strategies have an effect on the professional life quality of healthy people . accordingly , various coping strategies are directly investigated , so that they include all potential abilities of a person to engage in positive coping and to solve problems , usually by finding suitable solutions . therefore , efficient coping methods such as increasing people s self - confidence improves their problem solving skills and leads to greater satisfaction ( 27 , 28 ) . on the other hand , some studies have indicated that the hardier people are , the less they evaluate stressful conditions as threatening , and they take advantage of effective coping strategies . when people do not have the ability to control themselves emotionally , have less incentive to try and remain stable when faced with routine stresses , and since they have a low attempt and stability , they can not easily solve their problems . however , it should be noted that , in some situations , a person is not able to control his or her negative emotions , so methods and strategies for coping with emotions can be useful compared to not applying these strategies ( 29 - 31 ) . according to the positive relationship between problem - focused strategies and professional life quality , it seems that holding training classes about applying problem - focused coping strategies and giving self - confidence to participants would have a positive and appropriate result in promoting the psychological health of employees and , consequently , in increasing their efficiency . planning to train managers about coping strategies , hardiness , and professional life quality is imperative , so that effective strategies for improving occupational conditions and decreasing occupational stress of employees can be compiled in ways such as increased employee participation in decision - making , increased employee support , and a decrease in employee workloads ( 28 , 32 - 35 ) the main limitation of our study is its small sample size .

backgrounddue to the epidemiologic transition and a rise in the prevalence of non - communicable diseases different coping strategies have been studied and developed . these strategies may help the affected people to conduct a normal life style.objectivesthis research was conducted in qazvin , iran to determine the relationship between coping strategies , hardiness , and occupational life quality in type ii diabetic patients and healthy people.patients and methodsquestionnaires such as valton s on occupational life quality , billings and moos examination of coping strategies , and kobasa s investigation of hardiness were applied to collect the data needed for the present study . in this regard , 80 people were randomly selected from employees of offices in qazvin , iran.resultsthe results of this research indicated that there is a significant relationship between problem - focused strategies , emotion - focused strategies , hardiness , and occupational life quality in people suffering from type ii diabetes and healthy people ( p 0.05 ) . these results also indicated that hardiness does not predict occupational life quality of people suffering from type ii diabetes.conclusionsthe results of the present study give some evidence that allows us to conclude that hardiness and coping strategies affect occupational life quality for both people suffering from type ii diabetes and healthy people . therefore , it is proposed that people strengthen their hardiness and coping strategies , in order to improve their occupational life quality .

diverticula of the jejunum and ileum ( excluding meckel 's diverticulum ) are rare clinicopathological entities and most patients are asymptomatic . due to the rarity of the condition , the diagnosis is difficult and often delayed until complications arise . kayexalate ( sodium polystyrene sulfonate ) in sorbitol is used frequently for treatment of hyperkalemia and has been associated with colonic necrosis and upper gastrointestinal injuries in a subset of uremic patients . necrosis of the gastrointestinal ( gi ) tract due to kayexalate in sorbitol is underrecognized by pathologists as well as clinicians . small intestinal damage related to kayexalate has been described only infrequently in the terminal ileum . we report a case of jejunal diverticulosis with mucosal injury and diverticulitis in a uremic patient treated with kayexalate . to our knowledge the two conditions and the potential role of kayexalate in the pathogenesis of diverticulitis are discussed . a 87-year - old male patient under dialysis for a terminal chronic renal insufficiency was admitted to the surgery department for correction of an occlusion located at the vascular access of his left upper limb fistulae . there was a history appendectomy , cholecystectomy , radical prostatectomy and of ischemic cardiopathy with instable angor that had been relieved by stenting of the left circumflex coronary artery placed 2 months earlier . the patient received sorbisterit - calcium ( calcium - polystyrol ) with sorbitol among other medications . during the current admission , he developed abdominal pain that was more severe in the right hemi - abdomen . an abdominal ct scan was performed and revealed a small intestinal diverticulosis with 5 giant diverticula ( fig . there was an infiltration of mesenteric fat around 2 diverticula measuring 5.5 and 3 cm , which was suggestive of diverticulitis . , an exploratory laparotomy was performed with resection of a 25-cm - long jejunal segment ( fig . the serosal surface was partially covered with a fibrinous exudate but there was no evidence of perforation . the smallest measured 4 3.5 1.2 cm and the largest 6.5 4.5 4.4 cm ( fig . histological examination showed that the diverticula were in fact pseudo - diverticula , which consisted of mucosa and muscularis mucosae invaginating through the muscularis propria ( fig . however , some pseudo - diverticula also contained some thin and discontinuous smooth muscle bundles in addition to the muscularis mucosae ( fig . multiple ulcerations of various depth and areas of transmural necrosis were seen in most diverticula ( fig . numerous irregular , sharply angulated ( broken - glass like ) crystals were seen adherent on the mucosal side and on the ulcerations ( fig . they were basophilic on routine stain , with a mosaic or striped pattern , and stained bright red on periodic acid schiff ( pas ) stain . they were also found in the necrotic wall , and rarely in the exudate on the peritoneal surface consistent with microscopic perforations . the serosal surface was partially covered with a fibrinous exudate but there was no evidence of perforation . the smallest measured 4 3.5 1.2 cm and the largest 6.5 4.5 4.4 cm ( fig . histological examination showed that the diverticula were in fact pseudo - diverticula , which consisted of mucosa and muscularis mucosae invaginating through the muscularis propria ( fig . however , some pseudo - diverticula also contained some thin and discontinuous smooth muscle bundles in addition to the muscularis mucosae ( fig . multiple ulcerations of various depth and areas of transmural necrosis were seen in most diverticula ( fig . numerous irregular , sharply angulated ( broken - glass like ) crystals were seen adherent on the mucosal side and on the ulcerations ( fig . they were basophilic on routine stain , with a mosaic or striped pattern , and stained bright red on periodic acid schiff ( pas ) stain . they were also found in the necrotic wall , and rarely in the exudate on the peritoneal surface consistent with microscopic perforations . to our knowledge , this is the first description of an association between jejunal ulcerated diverticulitis and kayexalate intake in a uremic patient . in this unusual case , kayexalate might be just an innocent bystander but could also be on the other hand implicated to some extent in the pathogenesis of the mucosal injury and diverticulitis . before discussing the possible role of kayexalate in the pathogenesis of the diverticulitis , we will first review the two conditions ( ileo - jejunal diverticulosis and kayexalate related gi tract injury ) separately . diverticula of the jejunum and ileum are rare clinical entities in contrast to meckel 's diverticulum , which is fairly common [ 1 , 4 , 5 ] . reported incidence of ileo - jejunal diverticula ranges from 0.3 to 4.5% in autopsy series and from 0.5 to 2.3% in radiological studies and depends largely on examination technique . highest incidence was found either when filling the small bowel with water under moderate pressure in autopsy studies or when using enteroclysis in radiological studies . they are more frequently encountered in the elderly , with a peak incidence in the sixth and seventh decades , and have a slight male predominance . they are preferentially localized to the jejunum ( 80% ) , to a lesser degree to the ileum ( 15% ) and sometimes they involve both parts ( 5% ) . they can measure from a few millimeters up to more than 10 cm and tend to be more numerous and larger in the proximal jejunum [ 7 , 8 ] , such as in our case . strictly speaking , in most instances they are not true diverticula such as meckel 's diverticula , which comprise all parts of the bowel wall , but they are pseudo - diverticula formed by herniation of mucosa and submucosa through the muscular layer of the bowel wall on the mesenteric border of the bowel where the arteries enter the intestine . histologically they are lined by mucosa , muscularis mucosae and submucosa and lack a muscularis propria . in contrast , meckel 's diverticula are located on the side opposed to the mesentery , are delineated by mucosa , submucosa and muscularis propria and may contain heterotopic mucosa . although they can be congenital the vast majority is acquired [ 8 , 9 ] . the cause of small bowel diverticulosis seems to be motor dysfunction of the smooth muscle or the myenteric plexus . the resulting intestinal dyskinesia might increase intraluminal pressure that will push the mucosa and submucosa through the weakest site of the bowel wall , which are the entry points of the blood vessels from the mesentery [ 8 , 9 , 10 ] . the predominance of diverticula in the jejunum is attributed to the greater diameter of the penetrating jejunal arteries . the diverticula usually remain asymptomatic ( over 80% ) and become evident as an incidental finding at the time of surgery , during a radiographic examination or at autopsy . their clinical importance is based on the fact that like colonic diverticulosis they may cause serious complications , such as infection , hemorrhage , intestinal obstruction and ileovesical fistula . complications related to the diverticula occur in about 15 to 20% of patients and complications or symptoms requiring surgery have been reported to occur in up to 10% . the most frequent acute complication of the jejunoileal diverticula is probably diverticulitis ( with or without perforation ) , occurring in 2.3 to 6.4% of cases . the occurrence of gi necrosis due to kayexalate in sorbitol is well documented [ 2 , 3 , 11 , 12 , 13 , 14 , 15 ] and supported by experimental evidence . however , the true incidence of colonic necrosis after kayexalate intake is unknown . gerstmann et al . noted a 0.27% overall incidence , with a higher ( 1.8% ) incidence in the postoperative period . factors that contribute to this entity include uremia , hypovolemia , peripheral vascular disease , and immunosuppressive therapy . one of the first studies from lillemoe et al . reported five uremic patients who developed catastrophic colonic necrosis that was associated temporally with the use of kayexalate in sorbitol and contributed to death in four of the five patients . that study also provided experimental evidence implicating sorbitol rather than kayexalate as the agent responsible for colonic necrosis in a rat model . no pathologic changes developed in rats receiving enemas of kayexalate in water , but transmural colonic necrosis developed in six of 10 rats receiving sorbitol enemas . production of prostaglandins may be stimulated by sorbitol and could play a part in causing mucosal injury . previous reports of gi injury associated with administration of kayexalate in sorbitol have focused almost exclusively on colonic or ileocolonic necrosis in patients with hyperkalemia [ 3 , 11 , 12 , 13 , 14 , 15 ] . more recently , abraham et al . have shown that kayexalate can also induce upper gi tract injury . in the differential diagnosis , kayexalate crystals also need to be distinguished histologically from cholestyramine crystals that look very similar . the distinction is important since , in contrast to kayexalate , cholestyramine has not been associated with gi tract injury to date . both consist of irregular , sharply angulated ( broken - glass like ) fragments of an amorphous substance , but the former are slightly basophilic on routine stain with a mosaic or striped pattern . the latter appear bright red on routine stain and are more homogeneous , without a mosaic or striped pattern ( fig . it can be difficult to define the role of kayexalate in sorbitol in the development of mucosal injury and diverticulitis in elderly patients who presents with multiple medical problems and who are also at risk for ischemic complications . in our case , while it is possible that kayexalate might be just an innocent bystander in the pathogenesis of the diverticulitis , we believe that kayexalate likely played a role in the diverticulitis and mucosal injury either as a causative agent or as a cofactor . the pattern of ulceration and diverticulitis present in our case is not the one that is usually seen in diverticulitis in the colon or in a meckel 's diverticulum . the ulcerations were often associated with wall necrosis and there was mild acute inflammation which might point to a local ischemia . in contrast , the mucosae next to the diverticulitis did not show any sign of ischemia , which suggests that bowel segment ischemia is not the cause of the diverticulitis . rashid and hamilton reported similar findings to ours in their study about colonic specimens from nine patients with kayexalate - sorbitol induced colonic necrosis . they described in the involved segments mucosal ulceration , dusky mucosal coloration , mural oedema , serosal fibrinous exudate , mucosal or transmural necrosis and perforation with luminal kayexalate crystals [ 3 , 11 ] . in the gi tract , diverticula are likely to be sites vulnerable to kayexalate injury since the content in the lumen including kayexalate crystals and sorbitol is trapped inside the diverticula and is more likely to stay in contact with the mucosa for prolonged time . the same is also true for any mechanical factors such as tumors or other obstructing lesions that delay gi transit and prolong contact between the mucosa and sorbitol , hence predisposing to injury . furthermore , impairment of blood - flow by the pseudodiverticula , which compress the branches of the mesenteric blood vessels at the site of passage through the muscularis propria , may add additional stress to the mucosa . since the pseudodiverticula wall is also significantly thinner than the normal bowel wall , the risk of perforation and peritonitis is also increased . in conclusion , in addition to the more commonly reported risk of colonic necrosis , kayexalate in sorbitol could induce injury to other parts of the gi tract , particularly in sites where a prolonged contact with the mucosa might be expected . although the histologically visible kayexalate crystals are not directly responsible for mucosal damage , their recognition as a marker for the patient 's ingestion of kayexalate in sorbitol is a clue in providing the correct diagnosis in patients with mucosal injury , which is essential for appropriate treatment . diverticula of the jejunum and ileum are rare clinical entities in contrast to meckel 's diverticulum , which is fairly common [ 1 , 4 , 5 ] . reported incidence of ileo - jejunal diverticula ranges from 0.3 to 4.5% in autopsy series and from 0.5 to 2.3% in radiological studies and depends largely on examination technique . highest incidence was found either when filling the small bowel with water under moderate pressure in autopsy studies or when using enteroclysis in radiological studies . they are more frequently encountered in the elderly , with a peak incidence in the sixth and seventh decades , and have a slight male predominance . they are preferentially localized to the jejunum ( 80% ) , to a lesser degree to the ileum ( 15% ) and sometimes they involve both parts ( 5% ) . they can measure from a few millimeters up to more than 10 cm and tend to be more numerous and larger in the proximal jejunum [ 7 , 8 ] , such as in our case . strictly speaking , in most instances they are not true diverticula such as meckel 's diverticula , which comprise all parts of the bowel wall , but they are pseudo - diverticula formed by herniation of mucosa and submucosa through the muscular layer of the bowel wall on the mesenteric border of the bowel where the arteries enter the intestine . histologically they are lined by mucosa , muscularis mucosae and submucosa and lack a muscularis propria . in contrast , meckel 's diverticula are located on the side opposed to the mesentery , are delineated by mucosa , submucosa and muscularis propria and may contain heterotopic mucosa . although they can be congenital the vast majority is acquired [ 8 , 9 ] . the cause of small bowel diverticulosis seems to be motor dysfunction of the smooth muscle or the myenteric plexus . the resulting intestinal dyskinesia might increase intraluminal pressure that will push the mucosa and submucosa through the weakest site of the bowel wall , which are the entry points of the blood vessels from the mesentery [ 8 , 9 , 10 ] . the predominance of diverticula in the jejunum is attributed to the greater diameter of the penetrating jejunal arteries . the diverticula usually remain asymptomatic ( over 80% ) and become evident as an incidental finding at the time of surgery , during a radiographic examination or at autopsy . their clinical importance is based on the fact that like colonic diverticulosis they may cause serious complications , such as infection , hemorrhage , intestinal obstruction and ileovesical fistula . complications related to the diverticula occur in about 15 to 20% of patients and complications or symptoms requiring surgery have been reported to occur in up to 10% . the most frequent acute complication of the jejunoileal diverticula is probably diverticulitis ( with or without perforation ) , occurring in 2.3 to 6.4% of cases . the occurrence of gi necrosis due to kayexalate in sorbitol is well documented [ 2 , 3 , 11 , 12 , 13 , 14 , 15 ] and supported by experimental evidence . however , the true incidence of colonic necrosis after kayexalate intake is unknown . noted a 0.27% overall incidence , with a higher ( 1.8% ) incidence in the postoperative period . factors that contribute to this entity include uremia , hypovolemia , peripheral vascular disease , and immunosuppressive therapy . one of the first studies from lillemoe et al . reported five uremic patients who developed catastrophic colonic necrosis that was associated temporally with the use of kayexalate in sorbitol and contributed to death in four of the five patients . that study also provided experimental evidence implicating sorbitol rather than kayexalate as the agent responsible for colonic necrosis in a rat model . no pathologic changes developed in rats receiving enemas of kayexalate in water , but transmural colonic necrosis developed in six of 10 rats receiving sorbitol enemas . production of prostaglandins may be stimulated by sorbitol and could play a part in causing mucosal injury . previous reports of gi injury associated with administration of kayexalate in sorbitol have focused almost exclusively on colonic or ileocolonic necrosis in patients with hyperkalemia [ 3 , 11 , 12 , 13 , 14 , 15 ] . more recently , abraham et al . have shown that kayexalate can also induce upper gi tract injury . in the differential diagnosis , kayexalate crystals also need to be distinguished histologically from cholestyramine crystals that look very similar . the distinction is important since , in contrast to kayexalate , cholestyramine has not been associated with gi tract injury to date . both consist of irregular , sharply angulated ( broken - glass like ) fragments of an amorphous substance , but the former are slightly basophilic on routine stain with a mosaic or striped pattern . the latter appear bright red on routine stain and are more homogeneous , without a mosaic or striped pattern ( fig . it can be difficult to define the role of kayexalate in sorbitol in the development of mucosal injury and diverticulitis in elderly patients who presents with multiple medical problems and who are also at risk for ischemic complications . in our case , while it is possible that kayexalate might be just an innocent bystander in the pathogenesis of the diverticulitis , we believe that kayexalate likely played a role in the diverticulitis and mucosal injury either as a causative agent or as a cofactor . the pattern of ulceration and diverticulitis present in our case is not the one that is usually seen in diverticulitis in the colon or in a meckel 's diverticulum . the ulcerations were often associated with wall necrosis and there was mild acute inflammation which might point to a local ischemia . in contrast , the mucosae next to the diverticulitis did not show any sign of ischemia , which suggests that bowel segment ischemia is not the cause of the diverticulitis . rashid and hamilton reported similar findings to ours in their study about colonic specimens from nine patients with kayexalate - sorbitol induced colonic necrosis . they described in the involved segments mucosal ulceration , dusky mucosal coloration , mural oedema , serosal fibrinous exudate , mucosal or transmural necrosis and perforation with luminal kayexalate crystals [ 3 , 11 ] . in the gi tract , diverticula are likely to be sites vulnerable to kayexalate injury since the content in the lumen including kayexalate crystals and sorbitol is trapped inside the diverticula and is more likely to stay in contact with the mucosa for prolonged time . the same is also true for any mechanical factors such as tumors or other obstructing lesions that delay gi transit and prolong contact between the mucosa and sorbitol , hence predisposing to injury . furthermore , impairment of blood - flow by the pseudodiverticula , which compress the branches of the mesenteric blood vessels at the site of passage through the muscularis propria , may add additional stress to the mucosa . since the pseudodiverticula wall is also significantly thinner than the normal bowel wall , the risk of perforation and peritonitis is also increased . in conclusion , in addition to the more commonly reported risk of colonic necrosis , kayexalate in sorbitol could induce injury to other parts of the gi tract , particularly in sites where a prolonged contact with the mucosa might be expected . although the histologically visible kayexalate crystals are not directly responsible for mucosal damage , their recognition as a marker for the patient 's ingestion of kayexalate in sorbitol is a clue in providing the correct diagnosis in patients with mucosal injury , which is essential for appropriate treatment .

small intestine diverticulosis is a rare entity that is asymptomatic in the majority of cases . however , it may cause serious complications , such as infection , hemorrhage , intestinal obstruction and diverticulitis . kayexalate ( sodium polystyrene sulfonate ) in sorbitol has been associated with colonic necrosis and less frequently with upper gastrointestinal injuries in a subset of uremic patients treated for hyperkalemia . we report a case of jejunal diverticulosis with mucosal injury and diverticulitis in a uremic patient treated with kayexalate and discuss the potential role of kayexalate in the pathogenesis of diverticulitis .

all patients seen and assessed in our neuroophthalmology departments with visual disorders are investigated to identify the cause and to define the most appropriate treatment . this investigation includes a series of blood investigations and imaging ; when these investigations found no evidence for a demyelinating disorder ( through mri features principally ) , systemic autoimmune condition ( e.g. , antinuclear antibodies , dna , aquaporin-4 , and myelin oligodendrocyte glycoprotein antibodies ) , or infectious or metabolic causes , a granulomatous cause was considered , and further investigations were undertaken . those in whom systemic features of sarcoidosis were identified during investigation and those already known to have active systemic sarcoidosis , in whom the diagnosis of a granulomatous optic neuropathy was considered by the authors to be highly probable , were recorded prospectively and included in the study . the records were kept according to data protection regulations in the neuroophthalmology departments and collated by 2 of us ( b.j.b . and d.p.k . ) . criteria for the diagnosis of sarcoidosis involved histologic proof ( in 40 cases ) of a granulomatous disorder currently active or requiring continuing treatment or ( in 12 cases ) adequate proof on clinical grounds that no alternative explanation existed for the systemic disorder in the minds of the treating physicians : for example , clear radiologic evidence for lung and mediastinal involvement together with clear evidence for another characteristic feature of the disease , such as hypercalcemia , raised serum angiotensin - converting enzyme ( ace ) or other tissue involvement , associated with a typical pattern of isotope uptake using gallium scintigraphy or pet . ethical approval for the study was obtained , and patients gave informed consent to be included . ethical approval for the study was obtained , and patients gave informed consent to be included . twenty - four were caucasian , 24 african or caribbean , and 4 were from south asia . the diagnosis of systemic sarcoidosis was made concurrently in 33 patients and in one subsequently . the remaining 18 had already been diagnosed , between 6 months and 20 years before the onset of the optic neuropathy . a histologic diagnosis was made from respiratory tissue ( transbronchial or mediastinal lymph gland biopsy ) in 28 cases , lymph gland biopsy in 3 , liver biopsy in 1 , orbital biopsy in 3 , and neurologic tissue in 5 . all fulfilled the criteria for the highly probable diagnosis of the wasog ( world association of sarcoidosis and other granulomatous diseases ) sarcoidosis organ assessment instrument . in 12 cases , the diagnosis had been deemed secure on clinical grounds by the treating respiratory physicians , with clear evidence on clinical grounds for a multisystem disorder typical of sarcoidosis as noted in the methods section above ; these were all patients presenting with an optic neuropathy already diagnosed with systemic sarcoidosis . the predominate systemic clinical syndrome was respiratory in 38 cases , the orbit and lacrimal glands in 6 , cervical lymph glands in 4 , the skin in 3 , the nose and sinuses in 3 , and the liver and joints in 1 each . the serum ace was elevated in 24 ( 57% ) of available data at the time of onset of the optic neuropathy ; the median serum ace was 111 ( 18205 ) u / l ( normal range 850 gallium scans were undertaken in 10 instances , in which 1 was normal , 6 showed a panda pattern of uptake in the lacrimal and parotid glands , 1 a lambda pattern of mediastinal uptake , and 2 a combination of both patterns . all patients presented with symptoms and signs of an optic neuropathy , defined as a reduction in central visual acuity and color vision , reduction in the visual field , and when seen , an abnormality of the pupillary response . patients with a subacute optic neuropathy that evolved over days were considered separately from those with a more progressive clinical course , in which the symptoms evolved over weeks . the former were considered to show an optic neuritis syndrome , the latter a progressive syndrome . forty - three cases showed an optic neuritis syndrome , of which 27 were unilateral . twelve of the bilateral cases showed a sequential optic neuropathy with a range of latency between the 2 episodes of 1 to 60 months . the median ( range ) acuity at presentation was 0.05 ( 01.0 ) in unilateral cases and 0.13 ( 01.0 ) in bilateral involvement . mri showing ( a ) swelling and high signal within the nerve , and ( b ) enhancement in a patient who presented with a painless subacute optic neuropathy to no perception of light over 5 days . with oral corticosteroid treatment ( c ) t1-weighted axial mri showing swelling and enhancement of the anterior half of the left optic nerve . ( d , e ) t1-weighted coronal mri showing 2 other cases in which the nerve is seen to be swollen and enhancing following administration of contrast . a wide range of visual field defects was noted ; most had central or centrocecal scotomas , but hemianopic and altitudinal defects were also seen . clinical characteristics and ocular findings at presentation in the 52 patients studied intraocular inflammation was seen at the same time as the optic neuropathy in 19 cases ( 36% ) : a granulomatous anterior uveitis was seen in 10 cases , an isolated vitritis in 1 , a panuveitis in 1 , isolated choroidal involvement in 1 , features of a retinal vasculitis in 2 , cotton wool spots in 2 , macular exudates in 1 , and episcleritis in 1 . in no case was the intraocular disease longstanding and associated with chronic glaucoma or retinal ischaemia . one patient underwent ct ; the remainder had mri studies of the orbits and brain , of which 13 were adjudged to be normal . twenty patients had abnormalities restricted to the optic nerve or sheath , of whom 14 showed enhancement of the optic nerve and sheath ( figure 1 ) with intrinsic lesions of one or both optic nerves , while the remainder showed high signal within the nerve without enhancement . in addition , 3 cases ( and the one who had ct ) showed intrinsic lesions within the nerve and thickening of the adjacent optic nerve sheath complex . the chiasmal lesion was associated with enhancement of the chiasm extending forward into one of the nerves . both patients with a perineuritis showed features of this , i.e. , the predominate feature was of enhancement and thickening of the sheath , not the whole complex ( figure 2 ) . ( b , c ) optic perineuritis : t1-weighted axial and coronal mri showing enlargement of and enhancement of the optic nerve and its sheath throughout its entire length . this was the presenting feature of the disease , which was diagnosed subsequently on histologic analysis of a cervical lymph node . a minority of patients whose clinical syndrome was one of a subacute optic neuropathy resembling an optic neuritis showed additional imaging features of more widespread involvement of the nervous system by the disease : 8 patients with unilateral optic neuropathies and 2 with bilateral involvement showed high signal within the nerves , enhancement of the complex , which also involved the orbital apex ( 5 cases ) ( figure 3a ) , the floor of the middle cranial fossa ( 4 cases ) ( figure 3b ) , and the frontal lobes ( 1 case ) . ( a , b ) orbital apex mass with a progressive painful optic neuropathy and sarcoidosis of the lungs , mediastinum , and heart . ( c , d ) two cases of progressive optic neuropathy with striking meningeal enhancement and swelling to form mass lesions . despite intensive treatment with corticosteroids and immunosuppression , in general , the initial acuity was better in those with normal imaging and no enhancement than in those in whom enhancement was seen ( figure 4 ) . histogram showing the relationships between mri features at onset / diagnosis ( abscissa ) and initial ( nadir ) visual acuity then best acuity after treatment ( ordinate ) . those whose clinical syndrome was of a progressive optic neuropathy ( 6 cases ) all showed meningeal enhancing lesions that had formed masses , in each case at the intracranial portion of the orbital apex ( figure 3 ) . csf results were available in 33 cases , including 2 with mass lesions and one with perineuritis . seven ( 21% ) showed csf protein levels greater than 0.6 g / dl ( median 0.95 g / dl , range 0.791.92 ) . five ( 15% ) showed a leukocyte pleocytosis ( > 5 cells / cm ) ( median 19 , range 670 ) . twenty - eight ( 85% ) had no oligoclonal bands , and 5 showed matched bands in both csf and serum . patients were treated in an individual way in 3 different hospitals so there was a wide range of medications , dosage regimens , and treatment durations . forty - nine were treated with corticosteroids , of whom 14 commenced with iv methylprednisolone followed by oral prednisolone , while the remainder received treatment with oral steroids from the outset . the daily dose of prednisolone varied between 30 and 80 mg per day . the duration of treatment varied greatly ( 4320 weeks ) . the median ( range ) length of follow - up was 8 ( 020 ) years . the median ( range ) change in visual acuity ( defined as the best acuity following treatment , even after relapse ) was + 0.46 ( 0.01 to + 1.22 ) in bilateral cases and + 0.175 ( 0.05 to + 1.25 ) in unilateral cases . the median final acuity in these 2 groups was 0.7 ( 01.25 ) and 0.5 ( 01.25 ) . those with a subacute syndrome showed a median improvement of + 0.225 ( range 0.01 to + 1.22 ) . those in the progressive subgroup showed no improvement in visual acuity with treatment although in each case there was resolution of meningeal enhancement over time . while there was a suggestion that mri abnormalities at onset ( figure 4 ) correlated with nadir acuity and an improvement following treatment , this was not significant ( p > 0.3 ) . considering treatment in those without a progressive disease course or perineuritis , 12 were treated with iv then oral steroids ; median ( range ) acuity was 0.02 ( 01 ) and increased to 0.75 ( 0.021 ) , median change + 0.3 , and those treated with oral steroids at onset 0.1 ( 01 ) increasing to 0.75 ( 01.25 ) , and median change + 0.5 ( p = 0.77 ) . for the most part , this occurred early on ; for example , patients treated with 60 mg prednisolone developed recurrence of symptoms and signs at 40 to 20 mg , and the signs settled with an escalation of treatment . immunosuppressive agents were added in 7 cases azathioprine in 4 and mycophenolate and methotrexate in one each . the patient treated with methotrexate had had repeated relapses , which stopped on the immunosuppressive drug . one patient had an intracranial relapse of neurosarcoidosis and her optic neuropathy worsened ; she was treated with cyclophosphamide then infliximab . in those who relapsed , the improvement in acuity following treatment was + 0.25 ( 0.15 to + 1.2 ) , and relapse did not appear to influence the outcome in any way . we have seen 6 additional patients who presented with an optic neuritis and were found during investigation to have a raised serum ace ( median 100 [ range 80178 ] u / l ) . the clinical syndrome was not different from the others described above ; half were caribbean , half caucasian , and half were bilateral . the median ( range ) acuity in each eye was 0.1 ( 01.0 ) . in each case , the affected discs were seen to be swollen . imaging was normal in 3 and showed high signal within the affected nerve in the other 3 . csf in 4 cases showed normal protein and negative oligoclonal bands in 3 ; in the other case , matched serum and csf bands were found . in this case one was considered to have a disc granuloma and was watched without treatment ; he improved over 2 years . these cases have been followed and none has developed further or recurrent neurologic or systemic symptoms . they appear to have neither sarcoidosis nor multiple sclerosis . twenty - four were caucasian , 24 african or caribbean , and 4 were from south asia . the diagnosis of systemic sarcoidosis was made concurrently in 33 patients and in one subsequently . the remaining 18 had already been diagnosed , between 6 months and 20 years before the onset of the optic neuropathy . a histologic diagnosis was made from respiratory tissue ( transbronchial or mediastinal lymph gland biopsy ) in 28 cases , lymph gland biopsy in 3 , liver biopsy in 1 , orbital biopsy in 3 , and neurologic tissue in 5 . all fulfilled the criteria for the highly probable diagnosis of the wasog ( world association of sarcoidosis and other granulomatous diseases ) sarcoidosis organ assessment instrument . in 12 cases , the diagnosis had been deemed secure on clinical grounds by the treating respiratory physicians , with clear evidence on clinical grounds for a multisystem disorder typical of sarcoidosis as noted in the methods section above ; these were all patients presenting with an optic neuropathy already diagnosed with systemic sarcoidosis . the predominate systemic clinical syndrome was respiratory in 38 cases , the orbit and lacrimal glands in 6 , cervical lymph glands in 4 , the skin in 3 , the nose and sinuses in 3 , and the liver and joints in 1 each . the serum ace was elevated in 24 ( 57% ) of available data at the time of onset of the optic neuropathy ; the median serum ace was 111 ( 18205 ) u / l ( normal range 850 gallium scans were undertaken in 10 instances , in which 1 was normal , 6 showed a panda pattern of uptake in the lacrimal and parotid glands , 1 a lambda pattern of mediastinal uptake , and 2 a combination of both patterns . all patients presented with symptoms and signs of an optic neuropathy , defined as a reduction in central visual acuity and color vision , reduction in the visual field , and when seen , an abnormality of the pupillary response . patients with a subacute optic neuropathy that evolved over days were considered separately from those with a more progressive clinical course , in which the symptoms evolved over weeks . the former were considered to show an optic neuritis syndrome , the latter a progressive syndrome . forty - three cases showed an optic neuritis syndrome , of which 27 were unilateral . twelve of the bilateral cases showed a sequential optic neuropathy with a range of latency between the 2 episodes of 1 to 60 months . the median ( range ) acuity at presentation was 0.05 ( 01.0 ) in unilateral cases and 0.13 ( 01.0 ) in bilateral involvement . mri showing ( a ) swelling and high signal within the nerve , and ( b ) enhancement in a patient who presented with a painless subacute optic neuropathy to no perception of light over 5 days . with oral corticosteroid treatment ( c ) t1-weighted axial mri showing swelling and enhancement of the anterior half of the left optic nerve . ( d , e ) t1-weighted coronal mri showing 2 other cases in which the nerve is seen to be swollen and enhancing following administration of contrast . a wide range of visual field defects was noted ; most had central or centrocecal scotomas , but hemianopic and altitudinal defects were also seen . intraocular inflammation was seen at the same time as the optic neuropathy in 19 cases ( 36% ) : a granulomatous anterior uveitis was seen in 10 cases , an isolated vitritis in 1 , a panuveitis in 1 , isolated choroidal involvement in 1 , features of a retinal vasculitis in 2 , cotton wool spots in 2 , macular exudates in 1 , and episcleritis in 1 . in no case was the intraocular disease longstanding and associated with chronic glaucoma or retinal ischaemia . one patient underwent ct ; the remainder had mri studies of the orbits and brain , of which 13 were adjudged to be normal . twenty patients had abnormalities restricted to the optic nerve or sheath , of whom 14 showed enhancement of the optic nerve and sheath ( figure 1 ) with intrinsic lesions of one or both optic nerves , while the remainder showed high signal within the nerve without enhancement . in addition , 3 cases ( and the one who had ct ) showed intrinsic lesions within the nerve and thickening of the adjacent optic nerve sheath complex . the chiasmal lesion was associated with enhancement of the chiasm extending forward into one of the nerves . both patients with a perineuritis showed features of this , i.e. , the predominate feature was of enhancement and thickening of the sheath , not the whole complex ( figure 2 ) . the lesion was biopsied and granulomatous inflammation identified . the optic neuropathy improved with steroids . ( b , c ) optic perineuritis : t1-weighted axial and coronal mri showing enlargement of and enhancement of the optic nerve and its sheath throughout its entire length . this was the presenting feature of the disease , which was diagnosed subsequently on histologic analysis of a cervical lymph node . a minority of patients whose clinical syndrome was one of a subacute optic neuropathy resembling an optic neuritis showed additional imaging features of more widespread involvement of the nervous system by the disease : 8 patients with unilateral optic neuropathies and 2 with bilateral involvement showed high signal within the nerves , enhancement of the complex , which also involved the orbital apex ( 5 cases ) ( figure 3a ) , the floor of the middle cranial fossa ( 4 cases ) ( figure 3b ) , and the frontal lobes ( 1 case ) . ( a , b ) orbital apex mass with a progressive painful optic neuropathy and sarcoidosis of the lungs , mediastinum , and heart . ( c , d ) two cases of progressive optic neuropathy with striking meningeal enhancement and swelling to form mass lesions . despite intensive treatment with corticosteroids and immunosuppression , neither case showed a meaningful improvement in visual acuity . in general , the initial acuity was better in those with normal imaging and no enhancement than in those in whom enhancement was seen ( figure 4 ) . histogram showing the relationships between mri features at onset / diagnosis ( abscissa ) and initial ( nadir ) visual acuity then best acuity after treatment ( ordinate ) . those whose clinical syndrome was of a progressive optic neuropathy ( 6 cases ) all showed meningeal enhancing lesions that had formed masses , in each case at the intracranial portion of the orbital apex ( figure 3 ) . csf results were available in 33 cases , including 2 with mass lesions and one with perineuritis . seven ( 21% ) showed csf protein levels greater than 0.6 g / dl ( median 0.95 g / dl , range 0.791.92 ) . five ( 15% ) showed a leukocyte pleocytosis ( > 5 cells / cm ) ( median 19 , range 670 ) . twenty - eight ( 85% ) had no oligoclonal bands , and 5 showed matched bands in both csf and serum . patients were treated in an individual way in 3 different hospitals so there was a wide range of medications , dosage regimens , and treatment durations . forty - nine were treated with corticosteroids , of whom 14 commenced with iv methylprednisolone followed by oral prednisolone , while the remainder received treatment with oral steroids from the outset . the daily dose of prednisolone varied between 30 and 80 mg per day . the duration of treatment varied greatly ( 4320 weeks ) . the median ( range ) length of follow - up was 8 ( 020 ) years . the median ( range ) change in visual acuity ( defined as the best acuity following treatment , even after relapse ) was + 0.46 ( 0.01 to + 1.22 ) in bilateral cases and + 0.175 ( 0.05 to + 1.25 ) in unilateral cases . the median final acuity in these 2 groups was 0.7 ( 01.25 ) and 0.5 ( 01.25 ) . those with a subacute syndrome showed a median improvement of + 0.225 ( range 0.01 to + 1.22 ) . those in the progressive subgroup showed no improvement in visual acuity with treatment although in each case there was resolution of meningeal enhancement over time . while there was a suggestion that mri abnormalities at onset ( figure 4 ) correlated with nadir acuity and an improvement following treatment , this was not significant ( p > 0.3 ) . considering treatment in those without a progressive disease course or perineuritis , 12 were treated with iv then oral steroids ; median ( range ) acuity was 0.02 ( 01 ) and increased to 0.75 ( 0.021 ) , median change + 0.3 , and those treated with oral steroids at onset 0.1 ( 01 ) increasing to 0.75 ( 01.25 ) , and median change + 0.5 ( p = 0.77 ) . for the most part , this occurred early on ; for example , patients treated with 60 mg prednisolone developed recurrence of symptoms and signs at 40 to 20 mg , and the signs settled with an escalation of treatment . immunosuppressive agents were added in 7 cases azathioprine in 4 and mycophenolate and methotrexate in one each . the patient treated with methotrexate had had repeated relapses , which stopped on the immunosuppressive drug . one patient had an intracranial relapse of neurosarcoidosis and her optic neuropathy worsened ; she was treated with cyclophosphamide then infliximab . in those who relapsed , the improvement in acuity following treatment was + 0.25 ( 0.15 to + 1.2 ) , and relapse did not appear to influence the outcome in any way . we have seen 6 additional patients who presented with an optic neuritis and were found during investigation to have a raised serum ace ( median 100 [ range 80178 ] the clinical syndrome was not different from the others described above ; half were caribbean , half caucasian , and half were bilateral . the median ( range ) acuity in each eye was 0.1 ( 01.0 ) . in each case , the affected discs were seen to be swollen . imaging was normal in 3 and showed high signal within the affected nerve in the other 3 . csf in 4 cases showed normal protein and negative oligoclonal bands in 3 ; in the other case , matched serum and csf bands were found . in this case one was considered to have a disc granuloma and was watched without treatment ; he improved over 2 years . these cases have been followed and none has developed further or recurrent neurologic or systemic symptoms . this work forms the largest series of patients with optic neuropathy in sarcoidosis identified and each has been followed prospectively . the age range was wide but the condition was more prevalent in the first half of life . the majority of patients were women and there were equal numbers in caucasian and african or caribbean ethnic groups . according to the epidemiologic findings in access , the ethnic subgroups in london are 60% caucasian , 14% african and caribbean , and 18% south asian , and our results therefore suggest a greater prevalence of the disease in african and caribbean people who live in london . in general , the clinical syndrome resembled an optic neuritis , although in some , the disease course was more slowly progressive . pain was a less prevalent feature than is seen in demyelinating optic neuritis , and the median visual acuity at nadir was equivalent , although more were no perception of light ( table 2 ) . sixty - four percent had a unilateral involvement , 28% presented at a later time with involvement of the second eye , while synchronous bilateral involvement was less common ( 9% ) . it is noteworthy that there was evidence for concurrent intraocular inflammation in only a minority of cases . predominately this was mild ( although more severe cases had been excluded because of the uncertainty of the pathogenesis of the optic neuropathy ) . indeed , in 64% of patients , there was no history of ocular involvement at any stage of the disease to date . imaging showed clear abnormalities , which accounted for the clinical features in 75% of cases . comparison of clinical features with results of the onsg spontaneous improvement occurred ( as has been noted previously ) , although the majority of patients were treated with steroids . following this , an often substantial improvement in vision took place , even after several weeks without treatment . relapse occurred in 25% of cases , necessitating recommencement or an escalation of treatment , and in the majority , this occurred early , as steroids were withdrawn . six cases ( 11% ) had a more progressive disease course and were shown to have a more widespread neurologic involvement , each showing evidence for adjacent meningeal inflammation . visual outcome in this subgroup was poor , even though the disease process required , in general , a higher and more prolonged dose of steroids , concurrent immunosuppression , and in 2 cases , a requirement for biological agents with refractory disease . evidence for neurosarcoidosis defined as the presence of intracranial meningeal enhancement arose in 16 cases overall all of those with a progressive course and 10 with a subacute course . involvement of the optic nerve in sarcoidosis may arise through a variety of mechanisms : inflammation of the nerve itself , compression or infiltration by an inflammatory mass adjacent to the anterior visual pathway ( e.g. , the orbital apex or pituitary gland ) , secondary involvement through ischemic complications of retinal and choroidal inflammation and glaucoma , granulomas within the disc itself , meningeal inflammation within the optic nerve sheath leading to an optic perineuritis , and finally as a consequence of hydrocephalus . it was seen in 5% of one series of 202 patients with ocular disease in sarcoidosis , but only one in a series of 649 patients with systemic sarcoidosis and none in another of 285 . in the literature before 2000 , 57 cases had been published , of which 35 involved the optic nerve itself , 9 compression due to an intracerebral lesion , 6 due to hydrocephalus , and 6 due to the complications of uveitis . many noted that bilateral involvement occurred and that imaging showed enlargement of the optic nerve others noted that the acuity was lower than that seen in a demyelinating optic neuritis . one study described 24 cases with optic neuropathy : 71% had not been known previously to have systemic sarcoidosis and 42% showed previous or current intraocular inflammation . another showed that 14 of 67 patients known to have neurologic sarcoidosis had some form of optic neuropathy . we have identified a small subgroup of patients in whom the serum ace is elevated at presentation and then subsequently normalizes without relapse . on further investigation , these patients do not have sarcoidosis and on follow - up show no sign of transformation into a more widespread disease . the serum ace appears to increase in a nonspecific way and appears not to indicate a systemic granulomatous disease . it is important to identify this subgroup , which requires no additional treatment , merely close follow - up and observation . this large series of patients with optic neuropathy in conjunction with sarcoidosis has shown that more than half are not previously diagnosed with sarcoidosis , only a third have concurrent intraocular inflammation , and the clinical characteristics and in most cases the imaging features are not substantially different from that seen in a regular demyelinating optic neuritis . however , patients should be identified early because the response to treatment may lessen with time , and the identification of more widespread disease , particularly within the nervous system , will require prolonged , often high - dose treatment with steroids , immunosuppressive drugs , and modern biological therapies . modern imaging investigations including pet may make identification of these cases easier , but a high index of clinical suspicion is still important , particularly in non - caucasian patients presenting with a painless optic neuritis . once identified , we recommend that patients be treated with high - dose ( oral or iv ) corticosteroids , followed by a slow taper watching carefully for relapse as the dose is reduced . repeated relapses , particularly preventing steroid dose reduction , should be treated with an additional immunosuppressive agent . the presence of neurosarcoidosis requires more prolonged higher - dose treatment with steroids and immunosuppression from the outset , with early recourse to biological agents should the clinical and radiologic features of the disorder fail to improve quickly . further study is required to identify precisely which treatments are best and over what period of time . dr . kidd was responsible for the study design , data acquisition , analysis and interpretation , and writing the manuscript . burton served on the scientific advisory board for bayer , novartis , received travel funding from novartis , bayer , is subeditor for eastern european journal of neurology , received research support from novartis alcon .

objective : to identify and follow a series of 52 patients with optic neuropathy related to sarcoidosis.methods:prospective observational cohort study.results:the disorder was more common in women and affected a wide age range . it was proportionately more common in african and caribbean ethnic groups . two clinical subtypes were identified : the more common was a subacute optic neuropathy resembling optic neuritis ; a more slowly progressive optic neuropathy arose in the remaining 17% . sixteen ( 31% ) were bilateral . concurrent intraocular inflammation was seen in 36% . pain arose in only 27% of cases . an optic perineuritis was seen in 2 cases , and predominate involvement of the chiasm in one . mri findings showed optic nerve involvement in 75% of cases , with adjacent and more widespread inflammation in 31% . treatment with corticosteroids was helpful in those with an inflammatory optic neuropathy , but not those with mass lesions . relapse of visual signs arose in 25% of cases , necessitating an increase or escalation of treatment , but relapse was not a poor prognostic factor.conclusions:this is a large prospective study of the clinical characteristics and outcome of treatment in optic neuropathy associated with sarcoidosis . patients who experience an inflammatory optic neuropathy respond to treatment but may relapse . those with infiltrative or progressive optic neuropathies improve less well even though the inflammatory disorder responds to therapy .

older adults with type 2 diabetes ( t2 dm ) are at increased risk for developing micro- and macrovascular complications ( e.g. , retinopathy , neuropathy , nephropathy , and cardiovascular disease ) . recent studies suggested that t2 dm is associated with higher risk of cognitive dysfunction , dementia , and depression in the elderly [ 2 , 3 ] . the cause of cognitive impairment and depression in type 2 diabetes is unknown , but it is most likely multifactorial . chronic hyperglycemia , cerebral microvascular disease , severe hypoglycemia , and an increased prevalence of macrovascular disease could play significant role in pathophysiology of brain disturbances . diabetes is associated with an increased release of inflammatory cytokines , and the excess inflammation may be neurotoxic . depression , mild cognitive impairment ( mci ) , and dementia are highly prevalent chronic conditions associated with social , medical , and economic burdens . although there are several epidemiological studies that have reported the prevalence of mild cognitive impairment ( mci ) or depressive syndrome in elderly diabetic population little is known about the comorbidity of these conditions [ 5 , 6 ] . data about identification potentially modifiable risk factors for cognitive impairment and depressive syndrome in elderly people with type 2 diabetes are also poor and could be important for future diabetes health care initiatives . therefore , the aim of the current study was to ( 1 ) estimate the prevalence of mci , depressive mood , and its comorbidity in the elderly subjects with t2 dm and ( 2 ) find potential demographic , clinical , and biochemical risk factors associated with cognitive impairment and depressive syndrome . a survey was conducted among unselected 276 elders who attended to the outpatient clinic belonging to the department of internal medicine and diabetology , university hospital no . 1 in lodz , poland . we included patients aged 65 and over with diabetes type 2 diagnosed minimum 1 year earlier , subjects who had been able to understand and cooperate with study procedures . the exclusion criteria were diagnosed depression or dementia , use of possible or known cognition - impairing drugs in the previous 3 months , presence of neoplasm , constant alcohol or substance abuse , severe visual , mobility , or motor coordination impairment , history of head trauma , inflammatory or infectious brain disease , and severe neurological or psychiatric illness . written consent was obtained from the participants at the beginning of the study . the first part of visit included a morning blood draw after a 1012-hour overnight fast , blood pressure measurements , height and weight assessment , and complete physical examination . then patients had eaten a breakfast followed by capillary glucose level measuring to ensure that participants were not hypoglycemic at the time of cognitive testing . weight and height were measured to calculate body mass index ( bmi = weight / height ( kg / m ) ) . the systolic and diastolic blood pressures ( mmhg ) were measured with the patient in sitting position after 5 minutes of rest . the detailed medical history of diabetes type 2 was taken and includes diabetes duration , currant treatment for diabetes and complications if present , family history of diabetes , comorbid diseases of the patient ( hyperlipidemia , hypertension , cardiovascular disease , lung disease , cancer , and gastrointestinal tract diseases ) , and their treatment . diabetic vascular complications were assessed based on the existence of nephropathy , retinopathy , neuropathy , cardiovascular disease ( cvd ) , and stroke . hypertension was defined as either a history of hypertension or use of any antihypertensive agents , and hyperlipidemia was defined as use of any lipid lowering agent or an untreated serum ldl cholesterol level 2.6 mmol / l or / and triglycerides 1.7 mmol / l . after overnight fasting , blood samples were taken by venipuncture to assess serum levels of glycosylated hemoglobin ( hba1c ) , total cholesterol , triglycerides , low - density lipoprotein cholesterol ( ldl - c ) , and high - density lipoprotein cholesterol ( hdl - c ) . all participants underwent the following tests : the montreal cognitive assessment ( moca ) to evaluate the cognitive impairment , long version of the geriatric depression scale ( gds-30 ) to assess the depressive mood , katz basic activities of daily living ( badl ) , and lawton instrumental activities of daily living ( iadl ) questionnaires to collect information on daily activities [ 9 , 10 ] . the moca tests 8 cognitive domains , visual - spatial ability , attention , executive function , immediate memory , delayed memory , language , abstraction , calculation , and orientation , for a maximum total score of 30 . the normal moca score is 26 , with one point added if the subject has fewer than 12 years of formal education . the moca is better than other tools to detect mci in the elderly patients with type 2 diabetes . mci was diagnosed based on criteria established in the 2006 european alzheimer 's disease consortium which are currently available standard test [ 12 , 13 ] . the cut - off points for moca scores ( 19/30 ) are recommended for the diagnosis of dementia in epidemiological studies . patients with score 19 and below were excluded from the study as dementia and sent to psychiatrist for further care . the criteria mentioned above included also absence of major repercussions on daily life ( in our study , measured by katz badl and lawton iadl ) . scores range from 0 to 9 was considered as normal and from 10 to 19 was considered to have depressive symptoms . patients with score 20 and above were excluded from the study as severe depressive symptomatology and sent to psychiatrist for further diagnosis . according to criteria mentioned above 276 older subjects with diabetes type 2 were selected into groups : patients with mci , patients without mci , patients with depressive mood , patients without depressive mood , patients with mci and depressive mood , and patients without mci and depressive mood . the study was operated in accordance with the world medical association 's declaration of helsinki . each participant was assigned a number by which he / she was identified to keep his or her privacy . the approval was obtained from the independent local ethics committee of medical university of lodz . the descriptive statistics for the categorical variables were tested using the chi - square and the continuous variables using the student 's t - test or the mann - whitney u tests whenever applicable . three multivariate logistic regression models were generated to identify the influential factors associated with the presence of mci , depressive syndrome , and both . the dependent factors were set as having mci or having depressive mood or having both mci and depressive mood . the univariate analysis included the variables that were found to be significant in the models . the independent variables entered in the model at step one were gender , age , education , marital status , smoking status , physical activity , duration of diabetes , the body mass index , hba1c and lipids levels , treatment of dm 2 , micro- and macrovascular complications , previous ha or use of ha drugs , hyperlipidemia , number of comorbid conditions , and presence of hypoglycemia . odds ratios ( or ) were computed and presented with the 95% interval of confidence ( ci ) . the nonsignificant predictors ( p > 0.05 ) were subsequently removed on a backward stepwise basis . the demographic and clinical characteristics of the study group have been presented in table 1 . the prevalence of mci in elderly patients with type 2 diabetes was 31.5% ( n-87 ) , and the prevalence of subjects with depressive syndrome was 29.7% ( n-82 ) . we also evaluated 25 participants ( 9.1% ) with both mci and depressive mood . compared with the group without mci , patients with mci were significantly older and less educated and had a longer duration of diabetes ; more were diagnosed with cardiovascular disease , hypertension , hyperlipidaemia , retinopathy , nephropathy , and other comorbidities and more had a history of hypoglycemia . the level of hba1c and triglycerides was higher and hdl was lower in mci group . patients with depressive syndrome were more likely to be female , single , and current and past smokers and had less physical activity , higher bmi , and a longer duration of diabetes ; more were diagnosed with neuropathy and other comorbidities , had a history of hypoglycemia , and were treated with insulin . the level of total cholesterol and ldl cholesterol was higher in group with depressive mood . the characterization of 25 subjects with both mci and depressive syndrome is presented in table 1 . the univariate logistic regression models revealed many factors which are associated with the diagnosis of mci , depressive syndrome , and both ( table 2 ) . finally we constructed three multivariate logistic regression models to determine the predictors of mci , depressive status , and both ( table 3 ) . the first model showed that variables which increased the likelihood of having been diagnosed with mci were higher level of hba1c , presence of previous cvd , hypertension , retinopathy , increased number of comorbidities , and less years of formal education . significant predictors of having a depressive mood included female gender , single marital status , current and past smoking status , lack of physical activity , higher bmi and total cholesterol level , increased number of comorbidities , presence of history of hypoglycemia , and insulin treatment . factors associated both with mci and depressive syndrome were as follows : female gender , single marital status , past smoking status , retinopathy , presence of previous cvd or stroke , increased number of comorbidities , and insulin treatment . type 2 diabetes is a complex metabolic disease , caused by reduced insulin sensitivity and relative insulin deficiency . coexisting disorders , including hypertension , dyslipidaemia , and obesity , contribute to the severity of type 2 diabetes . the cause of diabetes - related brain dysfunction is difficult to establish because of the prevalence of several comorbidities , each of which might affect it . there are many studies which had investigating prevalence of cognitive function and depression disorder in type 2 diabetes . however none of them had described the coexistence of mci and depressive symptoms in diabetic subjects and factors associated with the presence of both these conditions . the prevalence of mci in our study was 31.5% and it was comparable to other data in the worldwide literature . one population - based study reported that the incidence of mci in diabetic subjects was around 28% . a meta - analysis of longitudinal studies showed that diabetes increased the risk of mild cognitive impairment by 21% . data from large population studies suggest that chronic hyperglycaemia is a risk for cognitive impairment in diabetes type 1 and type 2 [ 15 , 16 ] . in agreement with many other results our study reported that higher level of hba1c was a risk factor for diagnosis of mci . the pathomechanism underlying this process is complex and can include increased formation advanced glycation end products ( ages ) , oxidative stress and inflammation , accumulation of sorbitol , and development of a hyperosmotic state in nerve cells that leads to edema and impaired brain function , impaired insulin homeostasis in the brain , and insulin resistance syndrome . chronic hyperglycaemia and long duration of diabetes are both associated with increased development of cognitive dysfunction , as is the presence of vascular risk factors ( e.g. , hypertension , hypercholesterolaemia , and obesity ) and microvascular and macrovascular complications . the edinburgh type 2 diabetes study showed that general cognitive ability was significantly lower in people with moderate - to - severe diabetic retinopathy than in those without retinopathy . in our study presence of retinopathy increased a likelihood of having mci or both mci and depressive mood . the underlying mechanism of the association between hypertension and cognitive impairment in diabetics might include cerebrovascular diseases and vascular dysfunction . patients with diabetes have an increased risk in thrombotic stroke , and vascular disease has long been hypothesized to contribute to abnormalities in cognition in such patients [ 4 , 19 ] . the hypertension can cause vascular occlusion and compromise cerebral infarction , alter the structure of cerebral blood vessels , and change blood supply to the brain [ 20 , 21 ] . we had also noticed that patients with mci had higher level of triglyceride and lower level of hdl cholesterol . the hyperlipidemia was significant predictor of diagnosis with mci . in agreement with our results , other studies had showed similar association and explained that hyperlipidemia itself causes denaturation of neurons responsible for cognitive function or accelerates the progression of atherosclerosis , which leads to mci [ 22 , 23 ] . previous studies have shown that diabetic patients with lower levels of education have more long - term complications , more cardiovascular diseases , and a greater risk of mortality . in our study we noticed also a high prevalence of depressive symptoms ( 29.7% ) among elderly patients with type 2 diabetes . since depression can cause cognitive deficits , patients with diagnosed depression other studies performed in elderly general population ( but not only diabetics ) had showed also high prevalence [ 6 , 25 ] . in the cardiovascular health study cognition study approximately 41% of participants had depressive mood . in the large population study performed among japanese elders the authors had found high coexistence rate for depression and mci and concluded that individuals with mci were more likely to develop depressive symptoms and vice versa . in agreement with many other results our study revealed significant predictors of having a depressive mood : female gender , single marital status , current and past smoking status , lack of physical activity , higher bmi , and total cholesterol level [ 26 , 27 ] . some researchers also indicated that depression is highly prevalent among diabetics and the risk of depression might be increased in the presence of other comorbid conditions . we found that the strongest predictors of being diagnosed with mci and depressive symptoms were micro- and macrocomplications of diabetes like presence of retinopathy , previous cardiovascular disease , and stroke . other data had also showed that diabetic complications and mortality were greater among depressed patients . the microvascular and macrovascular complications of diabetes are augmented by the presence of depression in diabetes thus contributing to the increased mortality rate in this population . it have been hypothesized that small vessel diseases in the brain ( white matter lesions and lacunae ) affect cognitive function in older diabetics without overt dementia or symptomatic stroke . the contribution of small vessel disease to depression has been also described . pathogenesis other researchers had found that depressive symptoms were associated with increased risk of mci , and this association was independent of underlying vascular disease . the authors had explained that depressive symptoms in the absence of overt cognitive impairment may reflect the early signs of a neurodegenerative disease or that depression leads to damage in the hippocampus through a glucocorticoid cascade . although there have been many significant demographic , clinical , and biochemical risk factors associated with cognitive impairment and depressive syndrome the causative mechanisms of diabetes on brain 's complication are still unclear . systematic screening and early intervention strategies could result in the identification of high - risk patients and the improvement of the effectiveness of the treatment .

the aim of the study was to estimate the prevalence of mild cognitive impairment ( mci ) , depressive syndrome cases , and its comorbidity , and to identify predictors of these conditions . methods . 276 diabetics elders were screened for mci and depressive symptoms . detailed information of history of diabetes , and data of bmi , hba1c , and blood lipids were collected . results . the prevalence of mci was 31.5% , depressive syndrome was 29.7% , and mci with coexisting depressive mood was 9.1% . the logistic regression analysis revealed that variables which increased the likelihood of having been diagnosed with mci were : higher hba1c level , previous cvd , hypertension , retinopathy , increased number of comorbidities , and less years of formal education . significant predictors of having a depressive mood included female gender , single marital status , current and past smoking status , lack of physical activity , higher bmi and total cholesterol level , increased number of comorbidities , history of hypoglycemia , and insulin treatment . factors associated with both mci and depressive syndrome were female gender , single marital status , past smoking status , retinopathy , previous cvd or stroke , increased number of comorbidities , and insulin treatment . conclusions . depressive symptoms , mci , and its comorbidity are common in elderly subjects with type 2 diabetes . systematic screening could result in the identification of high - risk patients .

cystinuria is an autosomal recessive disorder of amino acids transport affecting the epithelial cells of the renal tubules and gastrointestinal tract . it is characterized by increased urinary excretion of cystine and the dibasic amino acids ( ornithine , lysine and arginine ) , and manifested by recurrent renal stones due to the poor solubility of cystine in the urine . there are three different types of cystinuria according to the urine phenotype in heterozygotes : type i ( mim220100 ) , non - type i ( mim600918 ) , and mixed . in type i cystinuria , heterozygotes have a normal pattern of amino acid excretion in the urine ( 1 ) . in non - type i cystinuria , heterozygotes have a variable degree of hyperexcretion of cystine and diabasic amino acids . patients with mixed type cystinuria inherit type i and non - type i alleles from either parent . two genes responsible for cystinuria have been identified . the first locus gene , slc3a1 , located on chromosome 2p-16.3 - 21 encoding rbat ( related to b amino acid transporter ) , was identified to be the cause of type i cystinuria in 1994 ( 2 ) . a second locus gene that is responsible for non - type i cystinuria was mapped on chromosome 19q12 - 13.1 . encoding for b at ( b amino acid transporter ) by linkage analysis and later identified as slc7a9 by international cystinuria consortium ( 3 ) . at present , 79 mutations in slc3a1 were reported and over 50 mutations in slc7a9 have been described so far ( 1 ) . there have been several reports of cystinuria associated with neurological manifestations ; mental retardation , muscular dystrophy , hypotonia and dwarfism , paroxysmal dyskinesia , migraine , subacute combined degeneration of the spinal cord , and cerebellar atrophy ( 4 - 6 ) . in terms of pathogenetic correlation between cystinuria and various neurological conditions , it is still undetermined whether these two conditions share a common pathogenesis or are coincidental . we report a case of non - type i cystinuria associated with neurologic symptoms and identified new missence mutation g173r in slc7a9 gene . a 13-yr - old boy was admitted due to increasing difficulty in walking and clumsiness of the movement , noticed at about 5 yr of age by his mother . he held his head at 3 months , turned over at 5 months , and sat alone at 10 months of age . his neurodevelopments were delayed since then ; standing with support at 16 months , walk alone at 21 months of age . he used his first meaningful words at 2 yr of age , make phrases at 4 yr of age , and simple sentences at 5 yr of age . at the age of 5 yr , his mother noticed that he walked with a slow and broad - based gait , and all his movements were weak . however , he did not sought medical advice . his difficulty in walking was slowly aggravated , and he also had a difficulty in academic achievement recently . physical examination revealed minor dysmorphic features : long face , macrognathia , high arched palate , and large ear . neurologic examination disclosed mild higher cortical dysfunction , dysarthria , upper limb and gait ataxia , hyperreflexia with bilateral extensor toe signs . the sensory examination disclosed no abnormalities except that vibration and position sense were impaired in lower extremities . neuropsychological evaluation revealed mild mental retardation on the k - wisc - iii : full - scale intelligence quotient , 62 ; verbal scale intelligence quotient , 79 ; performance scale intelligence quotient ; 47 . somatosensory evoked potential of posterior tibial nerve stimulation revealed no cortical waves ( p1 ) suggestive of central conduction dysfunction through the posterior column of the spinal cord . however , spine magnetic resonance imaging ( mri ) did not disclosed structural lesions in the spinal cord . chromosome analysis and fragile x mental retardation 1 ( fmr1 ) gene studies are normal . cerebrospinal fluid examination , serum and cerebrospinal fluid lactate / pyruvate level , urine organic acid analysis , and plasma amino acid analysis were normal . quantitative amino acid analysis of the urine disclosed increased excretion of cystine ( 1,345 m / g cr ) and an excess of the dibasic amino acids ; ornithine , lysine , and arginine ( fig . his mother and sister were screened by quantitative amino acid analysis of the urine and showed variable degree of cystine and dibasic amino acids excretion ( fig . mutation analysis of slc3a1 and slc7a9 genes was performed on genomic dna samples , which were extracted from peripheral blood lymphocytes of the patient , his mother , and his sister . amplification of individual exons of the two genes was performed using intronic primers obtained from the literature ( 7 ) . direct sequencing of entire coding region of slc3a1 and slc7a9 genes of the patient revealed both a g ( normal ) and a ( mutant ) at nucleotide 535 in exon 5 , resulting in the presence of a gly ( codon gga ) at amino acid position 173 and a arg ( codon aga ) in heterozygote state ( fig . we identified the novel mutation , g173r and 6 known polymorphisms ( table 1 ) . to confirm that g173r is not a polymorphism which could be found in normal control , we screened this mutation in 50 unrelated healthy korean . the patients has only one mutation , g173r in slc7a9 gene , therefore we screened for slc3a1 mutations to ensure that no other mutations were missed . g173r was also detected in the genomic dna of the patient 's mother and sister . cystinuria is an inherited disorder of cystine and dibasic amino acids ; ornithine , lysine , and arginine . this disorder is mainly expressed by the formation of cystine stones in the urinary tract . more than 80% of patients develop their first stone within the first 2 decades . on the other hand cystinuira has been occasionally described in association with a various neurological manifestations ; mental retardation , muscular dystrophy , hypotonia and dwarfism , paroxysmal dyskinesia , migraine , subacute combined degeneration of the spinal cord , and cerebellar atrophy ( 4 - 6 ) . our patient is similar to these in that there is clinical evidence of posterior column , corticospinal tract involvement , and mental retardation . pathogenetic correlation between cystinuria and various neurological conditions is still undetermined , and a relevant explanation must be needed . if the amino acid transport systems of the brain closely resemble those of the kidney and gastrointestinal tract , one might expect that cystinuria could impaired brain functions ( 8 , 9 ) . experimental results have demonstrated that transport systems for entry of cystine and cysteine into brain differ from those of kidney and intestine ( 10 ) . it is unlikely that cystinuric patients are at risk of impaired cerebral functions because of difference in the amino acid transport systems of the nervous system ( 9 ) . however , scriver et al . ( 4 ) reported that homozygous cystinuria is statistically more prevalent in patients in mental hospitals than in the general population . this finding suggest that cystinuric patients are still at higher risk for impaired cerebral function . cystinuria is currently classified into three types according to the urinary phenotype in heterozygotes : type i ( mim220100 ) , non - type i ( mim600918 ) , and mixed . in non - type i heterozygotes showed a variable degree of urinary hyperexcretion of cystine and dibasic amino acids . probands excreting more than 1,300 m / g cr are considered as phenotypically homozygote ( 11 ) . i cystinuria , there is no uptake of cystine by intestinal mucosal cells and there is no rise in serum cystine following an oral cystine load . in non - type i cystinuria , the intestinal transport of dibasic amino acids is disturbed , but not abrogated . there is some increment in plasma cystine following oral cystine load especially in patients which were previously classified as type iii cystinuria by old classification scheme ( 9 ) . in this patient , quantitative amino acid analysis disclosed increased urinary cystine excretion ( 1,345 m / g cr ) within the range of phenotypic homozygote , and an excess of the dibasic amino acids ; ornithine , lysine , and arginine ( fig . his mother and sister also showed increased excretion of cystine and diabasic amino acids in urine ( fig . . therefore , patient was diagnosed as non - type i cystinuria . despite the classification of the disease described above , a new scheme based only on genetic aspects has recently been proposed : type a , caused by mutations in both alleles of slc3a1 ; type b , caused by mutations in both alleles of slc7a9 ; the possibility of type ab , with one mutation on each of the above genes ( 1 ) . we screened the family with patient for gene mutation by sequencing of the slc7a9 and slc3a1 gene , and identified a new missence mutation g173r in slc7a9 gene in heterozygote state . the present search for mutations covered the whole coding region and neighboring intron / exon boundaries . despite careful analysis of the slc3a1 and slc7a9 genes , there are still a number of patients that lack mutations or display only heterozygosity ( 1 ) . recently , two distinct recessive contiguous gene deletion syndrome , hypotonia - cystinuria syndrome and 2p21 deletion syndrome , associated with type i cystinuria have been described ( 12 , 13 ) . in these contiguous gene deletion syndromes , various neurologic manifesations such as mental retardation , hypotonia , developmental delay , neonatal seizure , facial dysmorphism , and mitochondrial respiratory chain deficiencies this contiguous gene deletion is already known to disrupt the coding region of at least 4 genes ( slc3a1 , prepl , ppm1b and c2orf34 ) . deletion of slc3a1 gene causes cytinuria , and loss of the other 3 genes are elucidated to contribute neurologic manifestations in these contiguous gene deletion syndromes . in this pedigree , patient 's mother and sister did not showed clinical symptoms of cystinuria , but biochemical and molecular genetic data disclosed them as heterozygote of non - type i cystinuria . they also did not showed neurologic symptoms similar to those of the patient even though they had same g173r mutation in slc7a9 gene . although the neurologic manifestations of the patient probably are not to be caused by g173r mutation itself , it may be explained by unidentified other genes associated with slc7a9 gene mutation including large gene deletion such as a contiguous gene deletion syndrome which may be transmitted to the patient from paternal side . extensive genetic evaluation must be needed in the patient and his father to answer this question . , we identified a new mutation , g173r , in the exon 5 of slc7a9 gene , in the patient with non - type i cystinuria associated with neurologic manifestations which presented as mental retardation and ataxia .

cystinuria is an inherited renal and intestinal disease characterized by defective amino acids reabsorption and cystine urolithiasis . it is unusually associated with neurologic symptoms . mutations in two genes , slc3a1 and slc7a9 , have been identified in cystinuric patients . this report presents a 13-yr - old boy with cystinuria who manifested difficulty in walking , ataxia , and mental retardation . somatosensory evoked potential of posterior tibial nerve stimulation showed the central conduction dysfunction through the posterior column of spinal cord . he was diagnosed non - type i cystinuria by urinary amino acid analysis and oral cystine loading test . we screened him and his family for gene mutation by direct sequencing of slc3a1 and slc7a9 genes . in this patient , we identified new missence mutation g173r in slc7a9 gene .

kaposi sarcoma ( ks ) is a low - grade malignant vascular tumor , with a viral etiology . the typical skin lesions were first described in 1872 by moritz kaposi , a dermatologist from hungary . while in most of the world the tumour was seen only sporadically , in regions from central and southern africa it was found to be endemic , with incidence comparable to those of the cancer of the colon in resource - rich countries . a dramatic increase in the incidence of this malignancy worldwide was associated with the hiv - aids epidemic , to the extent where ks became one of the aids - defining diseases . to distinguish it from the other known forms of the disease , that is , sporadic and endemic , the etiological agent , ks herpes virus ( kshv ) , was only discovered in 1994 , as a result of the work by chang and collaborators , who isolated and characterized the agent from tissue samples of epidemic ks . a study of seroprevalence of the virus found that 15.9% of healthy children were infected , while in those living with hiv the prevalence was 28.6% . this prevalence explains why , with the advent of the hiv epidemic , the incidence of ks soared to previously unknown levels . the odds ratio for the association of hiv - kaposi sarcoma in malawian children was 93.5 ( 95% ci 26.9 to 324.4 , p < 0.001 ) . the literature on epidemic ks in african children is scarce ; the disease in children is not as well described as in adults , and the diagnosis , treatment , and prognosis are insufficiently studied . this situation can be attributed to the rarity of specialized pediatric oncology services in southern africa , where the aids epidemic generates most of the cases of ks . in south african children , an increase was seen from 1998 where one case was diagnosed to 2008 where 26 cases were documented in the south african children 's cancer registry ( see table 1 ) . ks is recognized as an aids - defining disease due to the strong association between the two entities . since more than 3 million children with hiv were living in sub - saharan africa in 2010 , the incidence of hiv in namibia and south africa is comparable , but both namibia and south africa are struggling to meet the need for arv therapy in children [ 8 , 9 ] . south africa will fall short of achieving the millennium development goals with child mortality rates increasing after the layout of these goals . by the middle of 2011 , the number of patients receiving antiretroviral ( arv ) therapy in sa had increased to 1.79 million . in 2004 there were 4200 children ( below 15 years ) on arv therapy , compared to 152 000 in 2011 . the relative low rates of arv therapy initiation in children are probably attributed to lower rates of hiv testing in paediatric population . there is a disproportionately high incidence of hiv infection in the sub - saharan region . with the increase of arv therapy availability , however , secondary malignancies are on the decline and the incidence of ks is expected to decrease as well . the incidence of childhood cancer in south african children aged 014 years ranged between 33.4 and 47.2 per million from 2003 to 2007 , while in namibia the incidence was slightly lower at 29.4 per million . many more children are diagnosed with cancer in developed countries , leading to a much higher incidence , of 176 per million , in the united states in 2005 . since the risk of developing a malignancy increases with hiv infection , it was expected that the incidence of childhood cancer would soar in countries with a high hiv burden , but this is not reflected in the available epidemiological data from south africa . there is very little research published on childhood cancers in namibia and also a paucity of studies about treatment and survival of ks in the paediatric population of sub - saharan africa . this study proposed to analyse and compare kaposi sarcoma in children in two african hospitals : tygerberg hospital ( th ) in south africa and windhoek central hospital ( wch ) in namibia . differences between the 2 groups of children diagnosed with ks were looked into in order to provide better insight into the characteristics of the disease . the variables of patient demographics , frequency of ks , comorbidities , management , survival , and hiv profile were assessed to find differences in this complex disease in the southern african region . the study evaluated all cases of ks in two hospitals in the southern african region : tygerberg hospital ( th ) situated in cape town in the western cape province of south africa and windhoek central hospital ( wch ) in the neighboring namibia , from january 1 , 1998 until december 31 , 2010 . tygerberg hospital ( th ) is situated in the western cape province of south africa , serving a population of 3.6 million people . it is the largest hospital in the western cape province of south africa and the second largest hospital in the country . the department of paediatrics has 308 beds and a dedicated hematology oncology unit where an average of 50 new patients are admitted yearly . all children diagnosed with a malignancy in the hospital drainage area are referred from peripheral hospitals and clinics to this tertiary center . a dedicated paediatric tumor registry has been in existence for 25 years in south africa , collecting data from all cases of malignancy nationwide . data is sent from all pediatric oncology units and the private sector in the country to the tumor registry . all units have ethical approval to collect and submit data to the registry through a combined national ethics agreement . the prevalence of hiv in children and adults in the western cape province was 3.8% in 2008 . the prevalence of hiv in children ( 015 years ) in south africa was estimated to be 2,5% in the same year . there is no dedicated pediatric oncology tumor registry in namibia , but the unit collects data of all new pediatric malignancies presenting to them . in 2008 , the paediatric oncology units from both hospitals decided to enter into a twinning agreement leading to close cooperation , which includes discussion of new patients , adaption of protocols for specific types of malignancies , and uniformity of treatment regimens , addressing drug availability and sharing of data . the tygerberg children 's cancer registry was used to obtain information regarding newly diagnosed ks in children treated in th . data of children treated for ks in wch were obtained from the namibian tumour registry . all children aged 015 years with a diagnosis of ks were included in the study . patient folders were reviewed and the following information was extracted : age , sex , ethnic group , hiv status , date of diagnosis of hiv disease , degree of immunosuppression ( who criteria ) , use of antiretroviral therapy , cd4 count , hiv viral load values , and date last seen or date when the patient died . hiv infection was confirmed via the enzyme - linked immunosorbent assay for hiv - specific antibodies or pcr - hiv testing in those children below the age of 18 months . the clinical picture of ks including symptoms , signs , site of disease , histological results , and radiological investigations was recorded , as well as any comorbid conditions , either at time of diagnosis or developing thereafter . uniformity of histological evaluation was assured , since samples were processed by pathologists of the national health laboratory service ( nhls ) in south africa and namibia where similar laboratory protocols are applied . the modality of treatment used ( chemotherapy , arv therapy , radiotherapy and/or surgery ) was documented . the data from patients fulfilling inclusion criteria were collected on a specifically designed data collection form and entered into a microsoft access database . treatment outcomes were analyzed using statistica software using descriptive analysis and survival analysis by way of kaplan - meier curves . approval was obtained from the relevant human ethics research committees of the 2 participating centers ( institutional review board number : irb0005239 ) . as this was a retrospective study , consent to access patient files was obtained through the custodian of the data and not through individual patient consent . the records of 21 patients were reviewed : 13 from th and 8 from wch . in the th cohort , one case was diagnosed in 1998 , with a period of no new cases for 2 years . most cases were diagnosed during 2000 to 2006 , with one more child being diagnosed in 2008 . from 2009 onwards , no new cases were seen until the end of the study period . in the wch group , thereafter one case per year was diagnosed , until 2010 when 4 new cases were identified ( figure 1 ) . the mean age at diagnosis in th was 44.2 months ( range 10 to 89 months ) and in wch 90 months ( range 43 to 141 months ) . the commonest sign of disease was skin discoloration ( 38% ; 10/26 ) with 5 cases each from th ( 38% ) and wch ( 62.5% ) . the third most common presenting symptom or sign was respiratory distress due to intrathoracic ks lesions ( 4 patients ; 15.3% ) . two patients presented with oral lesions ( 7.6% ) , 1 with stridor ( 3.8% ) , and 1 with a neck abscess ( see table 2 ) . some patients presented with a combination of symptoms and signs . the skin was the commonest site of disease with more than half of the patients presenting with skin lesions ( 13 patients ) . lymph node involvement was the second most common , followed by intraoral lesions / salivary gland involvement , intrathoracic lesions , and 1 case where the cranium was involved ( see table 3 ) . the diagnosis of ks was confirmed histologically in 76.9% of cases in th and 88% in wch by the nhls laboratory services . in the remaining cases no data was available regarding kshv status as testing was not routine practice at these hospitals during the study period . 62% ( 8 patients ) were already on arv therapy at the time of the diagnosis , while 38% of the wch group were receiving arv therapy at diagnosis . although ks is a who stage iv defining disease , 27% of children in th and 87.5% in wch were already classified as having stage iv disease prior to the diagnosis of ks . radiological changes ascribed to ks were documented in 2 cases in the tygerberg group : one with bilateral pleural effusions and one with pulmonary and pleural infiltrations . radiological changes were documented in only 1 case of disseminated ks ( right lower lobe opacification and pleural effusion ) . pulmonary tuberculosis ( ptb ) was the predominant comorbid disease present in 38.4% ( n = 5 ) of the th group . ptb was diagnosed by a combination of symptoms , chest radiographic changes , and sputum or gastric aspirate microscopy and culture . both th and wch used similar chemotherapy protocols comprising vincristine ( 1.5 mg / m ) , adriamycin ( 20 mg / m ) , and bleomycin ( 15 iu / m ) given at 3 - 4-week intervals for a total of 6 cycles . local therapy is instituted in cases of limited mucocutaneous disease , but no case in this study required control locally . paclitaxel was not used in any child in this study as no case of relapsed ks was diagnosed . the survival in wh was 62.5% and in th 46,1% . in the combined study population the children that survived ( n = 11 ) were followed up for a mean period of 14 months . in the tygerberg group 3 deaths were attributed to respiratory insufficiency ( intrapulmonary bleeds , direct intrathoracic tumor infiltration , and chylothorax ) . in the wh group children suffered disease relapse , 6 months and 29 months after the initial diagnosis . in the first case of early relapse chemotherapy was recommenced ( rescue protocol ) and the patient was alive with disease during the last followup in 2012 . there are many differences in the clinical presentation of ks in southern africa , notably in the age of the children at diagnosis , frequency between the 2 study populations , and profile of hiv between the 2 countries . similarities however exist in , for example , clinical presentation and gender distribution , which are also in keeping with the ks that is described in children in the rest of africa [ 21 , 22 ] . twenty - one cases of ks were identified in these 2 african centers over the time period from 1998 until 2010 . this could reflect a missed group of children with ks at a younger age in the namibian population , who did not benefit from screening programs to facilitate early diagnosis . the majority of patients in the tygerberg group were diagnosed before 2006 , in contrast to an increase in ks cases in wch from 2006 onwards . this difference in patterns could be explained due to a difference in access to arv therapy between the 2 study groups . another possibility is that increased cancer awareness and screening programs could have led to more cases diagnosed in namibia since 2005 as well as better referral of hiv positive children to monitor hiv associated comorbidities and access to arv therapy . the annual incidence of cancer in south african children aged 014 years is estimated to be between 33.4 and 47.2 per million from 2003 to 2007 . in a recent unpublished study in the same age group , it was found that the incidence in namibian children is 29.4 per million , although this might be an underestimation due to the lack of a formal or consistent cancer registry in namibia . these figures are much lower than those in developed countries where the incidence of cancer in children in the united states in 2005 was estimated to be 176 per million . it is likely that a large proportion of childhood cancers are not diagnosed in the sub - saharan african region . in th between 1998 and 2010 there were 547 newly diagnosed patients with a malignancy ( average of 42 malignancies / year ) . this is less than the national south african data where ks comprised almost 5% of the total pediatric malignancies in 2008 . national data showed a progressive increase in ks frequency in 1998 from 1 patient into the next decade to 28 patients . in namibia from 2003 to 2010 , 191 new pediatric malignancies were diagnosed , of which 11 cases ( 5.7% ) were ks , similar to the national south african data . this apparent discrepancy in frequency of ks has to be correlated with the incidence and management of hiv in both populations . in south africa the hiv prevalence in children below 14 years decreased from 5.6% in 2002 to 2.5% in 2008 . this considerable reduction is largely attributed to the national prevention of mother to child transmission ( pmtct ) program and the involvement of the south african government health services . the western cape is the province in south africa with the lowest prevalence of hiv infection ( in patients 2 years ) at 1.9% in 2005 . in namibia the prevalence of hiv infection in children below 14 years was estimated to be 2.1% in 2010/2011 . estimates of the hiv epidemic in namibia in 2008 have suggested that namibia has not yet reached its peak prevalence , but it did have a rapid prevalence growth period , which ended in 2001 . in 2007/2008 77% of new infections were among women aged 1524 years , which is a critical area for the country to address in prevention of mother to child transmission . inequities do exist between urban and rural areas in the country and there are also interprovincial differences in achieving arv therapy for all in need . in 2010 unicef estimated that 54% of children who were living with hiv in south africa were receiving arv therapy . in the western cape in 2005 , access to arv therapy varied from 68 to 85% between primary care clinics and tertiary hospitals . coverage of arv therapy in namibia increased rapidly from 3% ( 2003 ) to almost 60% ( 2007 ) according to national published data , but the majority of children in this study were already classified as who stage iv at the time of diagnosis . this might represent a missed group of children , which were not accessing arv therapy from an early age . in a review of early infant testing of hiv in namibia and 3 other countries , chatterjee et al . reported a very high rate of loss to followup in namibia in the postnatal population who tested hiv - pcr positive . this could in part be an explanation of the difference in age at presentation of ks between wch and th . our study found that children from wch were being diagnosed at almost double the mean age of children in th . local treatment modalities comprise intralesional vincristine , intralesional interferon alpha , radiotherapy , and surgery in selected cases . in sub - saharan africa access to treatment poses more challenges and there are relatively few studies on treatment response in this region . both groups had a significant male predominance , corresponding to results obtained by amir et al . in tanzania . as in the other african studies , this predominance also tended to be stronger in younger children . a ugandan study of 73 children found increased lymph node presentation despite higher cd4 values . in the south african literature there is a strong focus on site of presentation . a small case series of 6 south african children described the radiological findings of pulmonary ks as an initial site of presentation . other series looked at frequency of skin lesions in children with ks and found that younger children frequently have lymphadenopathy as presenting feature . this study confirmed the importance of nodal lesions as presenting feature , but cutaneous lesions were still predominant in both study groups . this study looked at the difference in clinical presentation in 2 populations also differing in hiv and arv therapy profile . of note is a possible inverse relationship between the use of arv therapy and the frequency of ks . as to the clinical expression of ks there are similarities that also correlate well with those seen in the rest of africa . ks in th and wch differs in many aspects notably age at diagnosis , where children were diagnosed at double the age in the windhoek group . the number of new cases diagnosed was found to increase over the study period in wch while a decrease was seen in the th group . a high mortality rate was found in the combined study group , probably reflecting the initial lack of availability of arv therapy . the widespread use of arv therapy currently is making major changes in the mortality rate . an opportunity exists for prospective studies to find the best modality of treatment for ks in the sub - saharan region . completeness of the study was limited by incomplete record keeping particularly chemotherapy use in the namibian population group which used the same treatment protocol as th , but it was not always strictly followed . as this is a retrospective analytical study , some missing information can be expected . followup was incomplete in many patients due to patient adherence factors , as expected in a third world setting . the small number of cases recorded overall might not be representative of the entire population . in namibia it is estimated that 75% of childhood cancer cases are not seen at the paediatric oncology unit . a few cases are seen privately and not documented , but in all likelihood the majority is missed , which would have a big influence on the frequency .

introduction . in 2010 more than 3 million children with human immunodeficiency virus ( hiv ) were living in sub - saharan africa . the aids epidemic has contributed to an abrupt increase of the frequency of kaposi sarcoma ( ks ) , especially in southern africa . there is a need to describe the clinical features of this disease , its management , and its outcome in hiv positive children in southern africa . the aim of the study is to describe two different populations with hiv and ks from two african hospitals in namibia and south africa . material and methods . a retrospective descriptive study of patients with ks who presented to tygerberg hospital ( th ) and windhoek central hospital ( wch ) from 1998 to 2010 . demographic data , hiv profile , clinical picture of ks , and survival were documented . results . the frequency of ks declined from 2006 to 2010 in th but showed an increase in the same period in wch . children in th were diagnosed at a much younger age than those in wch ( 44.2 months versus 90 months ) . cutaneous lesions were the most common clinical presenting feature , followed by lymphadenopathy , intrathoracic and oral lesions . conclusions . the clinical characteristics of ks in south africa and namibia differ in many aspects between the 2 countries .

human infection with streptococcus suis ( s. suis ) , a zoonotic pathogen , has been reported mainly in pig - rearing and pork - consuming countries . meningitis is the most - common clinical manifestation and is often associated with deafness and vestibular dysfunction . a 57-year - old man was referred to the hospital with headaches , fevers , chills , and hearing impairment . spondylodiscitis occurred after 2 weeks of antibiotic treatment , and was successfully treated with a prolonged course of antibiotics for another 4 weeks . we report the first human case of s. suis infection in korea . in patients presenting with meningitis , s. suis should be considered if the characteristic features of prominent and early hearing loss are present . streptococcus suis ( s. suis ) , a gram - positive facultative anaerobe , is a major porcine pathogen that can be transmitted to humans by close contact with pigs.1 meningitis is the most - common presentation of s. suis infection , followed by sepsis , which has a higher mortality rate , particularly for splenectomized patients . other clinical manifestations include enteritis , endocarditis , arthritis , endophthalmitis , uveitis , and spondylodiscitis.2,3 permanent hearing loss or vestibular dysfunction are commonly occurring sequelae of s. suis infection , especially in patients with meningitis.2 since the first described human case of s. suis infection in denmark in 1968,4 more than 700 cases have been reported worldwide.5 the association between human infection and contact with pigs has been noted since the discovery of the disease,6 and most of the reported human s. suis cases originate from southeast asia , which is characterized by a high density of pigs and the consumption of uncooked or lightly cooked pig products . pig farming and consuming pig products are also common in korea , but there have been no reports of human infection with s. suis . a 57-year - old , previously healthy man suffered from fevers , chills , and abdominal pain for 5 days . examination at a local hospital revealed hearing impairment and an increased c - reactive protein level . he was referred to our hospital for the treatment of a persistent fever , headaches , hearing impairment , and dizziness . the medical history was nonspecific , except that he had reared pigs for several years until 2 years previously . at the time of admission , he had a body temperature of 36.9 , a pulse rate of 68 beats / min , a blood pressure of 140/80 mm hg , and a respiration rate of 20/min . a physical examination revealed neck stiffness and signs of meningeal irritation . routine laboratory tests showed a leukocyte count of 8.310/mm with 67.4% neutrophils , a platelet count of 10010/mm , and a c - reactive protein level of 15.4 mg / dl . the cerebrospinal fluid ( csf ) had a slight turbid color , a high opening pressure ( 250 mmh2o ) , an increased leukocyte count ( 380/mm : neutrophils , 25% ; lymphocytes , 25% ; and monocytes , 50% ) , an increased protein level ( 86 mg / dl ) , and a decreased glucose level ( csf glucose , 21 mg / dl ; blood glucose , 129 mg / dl ) . gram staining , acid - fast bacillus staining , koh mount , india ink preparation , and tuberculosis polymerase chain reaction of csf were negative . the organisms , which were gram - positive cocci in chains , were catalase - negative and were identified as s. suis by the vitek 2 gram - positive card system ( biomrieux ) . the strain was sensitive to penicillin , ampicillin , ciprofloxacin , nitrofurantoin , imipenem , teicoplanin , and vancomycin . before isolating s. suis , vancomycin and cefotaxime the patient 's consciousness and meningitis symptoms gradually improved , but the hearing impairment and dizziness did not . magnetic resonance imaging ( mri ) performed 3 days after admission revealed no abnormalities of the inner ear or brain parenchyma ( fig . audiometry carried out 6 and 14 days after admission revealed bilateral moderate - to - severe sensorineural hearing loss , especially in the high - frequency range . after completion of a 14-day course of antibiotic therapy , his symptoms - with the exception of hearing loss and dizziness - were improved . a follow - up csf examination showed improved pleocytosis ( leukocyte count , 9/mm ) . the csf parameters were as follows : leukocyte count , 13/mm ; protein level , 49 mg / dl ; and glucose level , 55 mg / dl ( blood glucose , 129 mg / dl ) . ceftriaxone was given intravenously under the impression of a relapse , but this aggravated the back pain . follow - up lumbar spine mri performed 1 week later showed progression of the spondylodiscitis ( fig . 2b ) . staining and culture of a tissue specimen obtained from the lumbar inflammation area revealed only inflammation ceftriaxone was changed to ampicillin plus sulbactam , which results in the back pain gradually subsiding . the patient was discharged in a stable condition after completion of a 4-week course of antibiotic therapy . the lower - back pain had completely resolved 1 month after discharge , but his hearing loss persisted . human s. suis infection is usually - but not always - acquired through occupational or household exposure to contaminated pigs or pig meat.5 the proportion of s. suis meningitis patients with a history of pig exposure was 41% in thailand3 and 33% in vietnam.7 in addition to pigs , s. suis can be isolated from ruminants , cats , dogs , and horses.5 the patient described herein had a history of rearing pigs for several years , but he had not been in close contact with pigs or other animals for the previous 2 years . he had no other known predisposing factors , such as asplenia , diabetes mellitus , alcoholism , malignancy , or structural heart disease.2 although most reports are sporadic cases of infection , like our case , an outbreak of s. suis infection did occur in sichuan province , china between july and august 2005.8 the clinical course of patients in that outbreak was more fulminant than in previous reports , and 38 of the 215 affected patients died . meningitis is the most - common clinical manifestation of s. suis infection,5 and the presenting features of meningitis are generally similar to those of other types of bacterial pyogenic meningitis . little is known about how s. suis invades the host and how it crosses the blood - brain barrier , but sepsis is known to be the second - most - common manifestation and a major cause of s .- suis - related death.8 s. suis was identified in the csf in our patient , but not in repeated blood cultures . furthermore , he had no signs of sepsis . in a previous report of s. suis meningitis , the pathogen was isolated from the csf in 66.7% of the cases , from blood in 50% , and from both the csf and blood in 25%.3 early hearing impairment and vestibular dysfunction are characteristic features in patients with s. suis meningitis.2 hearing loss is usually sensorineural , occurring in the high - frequency range,5 and causes deafness in more than one - half of patients , as in our case.2 hemorrhagic labyrinthitis , which is evident on mri , was recently suggested as the cause of deafness in patients with s. suis meningitis.9 however , we could not find any signal changes in the cochlea or vestibule on mri in our patient . thus , we believe that hemorrhagic labyrinthitis is not a unique pathogenic characteristic of deafness in s. suis meningitis . spondylodiscitis is also one of the less - common manifestations , and some patients need surgery for its treatment.5,7,10 spondylodiscitis in our patient occurred somewhat later than did the meningitis and hearing loss , and it was successfully treated with antibiotics ( no surgery was required ) . previous work has shown that s. suis is susceptible to penicillin , ceftriaxone , and vancomycin.11 the treatment principles of s. suis meningitis are the same as those for other causes of bacterial meningitis.5 the meningitis symptoms in our patient initially responded well to cefotaxime and vancomycin . spondylodiscitis occurred after the completion of 2 weeks of treatment , and it responded not to ceftriaxone but to prolonged treatment with ampicillin plus sulbactam . some patients with s. suis meningitis described in previous reports have experienced relapse after 2 weeks of treatment with penicillin or ceftriaxone . such cases of relapse occurred within 1 week of antibiotic cessation and responded to prolonged treatment for 4 - 6 weeks.3,5,12 clinicians should be aware that the treatment recommendations may not be successful for all patients and may therefore need to be individually tailored . while s. suis can be cultured from csf or blood samples with the aid of standard microbiological techniques , it is often misidentified or even undiagnosed.13 in patients presenting with meningitis , s. suis should be considered regardless of the patient 's occupational background if the characteristic features of prominent and early hearing loss are present . moreover , increased awareness among clinicians and appropriate education of people who handle pigs or pork - derived products are needed so that they appreciate the importance of s. suis as a human pathogen .

backgroundhuman infection with streptococcus suis ( s. suis ) , a zoonotic pathogen , has been reported mainly in pig - rearing and pork - consuming countries . meningitis is the most - common clinical manifestation and is often associated with deafness and vestibular dysfunction.case reporta 57-year - old man was referred to the hospital with headaches , fevers , chills , and hearing impairment . meningitis was confirmed and s. suis was isolated from the cerebrospinal fluid . spondylodiscitis occurred after 2 weeks of antibiotic treatment , and was successfully treated with a prolonged course of antibiotics for another 4 weeks . his hearing loss was irreversible despite the improvement of other symptoms.conclusionswe report the first human case of s. suis infection in korea . in patients presenting with meningitis , s. suis should be considered if the characteristic features of prominent and early hearing loss are present .

stress is a biologically important and ubiquitous circumstance that can influence brain functions . because of the importance of both the beneficial and deleterious effects of acute and chronic stresses on cognitive functions , they have been the subject of numerous studies during the past 8 decades . the response to stress involves the activation of the hypothalamic - pituitary - adrenocortical ( hpa ) axis and its final product glucocorticoids . the hippocampus , which has the highest density of glucocorticoid receptors in the brain , is involved in the regulation of the hpa and the behavioral respon - ses to stress . this sea - horse - shaped structure is a part of a medial temporal lobe system necessary for the formation of stable declarative memory in humans ( 1 - 3 ) and spatial memory in rodents ( 4 , 5 ) . focusing on the effects of stress on the hippocampus , a large body of work has uncovered effects of chronic and short - term stress on learning and memory ( 6 - 8 ) . these effects have been accompanied by morpho - logical changes : specifically , reduced dendritic arbors after chronic stress ( 9 - 11 ) and reduced spine density after acute stress ( 12 - 14 ) have been described . numerous studies have shown that stressful experiences and/or corticosterone can dramatically impair subsequent cognitive processes ( 15 ) , such as the acquisition ( 16 , 17 ) or the retrieval of informa - tion ( 18 , 20 ) . previous findings indicated that acute administration of glucocorticoids impairs memory retrieval ( 20 - 22 ) systemic injections of stress - level doses of corticosterone administered to rats shortly before retention testing impair retrieval in tasks that rely on spatial or contextual information , including water - maze and inhibitory avoidance ( 20 ) . furthermore , stress - level glucocorticoid adminis - tration to human subjects shortly before retention testing impairs hippocampus - dependent recall of previously learned verbal material ( 23 , 24 ) on the other hand , chronic stress is known to increase levels of adrenal glucocorticoids resulting in deleterious cognitive functioning ( 25 , 26 ) . chronic restraint stress causes alterations in biochemistry , pharmacology , and morphology within the hippo - campus , especially in ca1 and ca3 ( 27 - 29 ) , and cognitive alterations have been systematically reported after repeated exposures to stress ( 29 - 34 ) we are exposed to various forms of stress daily , a common occurrence in the lives of most individuals , which have both positive and negative effects on brain function . in its acute form , stress may be a necessary adaptive mechanism for survival and with only transient changes in the brain . although , prolonged stress causes overactivation and dysre - gulation of the hpa axis thus induces detrimental changes in the brain structure and function . therefore , chronic stress is often considered a negative modulator of the cognitive functions including the learning and memory processes . exposure to long - lasting stress reduces health and increases vulnerability to mental disorders ( 35 ) . the mechanisms of impairment of cognition and synaptic plasticity following stress are largely unknown . however , based on studies in adult and older animals and humans , a glucocorticoid cascade hypothesis is suggested : there is a relationship between cumulative exposures to high glucocorticoid levels and hippocampal atrophy ( 36 ) . in an attempt to clarify the mechanism by which glucocorticoid levels correlate with hippocampal atrophy , a this hypothesis suggests that prolonged exposure to glucocorticoids diminishes the ability of neurons to resist insults , thus increasing the rate at which they are damaged ( 37 ) . several studies have shown that chronic and acute stress produce adverse effects on learning and memory ( 20 , 38 - 40 ) . since two type of stress influence plasma glucocorticoid levels that are involved in learning and memory impairments , we , therefore , hypothesized that the combination of acute and chronic stresses could exert a greater deleterious effect on learning and memory than either factor alone . in the current study , this hypothesis was tested on hippocampus - dependent learning and memory using the morris water maze tasks . male wistar rats ( weighing 20020 g ) were purchased from pasteur institute of iran . animals were kept under standard laboratory conditions with a 12-hr light / dark cycle with ad libitum food and water throughout the experiments . the animals were randomly divided into 4 groups : control , acute , chronic , and chronic + acute stress groups . to induce acute stress , animals received three footshocks ( 0.8 ma for 1 sec with a 5-sec inter - shock interval ) 30 min before the probe test . for chronic stress , rats were daily restrained for 6 hr / day ( from 9 : 00 to 15 : 00 ) for a total of 21 days in well - ventilated plexiglass tubes without access to food and water . during restraint stress , control animals were handled . in the chronic + acute stress group , rats were restrained daily for 6 hr / day for a total of 21 days in plexiglass tubes and received three footshocks 30 min before the probe test . it is important to note that this really examines the effects of heterotypic stress and that the responses to heterotypic stress may not exhibit habituation as is often observed during exposure to homotypic stress . immediately after the chronic stress , 13 of animals in chronically stressed groups and the control group were decapitated , and trunk blood was collected for corticosterone assay ( see below ) . timelines of experiments the mwm used in our study was a black circular pool ( 140 cm diameter , 45 cm high ) filled with water ( 30 cm depth ) at 242 c . an invisible escape platform ( 10 cm diameter ) was placed in the middle of one of the quadrants ( 1.5 cm below the water surface ) equidistant from the side wall and middle of the pool . the behavior of the animal ( latency , distance and swim speed ) was monitored by a video camera , mounted in the ceiling above the center of the pool , and a computerized tracking system ( ethovision ; noldus it , the netherlands ) . the training session consisted of six trials per day for 6 consecutive days , which were started from one of the four start positions , used in a random sequence identically for every rat . a trial began by placing the rat into the water facing the wall of the pool at one of the starting points . when a rat failed to escape within 60 sec , it was guided to the platform by the experimenter . once the rat reached the platform , it was allowed to remain for 30 sec and then placed in a holding cage for an inter - trial interval of 30 sec . after the last trial , each animal was towel dried and returned to its home cage . retention of the spatial training was assessed 22 days after the last training session with a 60 sec free - swim probe trial using a new starting position . the parameters measured in the probe trial were time spent in the quadrant containing the platform during training ( target quadrant ) , time spent in the quadrant opposite to the training quadrant ( opposite quadrant ) , initial latency to cross the platform location , number of crossing platform location , proximity ( the average distance from the center of the platform during the probe test ) , swimming speed , and total swim distance . to measure corticosterone , the rats were decapitated at immediately after the probe test in four experimental groups and at the end of 21 days restraint stress ( see timeline of experiments in figure 1 ) , their trunk blood was collected in tubes with edta , centrifuged ( 3500 g , 15 min ) , and the plasma was stored at -70 c until used for the corticosterone assay . corticosterone levels were determined by an elisa assay ( cayman chemical , item number 500655 ) . data is expressed as the mean standard error of the mean ( sem ) . behavioral data were analyzed by one - way and two - way ( anova ) , followed by tukey s test for post hoc comparison of the means . for the analysis of the corticosterone levels , a one - way anova with lsd post hoc test was conducted . all analyses were performed using the statistical package for the social science ( spss ) software in a pc - compatible computer . male wistar rats ( weighing 20020 g ) were purchased from pasteur institute of iran . animals were kept under standard laboratory conditions with a 12-hr light / dark cycle with ad libitum food and water throughout the experiments . the animals were randomly divided into 4 groups : control , acute , chronic , and chronic + acute stress groups . to induce acute stress , animals received three footshocks ( 0.8 ma for 1 sec with a 5-sec inter - shock interval ) 30 min before the probe test . for chronic stress , rats were daily restrained for 6 hr / day ( from 9 : 00 to 15 : 00 ) for a total of 21 days in well - ventilated plexiglass tubes without access to food and water . during restraint stress , control animals were handled . in the chronic + acute stress group , rats were restrained daily for 6 hr / day for a total of 21 days in plexiglass tubes and received three footshocks 30 min before the probe test . it is important to note that this really examines the effects of heterotypic stress and that the responses to heterotypic stress may not exhibit habituation as is often observed during exposure to homotypic stress . immediately after the chronic stress , 13 of animals in chronically stressed groups and the control group were decapitated , and trunk blood was collected for corticosterone assay ( see below ) . timelines of experiments the mwm used in our study was a black circular pool ( 140 cm diameter , 45 cm high ) filled with water ( 30 cm depth ) at 242 c . an invisible escape platform ( 10 cm diameter ) was placed in the middle of one of the quadrants ( 1.5 cm below the water surface ) equidistant from the side wall and middle of the pool . the behavior of the animal ( latency , distance and swim speed ) was monitored by a video camera , mounted in the ceiling above the center of the pool , and a computerized tracking system ( ethovision ; noldus it , the netherlands ) . the training session consisted of six trials per day for 6 consecutive days , which were started from one of the four start positions , used in a random sequence identically for every rat . a trial began by placing the rat into the water facing the wall of the pool at one of the starting points . when a rat failed to escape within 60 sec , it was guided to the platform by the experimenter . once the rat reached the platform , it was allowed to remain for 30 sec and then placed in a holding cage for an inter - trial interval of 30 sec . after the last trial , each animal was towel dried and returned to its home cage . retention of the spatial training was assessed 22 days after the last training session with a 60 sec free - swim probe trial using a new starting position . the parameters measured in the probe trial were time spent in the quadrant containing the platform during training ( target quadrant ) , time spent in the quadrant opposite to the training quadrant ( opposite quadrant ) , initial latency to cross the platform location , number of crossing platform location , proximity ( the average distance from the center of the platform during the probe test ) , swimming speed , and total swim distance . to measure corticosterone , the rats were decapitated at immediately after the probe test in four experimental groups and at the end of 21 days restraint stress ( see timeline of experiments in figure 1 ) , their trunk blood was collected in tubes with edta , centrifuged ( 3500 g , 15 min ) , and the plasma was stored at -70 c until used for the corticosterone assay . corticosterone levels were determined by an elisa assay ( cayman chemical , item number 500655 ) . data is expressed as the mean standard error of the mean ( sem ) . behavioral data were analyzed by one - way and two - way ( anova ) , followed by tukey s test for post hoc comparison of the means . for the analysis of the corticosterone levels , a one - way anova with lsd post hoc test was conducted . all analyses were performed using the statistical package for the social science ( spss ) software in a pc - compatible computer . male wistar rats ( weighing 20020 g ) were purchased from pasteur institute of iran . animals were kept under standard laboratory conditions with a 12-hr light / dark cycle with ad libitum food and water throughout the experiments . the animals were randomly divided into 4 groups : control , acute , chronic , and chronic + acute stress groups . to induce acute stress , animals received three footshocks ( 0.8 ma for 1 sec with a 5-sec inter - shock interval ) 30 min before the probe test . for chronic stress , rats were daily restrained for 6 hr / day ( from 9 : 00 to 15 : 00 ) for a total of 21 days in well - ventilated plexiglass tubes without access to food and water . during restraint stress , control animals were handled . in the chronic + acute stress group , rats were restrained daily for 6 hr / day for a total of 21 days in plexiglass tubes and received three footshocks 30 min before the probe test . it is important to note that this really examines the effects of heterotypic stress and that the responses to heterotypic stress may not exhibit habituation as is often observed during exposure to homotypic stress . immediately after the chronic stress , 13 of animals in chronically stressed groups and the control group were decapitated , and trunk blood was collected for corticosterone assay ( see below ) . the mwm used in our study was a black circular pool ( 140 cm diameter , 45 cm high ) filled with water ( 30 cm depth ) at 242 c . an invisible escape platform ( 10 cm diameter ) was placed in the middle of one of the quadrants ( 1.5 cm below the water surface ) equidistant from the side wall and middle of the pool . the behavior of the animal ( latency , distance and swim speed ) was monitored by a video camera , mounted in the ceiling above the center of the pool , and a computerized tracking system ( ethovision ; noldus it , the netherlands ) . the training session consisted of six trials per day for 6 consecutive days , which were started from one of the four start positions , used in a random sequence identically for every rat . a trial began by placing the rat into the water facing the wall of the pool at one of the starting points . when a rat failed to escape within 60 sec , it was guided to the platform by the experimenter . once the rat reached the platform , it was allowed to remain for 30 sec and then placed in a holding cage for an inter - trial interval of 30 sec . after the last trial , each animal was towel dried and returned to its home cage . retention of the spatial training was assessed 22 days after the last training session with a 60 sec free - swim probe trial using a new starting position . the parameters measured in the probe trial were time spent in the quadrant containing the platform during training ( target quadrant ) , time spent in the quadrant opposite to the training quadrant ( opposite quadrant ) , initial latency to cross the platform location , number of crossing platform location , proximity ( the average distance from the center of the platform during the probe test ) , swimming speed , and total swim distance . to measure corticosterone , the rats were decapitated at immediately after the probe test in four experimental groups and at the end of 21 days restraint stress ( see timeline of experiments in figure 1 ) , their trunk blood was collected in tubes with edta , centrifuged ( 3500 g , 15 min ) , and the plasma was stored at -70 c until used for the corticosterone assay . corticosterone levels were determined by an elisa assay ( cayman chemical , item number 500655 ) . data is expressed as the mean standard error of the mean ( sem ) . behavioral data were analyzed by one - way and two - way ( anova ) , followed by tukey s test for post hoc comparison of the means . for the analysis of the corticosterone levels , a one - way anova with lsd post hoc test was conducted . all analyses were performed using the statistical package for the social science ( spss ) software in a pc - compatible computer . distance traveled , escape latency and swimming speed data of the animals during the 6 days of training in water maze are illustrated in figure 2 a , two - way anova ( group training days ) was used to analyze the escape latencies during training . all animals were able to improve their performance as shown by the reduction of escape latencies ( figure 2a ) . statistical analysis showed significant differences in escape latency between different training days ( f5,80=58.8 , p=0.000 , n=84 ) , no significant differences among groups ( f3,80=0.364 , p= 0.779 , n=84 ) , and no interaction between days and groups ( f15,80=1.12 , p= 0.338 , n=84 ) . traveled distance , average escape latency , and swimming speed during 6-day training in mwm average escape latency ( a ) , traveled distance ( b ) , and swimming speed ( c ) across all training days . as shown the mean latency and traveled distance to find the platform declined progressively in all animals . analysis showed distance traveled and escape latency on the 6 day of training were statistically different ( p < 0.001 ) from the first day of training . data is expressed as meansem of 84 animals . * * * p < 0.001 versus the first day of training in each group data related to the distance traveled to reach the platform followed similar to the latency pattern . all groups traveled shorter distances to reach the platform as training progressed ( figure 2b ) . two - way anova analysis showed significant differences in swimming distance between different training days ( f5,80=54.07 , p=0.000 , n=84 ) , no significant differences among groups ( f3,80=0.777 , p=0.513 , n=84 ) , and no interaction between days and groups ( f15,80=1.27 , p=0.221 , n=84 ) . the analysis of swimming speed also showed no significant differences as training days progressed ( f5,80=0.449 , p=0.813 , n=84 ) among groups ( f3,80=1.067 , p= 0.373 , n=84 ) and no interaction between days and groups ( f15,80= 1.442 , p= 0.129 , n=84 ) ( figure 2c ) . the aim of this study was to examine the effect of chronic+acute stress on memory retrieval in morris water maze task . exerted stress followed learning in mwm . the probe test was performed 22 days after last acquisition trials . in probe trials , time spent in target quadrant was used to evaluate long - term memory . a two - way analysis of variance ( anova ) , with zones as repeated measure , showed significant effects of group ( f3,50=3.02 , p= 0.043 , n=54 ) and zone ( f1,50=10.73 , p=0.007 , n=54 ) , and a significant interaction between group and zone ( f3,50=3.91 , p=0.017 , n=54 ) . between - group comparisons indicated that the acute stress group ( p<0.05 ) , the chronic stress group ( p<0.01 ) and the chronic + acute stress group ( p<0.01 ) spent significantly more time in the opposite quadrant and less time in the target quadrant as compared to the control group ( figure 3a ) . also , within group comparisons revealed that the control group spent significantly more time in the target quadrant than in the opposite quadrant ( p<0.05 ) , and all stress groups spent significantly more time in the opposite quadrant than in the target quadrant ( all , p < 0.001 ) . effects of stress on retention performance in a water maze task ( a ) all stress groups spent less time in the target quadrant and spent more time in the opposite quadrant compared to the control group ( b ) the average proximity ( c ) the platform location latency ( d ) the number of crossings in the area of the original location of the platform . stress impaired memory retention , as evidenced by the fact that the stressed rats had significantly larger proximity values , more platform location latency , and lower number of crossings compared to the control group . data are expressed as meansem * p<0.05 , * * p<0.01 , * * * p<0.001 and p<0.05 , p<0.01 compared with controls # # # p < 0.001 within group comparisons figure 3b represents the average proximity to the platform . one - way anova showed a significant difference among the four groups ( f3,54=3.427 , p= 0.023 , n=58 ) . the control group had a smaller average proximity than the acute ( p<0.05 ) , chronic ( p<0.05 ) , and chronic+acute stress groups ( p<0.01 ) . one - way anova on platform location latency data indicated a significant difference among the four groups ( f3,54=3.111 , p=0.034 , n=58 ) . acute stress ( p<0.01 ) , chronic stress ( p < 0.01 ) , and chronic+acute stress groups ( p<0.05 ) showed increased escape latency as compared with controls ( figure 3c ) . one - way anova on quadrant entries also displayed a significant difference between groups ( f3,54=3.281 , p=0.027 , n=58 ) . as represented in figure 3d , the control group had more crossings as compared to acute ( p<0.01 ) , chronic ( p<0.05 ) and chronic+acute stressed groups ( p<0.05 ) . these findings show that stress could impair memory retrieval . to control the differences in the mwm per - formance , we recorded swimming speed of animals ( figure 4a ) . one - way anova showed no significant differences of swimming speed among the four groups ( f3,54=1.90 , p= 0.14 , n=58 ) . also , there were no significant differences in total distance traveled in the four groups ( f3,54=1.97 , p= 0.13 , n=58 , figure 4b ) . effect of stress on total distance traveled and swimming speed in a water maze task during the retention test total traveled distance ( a ) , and swimming speed ( b ) in the probe trial . the figures show there were no significant differences in both total traveled distance and swimming speed in the four groups . data is expressed as mean sem of 15 - 16 animals in each group there was a significant difference in cortico - sterone levels between control and chronically stressed groups after 21 days stress ( 8.373.3 ng / ml in control versus 103.98.9 ng / ml in the chronically stressed group , p<0.01 ) . also , one - way anova analysis indicated that there were significant differences in corticosterone levels between groups ( figure 5 , f3,36=10.67 , p=0.000 , n=40 ) . as was expected , post hoc tukey analysis showed acute , chronic , and chronic+acute stress groups had significantly elevated levels of corticosterone in comparison to controls at the post - probe time , respectively ( p<0.01 , p<0.001 , p<0.001 , figure 5 ) . there were no significant differences in cortico - sterone levels among acute , chronic , and chronic+acute stress groups . distance traveled , escape latency and swimming speed data of the animals during the 6 days of training in water maze are illustrated in figure 2 a , two - way anova ( group training days ) was used to analyze the escape latencies during training . all animals were able to improve their performance as shown by the reduction of escape latencies ( figure 2a ) . statistical analysis showed significant differences in escape latency between different training days ( f5,80=58.8 , p=0.000 , n=84 ) , no significant differences among groups ( f3,80=0.364 , p= 0.779 , n=84 ) , and no interaction between days and groups ( f15,80=1.12 , p= 0.338 , n=84 ) . traveled distance , average escape latency , and swimming speed during 6-day training in mwm average escape latency ( a ) , traveled distance ( b ) , and swimming speed ( c ) across all training days . as shown the mean latency and traveled distance to find the platform declined progressively in all animals . analysis showed distance traveled and escape latency on the 6 day of training were statistically different ( p < 0.001 ) from the first day of training . data is expressed as meansem of 84 animals . * * * p < 0.001 versus the first day of training in each group data related to the distance traveled to reach the platform followed similar to the latency pattern . all groups traveled shorter distances to reach the platform as training progressed ( figure 2b ) . two - way anova analysis showed significant differences in swimming distance between different training days ( f5,80=54.07 , p=0.000 , n=84 ) , no significant differences among groups ( f3,80=0.777 , p=0.513 , n=84 ) , and no interaction between days and groups ( f15,80=1.27 , p=0.221 , n=84 ) . the analysis of swimming speed also showed no significant differences as training days progressed ( f5,80=0.449 , p=0.813 , n=84 ) among groups ( f3,80=1.067 , p= 0.373 , n=84 ) and no interaction between days and groups ( f15,80= 1.442 , p= 0.129 , n=84 ) ( figure 2c ) . the aim of this study was to examine the effect of chronic+acute stress on memory retrieval in morris water maze task . exerted stress followed learning in mwm . the probe test was performed 22 days after last acquisition trials . in probe trials , time spent in target quadrant was used to evaluate long - term memory . a two - way analysis of variance ( anova ) , with zones as repeated measure , showed significant effects of group ( f3,50=3.02 , p= 0.043 , n=54 ) and zone ( f1,50=10.73 , p=0.007 , n=54 ) , and a significant interaction between group and zone ( f3,50=3.91 , p=0.017 , n=54 ) . between - group comparisons indicated that the acute stress group ( p<0.05 ) , the chronic stress group ( p<0.01 ) and the chronic + acute stress group ( p<0.01 ) spent significantly more time in the opposite quadrant and less time in the target quadrant as compared to the control group ( figure 3a ) . also , within group comparisons revealed that the control group spent significantly more time in the target quadrant than in the opposite quadrant ( p<0.05 ) , and all stress groups spent significantly more time in the opposite quadrant than in the target quadrant ( all , p < 0.001 ) . effects of stress on retention performance in a water maze task ( a ) all stress groups spent less time in the target quadrant and spent more time in the opposite quadrant compared to the control group ( b ) the average proximity ( c ) the platform location latency ( d ) the number of crossings in the area of the original location of the platform . stress impaired memory retention , as evidenced by the fact that the stressed rats had significantly larger proximity values , more platform location latency , and lower number of crossings compared to the control group . data are expressed as meansem * p<0.05 , * * p<0.01 , * * * p<0.001 and p<0.05 , p<0.01 compared with controls # # # p < 0.001 within group comparisons figure 3b represents the average proximity to the platform . one - way anova showed a significant difference among the four groups ( f3,54=3.427 , p= 0.023 , n=58 ) . the control group had a smaller average proximity than the acute ( p<0.05 ) , chronic ( p<0.05 ) , and chronic+acute stress groups ( p<0.01 ) . one - way anova on platform location latency data indicated a significant difference among the four groups ( f3,54=3.111 , p=0.034 , n=58 ) . acute stress ( p<0.01 ) , chronic stress ( p < 0.01 ) , and chronic+acute stress groups ( p<0.05 ) showed increased escape latency as compared with controls ( figure 3c ) . one - way anova on quadrant entries also displayed a significant difference between groups ( f3,54=3.281 , p=0.027 , n=58 ) . as represented in figure 3d , the control group had more crossings as compared to acute ( p<0.01 ) , chronic ( p<0.05 ) and chronic+acute stressed groups ( p<0.05 ) . these findings show that stress could impair memory retrieval . to control the differences in the mwm per - formance , we recorded swimming speed of animals ( figure 4a ) . one - way anova showed no significant differences of swimming speed among the four groups ( f3,54=1.90 , p= 0.14 , n=58 ) . also , there were no significant differences in total distance traveled in the four groups ( f3,54=1.97 , p= 0.13 , n=58 , figure 4b ) . effect of stress on total distance traveled and swimming speed in a water maze task during the retention test total traveled distance ( a ) , and swimming speed ( b ) in the probe trial . the figures show there were no significant differences in both total traveled distance and swimming speed in the four groups . data is expressed as mean sem of 15 - 16 animals in each group there was a significant difference in cortico - sterone levels between control and chronically stressed groups after 21 days stress ( 8.373.3 ng / ml in control versus 103.98.9 ng / ml in the chronically stressed group , p<0.01 ) . also , one - way anova analysis indicated that there were significant differences in corticosterone levels between groups ( figure 5 , f3,36=10.67 , p=0.000 , n=40 ) . as was expected , post hoc tukey analysis showed acute , chronic , and chronic+acute stress groups had significantly elevated levels of corticosterone in comparison to controls at the post - probe time , respectively ( p<0.01 , p<0.001 , p<0.001 , figure 5 ) . there were no significant differences in cortico - sterone levels among acute , chronic , and chronic+acute stress groups . distance traveled , escape latency and swimming speed data of the animals during the 6 days of training in water maze are illustrated in figure 2 a , two - way anova ( group training days ) was used to analyze the escape latencies during training . all animals were able to improve their performance as shown by the reduction of escape latencies ( figure 2a ) . statistical analysis showed significant differences in escape latency between different training days ( f5,80=58.8 , p=0.000 , n=84 ) , no significant differences among groups ( f3,80=0.364 , p= 0.779 , n=84 ) , and no interaction between days and groups ( f15,80=1.12 , p= 0.338 , n=84 ) . traveled distance , average escape latency , and swimming speed during 6-day training in mwm average escape latency ( a ) , traveled distance ( b ) , and swimming speed ( c ) across all training days . as shown the mean latency and traveled distance to find the platform declined progressively in all animals . analysis showed distance traveled and escape latency on the 6 day of training were statistically different ( p < 0.001 ) from the first day of training . data is expressed as meansem of 84 animals . * * * p < 0.001 versus the first day of training in each group data related to the distance traveled to reach the platform followed similar to the latency pattern . all groups traveled shorter distances to reach the platform as training progressed ( figure 2b ) . two - way anova analysis showed significant differences in swimming distance between different training days ( f5,80=54.07 , p=0.000 , n=84 ) , no significant differences among groups ( f3,80=0.777 , p=0.513 , n=84 ) , and no interaction between days and groups ( f15,80=1.27 , p=0.221 , n=84 ) . the analysis of swimming speed also showed no significant differences as training days progressed ( f5,80=0.449 , p=0.813 , n=84 ) among groups ( f3,80=1.067 , p= 0.373 , n=84 ) and no interaction between days and groups ( f15,80= 1.442 , p= 0.129 , n=84 ) ( figure 2c ) . the aim of this study was to examine the effect of chronic+acute stress on memory retrieval in morris water maze task . the probe test was performed 22 days after last acquisition trials . in probe trials , a two - way analysis of variance ( anova ) , with zones as repeated measure , showed significant effects of group ( f3,50=3.02 , p= 0.043 , n=54 ) and zone ( f1,50=10.73 , p=0.007 , n=54 ) , and a significant interaction between group and zone ( f3,50=3.91 , p=0.017 , n=54 ) . between - group comparisons indicated that the acute stress group ( p<0.05 ) , the chronic stress group ( p<0.01 ) and the chronic + acute stress group ( p<0.01 ) spent significantly more time in the opposite quadrant and less time in the target quadrant as compared to the control group ( figure 3a ) . also , within group comparisons revealed that the control group spent significantly more time in the target quadrant than in the opposite quadrant ( p<0.05 ) , and all stress groups spent significantly more time in the opposite quadrant than in the target quadrant ( all , p < 0.001 ) . effects of stress on retention performance in a water maze task ( a ) all stress groups spent less time in the target quadrant and spent more time in the opposite quadrant compared to the control group ( b ) the average proximity ( c ) the platform location latency ( d ) the number of crossings in the area of the original location of the platform . stress impaired memory retention , as evidenced by the fact that the stressed rats had significantly larger proximity values , more platform location latency , and lower number of crossings compared to the control group . data are expressed as meansem * p<0.05 , * * p<0.01 , * * * p<0.001 and p<0.05 , p<0.01 compared with controls # # # p < 0.001 within group comparisons figure 3b represents the average proximity to the platform . one - way anova showed a significant difference among the four groups ( f3,54=3.427 , p= 0.023 , n=58 ) . the control group had a smaller average proximity than the acute ( p<0.05 ) , chronic ( p<0.05 ) , and chronic+acute stress groups ( p<0.01 ) . one - way anova on platform location latency data indicated a significant difference among the four groups ( f3,54=3.111 , p=0.034 , n=58 ) . acute stress ( p<0.01 ) , chronic stress ( p < 0.01 ) , and chronic+acute stress groups ( p<0.05 ) showed increased escape latency as compared with controls ( figure 3c ) . one - way anova on quadrant entries also displayed a significant difference between groups ( f3,54=3.281 , p=0.027 , n=58 ) . as represented in figure 3d , the control group had more crossings as compared to acute ( p<0.01 ) , chronic ( p<0.05 ) and chronic+acute stressed groups ( p<0.05 ) . these findings show that stress could impair memory retrieval . to control the differences in the mwm per - formance , we recorded swimming speed of animals ( figure 4a ) . one - way anova showed no significant differences of swimming speed among the four groups ( f3,54=1.90 , p= 0.14 , n=58 ) . also , there were no significant differences in total distance traveled in the four groups ( f3,54=1.97 , p= 0.13 , n=58 , figure 4b ) . effect of stress on total distance traveled and swimming speed in a water maze task during the retention test total traveled distance ( a ) , and swimming speed ( b ) in the probe trial . the figures show there were no significant differences in both total traveled distance and swimming speed in the four groups . there was a significant difference in cortico - sterone levels between control and chronically stressed groups after 21 days stress ( 8.373.3 ng / ml in control versus 103.98.9 ng / ml in the chronically stressed group , p<0.01 ) . also , one - way anova analysis indicated that there were significant differences in corticosterone levels between groups ( figure 5 , f3,36=10.67 , p=0.000 , n=40 ) . as was expected , post hoc tukey analysis showed acute , chronic , and chronic+acute stress groups had significantly elevated levels of corticosterone in comparison to controls at the post - probe time , respectively ( p<0.01 , p<0.001 , p<0.001 , figure 5 ) . there were no significant differences in cortico - sterone levels among acute , chronic , and chronic+acute stress groups . extensive evidence from animal and human studies indicate that stress and glucocorticoids influence cognitive function ( 41 - 43 ) . previous studies have shown stress levels of glucocorticoids impair memory retrieval in animals as well as humans ( 39 , 44 - 47 ) . in this study moreover , the impairing effect of chronic stress was not affected by acute stress . during learning , all animals were able to improve their performance as shown by the reduction of escape latencies and traveled distance . statistical analysis showed no significant difference in swi - mming speed , distance traveled , and escape latency among the four groups during training days . mean plasma corticosterone levels for stressed groups , immediately following immobilization stress and immediately after probe test ( a ) : meansem corticosterone levels of 10 animals immediately following the immobilization stress on the last day of the stress treatment . ( b ) : meansem of corticosterone levels in stressed groups immediately following the probe test * * p < 0.01 , * * * p<0.001 versus controls thus , memory performance impairment in stress exposure groups was due to disruption of memory retrieval . findings indicated chronic stress impaired memory retention as the stressed animals spent significantly less time in target quadrant , had longer platform location latencies , and larger average of proximity than their non - stressed control group . also , corticosterone levels significantly increased in chronically stressed animals as measured immediately after the period of 21 days stress and after the probe test . mclaughlin et al ( 2007 ) reported the use of chronic restraint stress , with wire mesh , for 6 hr / day for 21 days as a reliable and efficient method to produce psychological stress and to cause ca3 dendritic retraction and spatial memory deficits in male sprague dawley rats ( 48 ) . in this study , we used the same method for chronic stress and found that corticosterone levels significantly increased and spatial memory retrieval reduced in the stressed rats as compared to the control groups . the results of previous studies are in agreement with our finding that chronic stress has an impairing effect on learning and memory ( 40 , 49 , 50 ) . most studies on the relationship between chronic stress and spatial memory have focused on the hippocampus because of its crucial role in spatial learning and its well - known susceptibility to stress ( 33 ) . the hippocampus plays a critical role in spatial memory s ability because damage to the hippo - campus corresponds with spatial memory impairments in both animals ( 30 , 50 , 51 ) and humans ( 52 , 54 ) . stress resulted in enhanced release of catecholamines and glucocorticoids due to the activation of sympathoadrenal and hypothalamic - pituitary - adrenal axes ( 55 ) . the impairing effects of chronic stress on learning and memory are mainly mediated via elevated levels of glucocorticoids . chronic stress may affect hippocampal function through such mechanisms as ca3 neuronal remodeling ( 56 ) , suppression of synaptic activity ( 45 , 57 ) , and altered neurogenesis ( 51 , 58 , 59 ) . these changes in the hippocampus following chronic stress or elevation of glucocorticoids have been related to changes in spatial learning and memory ( 60 ) . also , acute stress increased average proximity and escape location latency compared with the controls . the finding that plasma levels of corticosterone , from blood collected immediately after the probe trial , was elevated in the acutely stressed group compared with the control group , suggests that increased adrenocortical function induced by the stressor may have disrupted memory retrieval . there is evidence that glucocorticoids impair retention performance when rats or human subjects are tested shortly after training when circulating levels of glucocorticoids are still elevated ( 44 - 46 ) these effects on retention performance suggest that the retention impairment is directly related to increased adrenocortical function . glucocorticoids can affect retention performance by selectively influencing memory retrieval proce - sses and these effects appear to depend on gr activation . in one study ( 20 ) , rats that were given an aversive experience of footshock exposure 30 min before retention testing , failed to remember the platform location as indicated by equal swim times in both target and opposite quadrants . stress effects on memory retrieval are time - dependent and retention performance was not impaired when rats were tested either 2 min or 4 hr after footshock exposure . this time course on retention impairment correlated with plasma corticosterone levels that peak 30 min after stress exposure and return to baseline within 4 our result in this study was in agreement with findings of de quervain et al that indicated foot - shock exposure 30 min before retention testing impaired memory retention ( 20 ) . also , our study indicated chronic plus acute stress impaired memory retention as the stressed animals spent significantly less time in the target quadrant and had longer platform location latencies and average of proximity than their non - stressed control group . also , corticosterone levels increased signi - ficantly more in these animals than controls at immediately after the probe test . as mentioned before , animal studies ( 20 ) , as well as studies on healthy human volunteers ( 23 ) , have demonstrated that retrieval of learned information is susceptible to glucocorticoid - induced impairment . by administrating cortisone at different times to different groups of healthy volunteers ( one hour before word presentation to test consolidation ; and one hour before the delayed recall test to test retrieval ) , it was found that only declarative memory was affected by acute exposure to glucocorticoids ; there was no effect on acquisition or consolidation . in our study , there was no significant difference in platform location latency , average of proximity , time in target quadrant , and number of crossings between acute , chronic , and chronic + acute stress groups . furthermore , there was no significant difference in corticosterone levels between chronic stress and chronic + acute stress groups . wright et al reported novel findings that chronic stress impairs spatial memory through changes in the hpa axis and that attenuating corticosterone levels can restore spatial memory ( 61 ) . these findings are consistent with the hypothesis presented by roozendaal et al ( 2001 ) and roozendaal ( 2002 ) that elevated corticosterone levels at the time of memory assessment may mediate spatial memory impairment in rats with a compromised hippocampus ( 62 , 63 ) . our data may indicate that enhanced corticosterone secretion as a result of exposure to behavioral tasks ( probe test ) may mediate this effect . corticosterone levels in chronically stressed animals immediately after the probe test was significantly more than chronically stressed group immediately after termination of stress . one limitation of the results in this study that needs to be addressed is that corticosterone levels must be measured in all groups immediately after ending of stress and after the probe test , but we measured it only in the chronically stressed and the control groups . chronic stress and the subsequent corticosterone hypersecretion increase adrenal weight ( 64 ) , which can release additional corticosterone in response to a stressor . there is evidence that chronic stress increased total corticosterone levels in response to the y - maze procedure and down - regulated hippo - campal gr mrna expression , which may have functional consequences at the level of receptor capacity . gr reduction or changes in other brain areas may have been responsible for the enhanced corticosterone response to the y - maze procedure because gr are proposed to mediate corticosterone release during the stress response and diurnal rhythms ( 65 , 67 ) , and alterations in extrahippo - campal brain areas have been shown to be involved in enhanced corticosterone response to novel stressors after chronic stress ( 68 , 69 ) . the reduction in gr mrna and an enhanced corticosterone response to the y - maze procedure in chronically stressed rats is consistent with the hypothesis that stress - induced corticosterone elevations on the day of memory assessment impairs spatial memory in chronically stressed rats . in our study , probably alterations of rats hippocampus in both chronic and chronic plus acute stress groups and similar corticosterone levels after the probe test are related to similar memory retrieval impairment in the two groups . in this study , we expected that prior chronic stress enhances hippocampal vulnerability to acute stress , thereby further increasing spatial memory retrieval impairment induced by acute stress . however , there were no significant differences in memory retrieval impairment of three stress exposure groups , suggesting that memory impair - ment has reached a ceiling effect in the chronic plus acute stress group , and so acute stress could not further increase memory retrieval impairment . also , one possibility for interpretation of this finding might be that other adaptation systems are activated during chronic stress that can prevent acute stress effects , thus that we did not observe additive effects . on the other hand , brain regions other than the hippocampus may have been affected by chronic restraint stress paradigm and could have contributed to the memory impairment , since corticosteroid receptors are present ubiquitously in the brain and consequently , every region has the potential to be affected by chronic stress . it will be interesting to see whether chronic restraint stress also induces morphological changes in regions other than the hippocampus . in addition , the interaction of glucocorticoid with several neurotransmitter systems in the brain such as adrenergic ( 70 , 71 ) , dopaminergic ( 21 ) , and opioidergic ( 22 , 72 ) systems may influence memory retrieval . chronic + acute stress same as acute and chronic stress alone impair retrieval of spatial memory . a similar effect of three types of stressors on memory retrieval suggests that the extent of memory impairment by stress is not influenced by prior stress experience . future studies may provide a clearer indication of the exact areas of the memory process that are impaired by excess levels of glucocorticoids and the mechanisms by which it happens .

objective(s):due to the prevalence and pervasiveness of stress in modern life and exposure to both chronic and acute stresses , it is not clear whether prior exposure to chronic stress can influence the impairing effects of acute stress on memory retrieval . this issue was tested in this study.materials and methods : adult male wistar rats were randomly assigned to the following groups : control , acute , chronic , and chronic + acute stress groups . the rats were trained with six trials per day for 6 consecutive days in the water maze . following training , the rats were either kept in control conditions or exposed to chronic stress in a restrainer 6 hr / day for 21 days . on day 22 , a probe test was done to measure memory retention . time spent in target and opposite areas , platform location latency , and proximity were used as indices of memory retention . to induce acute stress , 30 min before the probe test , animals received a mild footshock.results:stressed animals spent significantly less time in the target quadrant and more time in the opposite quadrant than control animals . moreover , the stressed animals showed significantly increased platform location latency and proximity as compared with control animals . no significant differences were found in these measures among stress exposure groups . finally , both chronic and acute stress significantly increased corticosterone levels.conclusion:our results indicate that both chronic and acute stress impair memory retrieval similarly . additionally , the impairing effects of chronic stress on memory retrieval were not influenced by acute stress .

worldwide , cervical cancer is the second most common ( 12% ) cancer in women , however , in developing countries ; it is the most common cancer among women . with 528,000 new cases detected every year , cervical cancer is most notable among lower resource countries of sub - saharan africa . it is also the fourth most common cause of cancer death in women worldwide with 266,000 deaths in 2012 . india bears about one fifth of the world 's burden of cervical cancer , and > 100,000 new cases are detected every year in india , which causes 20% of all female deaths in india . according to 3 year report ( 2009 - 2011 ) of population based cancer registries in india , cancer cervix continues to be leading site of cancer in india and sikkim , and two - thirds of the cases are reported in advanced stage . the key to reducing cervical cancer morbidity and mortality is early detection and treatment of cervical precancerous lesions . among all malignant tumors , cervical cancer is the one that can be most effectively controlled by organized screening programs . an organized screening program can reduce incidence and mortality by 80% as shown in developed countries . despite being effective , most of the women in developing and under - developed countries do not have access to pap smear screening . also , both early detection and screening remains a major area of concern coupled with poor literacy and low level of awareness amongst indian women . because of low doctor patient ratio in india , nursing staff are the major workforce in rural public health centers and sub centers of india . the staff nurses are responsible , as primary gate keepers for giving information about cervical cancer and creating and conducting pap smear screening tests among rural indian women . to have a successful cancer control program , nursing staff must be aware of facts about cervical cancer and screening tests themselves . furthermore , negative attitude toward and inaccurate knowledge of cervical cancer and screening methods among health care providers especially among nurses can pose substantial barriers to cervical control program in india and other developing countries . moreover , if nurses themselves undergo screening tests regularly , they can be role models for other females in carrying out cervical cancer screening tests . it is with this in mind , the aim of this study was to assess knowledge of cancer cervix , screening methods , attitude toward and practice of pap smear screening among nursing staff working in sikkim . the population of sikkim is different in their culture , religion , customs and traditions from other states of india . the three different communities living in sikkim - the nepalis , bhutias and lepchas , constitute a homogeneous blend . there are two referral hospitals in sikkim , central referral hospital - teaching hospital of sikkim manipal institute of medical sciences ( smims ) , and the sir thutob namgyal memorial hospital at gangtok , four district hospitals , 24 primary health centers and 147 sub centers in sikkim . initially major hospitals nursing staff ( including trainee nursing staff , that is , nursing students , who were in their final year internship and yet to obtain their full registration ) were invited in a group in a big hall and explained about the nature and purpose of the research . the staff nurses who agreed to participate were given a consent form along with a predesigned , pretested , self - administered multiple response questionnaires with both closed and open ended questions . the staff nurses , who could not come in groups and those who were working small hospitals ; to them , an invitation letter along with consent form and questionnaire was sent in a sealed envelope to participate . those who agreed to participate were requested to fill the questionnaire along with consent form and send back to the principal author . the questionnaire items consisted of various basic facts about cervical cancer and screening , attitude of nursing staff toward screening , and about their practice habits . some items were modified , and some were adapted from review articles and added to the questionnaire by the principal investigator to suit the context of the study . the data collected were thoroughly screened and entered into ms - excel ( microsoft , redmond , wa , usa ) spread sheets for analysis . the data were analyzed by computer software instat graph pad version 3 ( graphpad software , la jolla , ca , usa ) . descriptive statistics chi - square tests were done , and the significance of tests was decided at p = 0.05 . a total of 396 questionnaires were distributed during the study period of which 354 nursing staff returned the questionnaire in completed form with a response rate of 89.4% . majority of the nursing staff were between 21 and 40 years of age , married , hindu and belonged to nepali community . among the participants , 28.8% were trainee staff . socio demographic characteristics of 354 nursing staffs from the state of sikkim , india who participated in the survey to the question are you aware of cancer cervix 320 of 354 nursing staff ( 90.4% ) responded that they knew about it . table 2 shows the detailed knowledge of the participants who had heard of cancer cervix . three - quarter of the nursing staff were not aware that commonest site of cancer cervix is ectocervix . although almost half of the participants were aware of cervical intraepithelial neoplasia ( cin ) , they had poor knowledge about association of cin with cancer cervix . most common risk factors identified by participants were multiple sex partners ( 52% ) , early age of coitus ( 35% ) and multiple births ( 24% ) . most common symptom responded by participants were offensive foul - smelling vaginal discharge ( 63% ) . only half of the participants knew that the treatment of cancer cervix is radiotherapy or surgery . detailed knowledge among 320 nursing staff who ever heard of cancer cervix of the 320 participants those who heard of cancer cervix , 253 ( 79.1% ) were aware of cancer cervix screening or a pap smear screening [ table 2 ] . of these 62.5% ( n = 158 ) responded that they had heard about it from doctors . other source of knowledge were medical text book 47.4% ( n = 120 ) , television , radio or internet 22.1% ( n = 56 ) , print media 14.6% ( n = 37 ) and friends , spouse and colleagues 5.1% ( n = 13 ) . table 3 presents detailed knowledge of and attitudes toward pap smear screening of the participant nursing staff . that pap smear screening should start at 21 years or 3 years after sexual debut was known by one - third nursing staff , while recommended pap smear interval of 3 years were known to 60% of the staff [ table 2 ] . unadjusted associations between participant 's socio demographic characteristics and awareness of pap smear screening and results of multivariate binary logistic regression analysis to determine factors independently associated with awareness of pap smear screening among the participants [ table 3 ] presents the unadjusted associations between participants socio - demographic characteristics and awareness of pap smear and results of multivariate analysis of selected independent variables and their associations with awareness of pap smear . age was found to be a significant predictor of awareness of pap smear screening among nursing staff . awareness was significantly more prevalent among older staff ( p < 0.007 ) and awareness increased with advancing age . married nursing staff were significantly more likely to be aware of screening , and nursing staff of christian and buddhist religion were 1.25 times and 2.03 times more likely to be aware of screening methods than hindu religion respectively . when the nursing staff was categorized by community , 85% bhutia staff were aware of pap smear that made them most aware of pap smear screening . bhutia staff was 1.96 times , and lepcha staff was 1.39 times more likely to be aware of pap smear . sexually active staffs were significantly more aware of pap smear screening [ table 3 ] . only 16.6% ( n = 42 ) of the 253 nursing staff who were aware of pap smear had actually undergone a pap test . of these three - fourth ( 76% ) staff underwent test only once , 21.4% twice , while only one ( 2.4% ) staff underwent the test thrice . thirty one percentage ( n = 13 ) nursing staff were not satisfied with the test [ table 4 ] . practice of pap smear among 253 nursing staffs who were aware of pap smear screening test among the participant staff nurses , who were aware of pap smear test 83.4% ( n = 211 ) had never undergone a test . the most common reason offered were - they felt that they were not at risk ( 41% ) . twenty five percentage ( n = 53 ) nurses offered uncomfortable pelvic examination as the reason for not undergoing the test , while 16.6% ( n = 35 ) never underwent pap smear for fear of a bad result [ table 4 ] . the findings of the present study show that knowledge of cancer cervix and pap smear screening is low among the participant nursing staff . this study also shows that awareness about cancer cervix and pap smear screening is lower among sikkimese nursing staff than staff from high income countries , but slightly higher than that had been reported from other indian states . an important finding is the low level of awareness of cancer cervix and pap smear screening among younger nursing staff . when 80% cervical cancer cases occur in developing countries including india and increasing importance has been given for controlling cervical cancer , it is worrying to find a low level of awareness about cancer cervix and pap smear screening among young nursing staff . this also implies that more importance in teaching curriculum and training programs should be incorporated about cancer cervix and screening . cancer cervix is preventable , and one of the important aspects in prevention is detection of the premalignant lesions by screening . although 49% of the participants had heard of cin , only 17% knew cin occurs in younger women , which if left untreated may progress to advanced carcinoma . in our study , 52% participants were aware that multiple sexual partner is an important risk factor for cervical cancer , while in a study of ali et al . 45% nursing staff mentioned multiple partners and other promiscuous behavior as the most common risk factor . the most common symptom of cancer cervix reported by nursing staff in our study were offensive foul - smelling discharge ( 63.8% ) , irregular bleeding ( 50.6% ) and postmenopausal bleeding ( 26% ) while in a study by nganwai et al . 77.7% and 92.4% knew that common symptoms of cervical cancer include postcoital bleeding , inter - menstrual bleeding and abnormal blood - stained vaginal discharge . about 57% knew that the cervical cancer could be treated by surgery while only 50% aware of radiotherapy as treatment options . these results are not surprising since most patients suspected or diagnosed of having cancer are referred to higher oncology centers for confirmation of diagnosis and treatment . the nurses , therefore , do not get to see the management of cervical cancer . only 79% of the staff nurses , who had known cervical cancer , was aware of pap smear . where 75% nursing staff knew that pap smear is a screening test for cervical cancer . most of the nurses ( 67% ) were not aware of recommended timing of pap smear . comparatively a larger proportion of thai nurses could correctly identify the timing was found by nganwai et al . our finding that elderly and married nurses were more likely to be aware and undergo screening was also reported by other authors . the proposed reason is that elderly and married women may receive more frequent gynecological care and the common belief that cervical cancer only occurs in older women , making them more responsive to their health . an interesting finding of the present study was that the buddhist and christian 's staff were more aware of cervical cancer screening compared to hindus . this is possibly due to the fact that the orthodox christians and buddhists may have benefitted from the health awareness programs or discussions at their religious institutions . therefore , knowledge and awareness of cervical cancer are the most important for general women , who are educated by nurses . in the literature , some studies have shown that nurses who knew very well about symptoms , risk factors , and screening methods of cancer were more likely to use cervical cancer screening methods . however , although 72% nursing staff were aware of pap smear only 16.6% ( 11.9% of total nurses participated ) had ever undergone a pap smear test . a more disappointing low level of pap smear test was reported by other authors from developing countries , 7% by shekhar et al . in indian nurses and 5.5% by udigwe among nigerian nursing staff . all the staff nurses , who underwent pap smear in our study were married . because sexual relationships outside marriage are not culturally accepted in india , unmarried sexually active women would refuse to undergo screening out of fear of the potential social stigma they would suffer if they had a test perceived to be used for married women . among the eligible respondents 83.4% , had never undergone pap smear test . most common ( 41% ) had never screened themselves because they did not feel at risk to the disease . additionally , one fourth of the eligible nursing staff offered uncomfortable pelvic examination as the reason for not undergoing test could be attributed to their routines , but this would not explain the reluctance to get screened themselves despite the availability of a free service almost any time they wished to . the belief that cervical cancer is a terminal illness and death is inevitable when cancer is detected has been identified as a barrier to participation in cancer screening , detection and treatment . it is unlikely that this staff will ever motivate others or advise them until their doubts are cleared . the lack of depth on knowledge of cervical cancer in staff nurses can be explained by their training curriculum . in the present study also , only 47% participants obtained knowledge of cervical screening from their curriculum book . until recently , cervical cancer prevention issue has been the concern of physicians . in the proposed cervical cancer prevention strategies , there is an urgent need to integrate cervical cancer prevention issues in the nurses training curriculum . the attitudes that screening has to be done only by doctors or gynaecologists needs to change . studies have shown it is possible to train nurses or other health care workers to screen for cervical cancer , and they play an important role in successful screening against cancer cervix . this calls for a re - orientation course for nurses to give more importance on cervical cancer screening methods . the strength of the present study is that it is the first to assess knowledge and practice about cervical cancer screening among nursing staff in eastern india , where incidence of cervical cancer is high . on the other hand , firstly , some questions were recognition , and some were recall type . both the recall and recognition questions have limitations . recall underestimates awareness because it is limited by memory , while recognition overestimates awareness because participants find it easy to guess . secondly , the method used for estimating the practice of pap smears were self - reported history , which may not give the actual picture due to inaccurate recall or desirability bias . knowledge about cancer cervix , screening and practice of pap smear is low among nursing staff working in indian state of sikkim . there is an urgent need for integration of cervical cancer prevention issues in the nurses existing training curriculum . nursing staff , if properly aware of cervical cancer and screening methods , can educate the women in the community and increase health - seeking behavior among eligible women .

objectives : to assess baseline knowledge of cancer cervix , screening and practice of pap smear screening among sikkimese staff nurses in india.materials and methods : between april 2012 and february 2013 , a predesigned , pretested , self -administered multiple responses questionnaire survey was conducted among staff nurses working in various hospitals of sikkim . questionnaire contained information about their demographics , knowledge of cervical cancer , its risk factors , screening methods , attitudes toward cervical cancer screening and practice of pap smear amongst themselves.results:overall , 90.4% nurses responded that they were aware of cancer cervix . three quarter of the staff nurses were not aware of commonest site being cancer cervix in women . of the 320 participants , who had heard of cancer cervix , 253 ( 79.1% ) were aware of cancer cervix screening . pap smear screening should start at 21 years or 3 years after sexual debut was known to only one - third of the nursing staff . age was found to be a significant predictor of awareness of pap smear screening among nursing staff . awareness was significantly more prevalent among older staff ( p < 0.007 ) . married nursing staffs were significantly more likely to be aware of screening methods , and nursing staff of christian and buddhist religion were 1.25 times and 2.03 times more likely to aware of screening methods than hindu religion respectively . only 16.6% nurses , who were aware of a pap smear ( 11.9% of the total sample ) , had ever undergone a pap smear test . most common reason offered for not undergoing pap smear test were , they felt they were not at risk ( 41% ) , uncomfortable pelvic examination ( 25% ) and fear of a bad result ( 16.6%).conclusion : knowledge of cancer cervix , screening and practice of pap smear was low among sikkimese nursing staff in india . there is an urgent need for re - orientation course for working nurses and integration of cervical cancer prevention issues in the nurses existing curriculum in india and other developing countries .

posterior cervical fixation by lateral mass screw insertion and rod fixationhas been frequently used to manage instability caused by trauma , extensive laminectomy state , and destruction by tumors . this technique was first described by roy - camille et al.21 ) , and several modified techniques of lateral mass screw insertion had been introduced to avoid the related complications . among them , the most common techniques for lateral mass screw insertion were the roy - camille and magerl 's techniques8,14,19 ) . based on these techniques , new insertion angle techniques for deep screw insertion were modified , and their clinical and radiological safety data had been reported previously . but , yoon 's method , one of the modified magerl 's technique , was not precisely studied in long - term follow - up yet , although it was reported safe method27 ) . for this reason , this study assessed the radiological efficacy of the cervical lateral mass screw insertion and rod fixation by yoon 's method with minimum 2 years follow - up . this retrospective analysis study reviewed total 50 consecutive patients who were treated by lateral mass screw insertion and rod fixation between january 2005 and december 2007 . the cases presented cover multiple pathologies including 14 cases of fracture / dislocation , 3 cases of spondylosis , 5 cases of ossification of posterior longitudinal ligament ( opll ) , 16 cases of cord contusion , and 12 cases of tumors . these all patients were operated on by a surgeon ( yoon sh ) , and total 323 screws of all patients were treated with the modified magerl 's method according to the modified trajectory described by yoon et al27 ) . cervical lateral mass screw insertion and rod fixation was done as usual method of posterior cervical fusion . all patients were taken a prone position with the head hold sligh- tly flexed using three - pin skull fixation . standard- midline incision was performed and all cervical lateral masses of interest were exposed to facilitate fusion and allow for accurate screw trajectory . the lateral masses were drilled and tapered using the technique of lateral screw insertion trajectories described by a modified magerl 's technique ( yoon 's method ) . the entry point wasinitiated at a point 1 mm medial and 1 mm superior to the mid - portion of the lateral mass , and preceded along a course 20 - 30 cephalic and about 20 laterals from c3 to c7 . there were 12 cases of transpedicle screw insertion on the thoracic spine due to a c6 - 7 level injury , but thoracic screws were not included in this study . arthrodesis was completed by burring the exposed bone surfaces and placing allobone graft into and around the lateral mass . after surgery , philadelphia collar was routinely applied to all patients for neck protection and motion limitation to promote the cervical fusion until 6 to 8 weeks after the operation . postoperative assessment of lateral mass screw insertion and rod fixation was performed according to the previous study17 ) . at 1 , 6 , 12 and 24 months after surgery , routine anterior - posterior and flexion - extension lateral radiographs were obtained for each visit to our clinic , andwe checked the sagittal angle of lateral mass screws on both sides and the presence of screw loosening or breakage . a thin section ct scan focused on the lateral mass fixated area during immediate and last follow - up periods , and the lateral angle of the lateral mass screws on both sides and the position of the screw tip in relation to the vertebral foramen were recorded . the screws with the tip penetrated into the vertebral foramen ( foraminal invasion ) or into the spinal canal ( cord invasion ) were defined as mal - position of screw . cervical lordosis or the instrument level angle were checked by the sagittal cobb 's angle between c2 and c7 ( fig . 1a ) or the upper and lower endplate of lateral mass fixated cercvical levels ( fig . 1b ) in the neutral cervical lateral radiographies . cervical or segmental kyphosis of instrument level were defined as more than 10 degrees of the sagittal cobb 's angle between c2 and c7 or 10 degrees up increment in sagittal cobb 's angle between the upper and lower level of lateral mass fixated levels . in this study , the criterion for fusion is the presence of bony trabecular continuity between the vertebral bodies , and non - union was defined as a visible gap , graft collapse , and motion of greater than 5 with dynamic radiographies . the age of patients at the time of operation ran ged from 16 to 80 years ( mean 52 years ) . mean follow - up period was 32 months ( range 24 months to 52 months ) . there were no statistically difference between the surgical levels , composition of cervical disease , and sizes of cervical lateral masses of lesions by the age or sex of patients . all patients were stabilized with screws and rods , and total screw cases were 323 screws implanted in 50 patients . cervical lordosis by cobb angle between c7 and c7 was 18.8110.72 in immediate post - operated status , and it significantly increased to 14.768.47 in 24 months follow - up ( p<0.001 ) . the instrument level cobb angle was not significantly change in immediate post - operated and 24 months follow - up status ( 7.014.38 , 6.992.80 , respectively ) . flexibilities checked by proximal and distal kyphosis were markedly increased from immediate operated status to follow - up . proximal kyphosis was increased from 4.002.29 to 5.913.17 ( p<0.001 ) , and distal kyphosos was increased from 3.172.89 to 4.452.84 ( p<0.001 ) . junctional kyphosis between immediate postoperative and last follow- up lateral x - ray was observed in 4.0%(2 of 50 cases ) including one case with non - fusion . unsuccessful bone fusion occurred in 4.0%(2 of 50 cases ) , but did not need further operation . among the 323 screws , screw pull - out was observed in 2 patients ( 4.0% ) with 0.9% of case by screw rate ( 3 of 323 screws ) ( fig . 2 ) , foramen invasion without definite vertebral artery injury was exhibited in 1.2%(4 of 323 screws ) ( fig . 3 ) . the radiographs revealed facet injury occurred in 2 patients ( 0.6% ) ( fig . 4 ) . one patient experienced screw pull - out , foraminal stenosis , and aggra- vation of adjacent disc protrusion simultaneously . other complications suchas pseudoarthrosis and vertebral artery injury were not observed during this follow - up . since roy - camille first introduced cervical lateral mass screw fixation in 197921 ) , many modified techniques for screw insertion , including magerl , anderson , an , and yoon have been published in an effort to reduce the risk of neurovascular and facet injury3,4,6,7,9,10,11,14,24,27 ) . previously introduced lateral mass screwingmethods have reported some neurovascular complications related to the trajectory of screw . heller et al . reported that biomechanical limitations of lateral mass screwing came from the small amount of bonypurchase availability such as screw loosening or avulsion12,13 ) . currently , many researchers have introduced a modifiedtrajectory of the lateral mass screw to supplement the limitation of this method . heller et al . reported the result of 654 screws inserted in 78 patients12 ) . complication rates as a function of the number of inserted screws which included nerve root injury , facet violations , vertebral artery injury , broken screw , screw avulsion , and screw looseningwere below 1.1% . the authors had introduced a modified lateral mass screwing method , a reduced sagittal ( 43.94.5 ) , and a lateral angle of screw(19.63.5 ) performed with a concept based on magerl 's method which resulted in a deeper depth of the screw(13.52.1mm)27 ) . the principle of this technique involved deeper insertion of screws safely while avoiding cord or root violation . this modified from magerl 's technique ascertained evidence that our consecutive lateral mass screwing method was safe and efficient . in 50 patients , there were kyphotic changes in 4.0%(2 of 50 cases ) and among 323 screws , screw pull - out ( 4% of 50 cases or 0.9% of total screws ) , foraminal invasion ( 1.2% of total screws ) , and facet injury ( 0.6% of total screws ) occurred . there was no vertebral artery injury on postoperative ct scan or screw loosening on dynamic x - ray follow up . this study showed that lateralmass fixation is a safe and reliable method of posterior stabilization with a modified trajectory of the lateral mass screw . junctional kyphosis related to the thoracolumbar spinal fusion has been considered a major drawback and has been reported in several studies5,16,25 ) . however , even though there is a long level fusion , posterior lateral mass screw fixation of the cervical spine has a lower rate of junctional problem than that of th elumbar spine . heller et al . reported that the adjacent segment degeneration after cervical lateral mass screw fixation was 3.8%13 ) , and lali described the occurrence of adjacent segment degeneration as one in 143 patients ( 0.6%)22 ) . in the current series , we found that junctional problems in cervical lateral mass fixation occurred at a rate of 4.0% . the main cause of this may be lower motion of the upper - lower lateral mass screw on the cervical spine than that on the lumbar spine or lower weight of loading on the cervical spine than on the lumbar spine . actually , the modern posterior cervical stabilizing techniques was divided into pedicle screw fixation and lateral mass screw fixation . pedicle screw fixation of the cervical spine has been considered as an effective procedure for stabilizing unstable spine injury1 ) . most researchers report a high success rate of stabilization of the cervical spine after pedicle screw fixation however , it also has the potential to injurethe nerve root or vertebral artery and there is a high mal - position rate of screw and therefore , the safety of this technique is still in doubt20,26 ) . for example , the pedicle perforation rate was 6.2%(45 out of 667 screws ) and neurovascular complications occurred in two screws . yoshimoto et al . , yukawa et al . , and kasts et al . reported the pedicle perforation rates were 11.1%(15 out of 134 screws),14.3%(59 out of 417 screws ) , and 30%(28 out of 94 screws ) , respectively , including eight screws with critical breaching2,15,28,29 ) . lateral mass screw fixation of cervical spine also provides an effective bone fusion for stabilizing unstable spinal injury compared to pedicle screw fixation . they concluded that bone fusion be- tween c1 lateral mass and c2 pedicle screws were achieved in all 319 cases ( 100% ) . liu et al . reported that 38 consecutive patients with symptomatic anterior cervical pseudarthrosis were managed successfully with posterior lateral mass screw fixation and fusion ( 100%)18 ) . in our study , kyphosis and/or unsuccessful bone fusion occurred in 6.0% ( 3 of 50 cases ) , but did not need further operation . further , the rate of screw pull - outs was 4.0%(2 of 50 cases , 3 of the 323 screws ) . lateral mass screws may be considered a safe and efficient method for stabilization because this method shows a relatively lower complication rate than pedicle screw fixation and there is not much difference between the two with respect to the rate of bone fusion or screw failure ( biomechanical stability ) . we believe that this study has not provided a newer technique for lateral mass screwing as compared to previous method . therefore , it should include follow - up on both clinical and anatomical bases using cadavers for comparison in future studies . the lateral mass screw insertion and rod fixation by the modified magerl 's method ( yoon 's method ) is a safe and reliable technique with low rate of complication related to instruments in minimum 2 years follow - up .

objectivecervical lateral mass screw insertion and rod fixation is a useful method for stabilizing the cervical disease , so various modified techniques were present . many surgeons had reported the biomechanical safety according to the screw positioning method in the cervical spine , but the modified magerl 's method ( yoon 's method ) was not well studied . so , this study assessed the radiological efficacy of the modified magerl 's method with long - term follow-up.methodsthis study retrospectively reviewed 323 lateral mass screws of 50 patients who had followed - up at least 2 years . radiologic data were analyzed as parameters of complications after operation , including kyphotic or lordotic change , bone fusion , pull - out or malposition of screw , foraminal stenosis , adjacent disc degeneration or aggravation , pseudoarthrosis , and vertebral artery injury.resultsthe mean follow - up period was 32 ( 24 to 52 ) months . there were kyphotic changes in 4.0%(2 of 50 cases ) . unsuccessful bone fusion occurred in 4.0%(2 of 50 cases ) . among the 323 screws , screw pull - out ( 4.0% . 2 of 50cases , 3 of 323 screws ) , foraminal invasion ( 1.2% of total screws ) , and facet injury ( 0.6% of total screws ) occurred.conclusionthe lateral mass screw insertion and rod fixation by the modified magerl 's method ( yoon 's method ) is a safe and reliable technique with low rate of complication related to instruments in minimum 2 years follow - up .

a 31-year - old - male , a diagnosed case of stargardt disease [ fig . 1a and b ] on follow - up since 6 years presented with gradual diminution of vision in the right eye since 2 months . his best - corrected visual acuity ( bcva ) had dropped from 20/40 , n6 to 20/400 , n36 in the right eye while left eye was 20/40 , n6 as before . fundus examination revealed the presence of a thick erm with macular edema [ fig.1c ] . 1-month later macular thickness increased to 796 . no obvious vascular pathology was noted that could account for increased thickness . the rapid increase in the thickness of the retina could have been due to an attempt at spontaneous posterior vitreous detachment which did not occur . 1d ] and restoration of the foveal contour on sd - oct [ fig.2b ] and unchanged autofluorescence pattern [ fig.2c and d ] . at 6 months follow - up , bcva had improved to 20/120 n18 . ( a and b ) colour fundus picture of the postpole of right and left eyes of case 1 showing numerous flecks in the perifoveal area and posterior pole ( c ) colour fundus picture of the right eye 6 years later showing a thick epiretinal membrane at the macula . ( d ) postoperative colour fundus picture of the right eye showing absence of epiretinal membrane with retinal pigment epithelial atrophy at the macula ( a ) preoperative optical coherence tomography ( oct ) of case 1 showing increased macular thickness secondary to thick epiretinal membrane . ( c ) preoperative autofluorescence image showing hypoautofluorescence corresponding to central area of retinal pigment epithelial atrophy . ( d ) postoperative autofluorescence image showing no change in the hypoautofluorescent area a 24-year - old myopic male , a diagnosed case of stargardt disease [ fig . 3a ] on follow - up since 3 years with a bcva of 20/400 , n36 in both eyes , presented with sudden onset metamorphopsia in the left eye of 1-week duration . though , bcva was unchanged , fundus showed an erm causing traction on the macula with associated edema and subretinal hemorrhage [ fig . choroidal neovascular membrane was ruled out on fundus fluorescein angiography ( ffa ) [ fig . the ill defined leak seen on ffa could be due to traction on the capillaries , which is one of the causes for leakage in erms on ffa . on review 1-month sd - oct revealed thick erm with a taut posterior hyaloid and increased retinal thickness of 1198 microns [ fig . 25 g pars plana vitrectomy with erm removal was done . with an uneventful postoperative period , 3d and 5b ] and a slight increase in the hypoautofluorescence due to unfolding of the retina was noted [ figs . ( a ) colour fundus picture of right eye of case 2 patient at 3 years follow - up showing beaten metal appearance at the macula . ( b ) colour fundus picture of the left eye showing thick epiretinal membrane causing macular traction and associated with subretinal hemorrhage . ( c ) colour fundus picture of the left eye 1-month later showing further increase in the macular traction with resolution of subretinal hemorrhage . ( d ) postoperative colour fundus picture at 5 month follow - up showing traction relief at macula with retinal pigment epithelial atrophy colour photograph and fundus fluorescein angiography ( ffa ) images of second patient showing the epiretinal membrane with traction and hemorrhage . ffa shows an ill defined leak from distorted capillaries , temporal to the fovea and late staining in the foveal region . no choroidal neovascular membrane can be made out ( a ) preoperative optical coherence tomography picture showing increased macular thickness due to thick epiretinal membrane . ( b ) postoperative optical coherence tomography picture showing restoration of foveal contour with foveal thinning . ( c ) preoperative autofluorescence image showing an area of hypoautofluorescence corresponding to area of retinal pigment epithelium atrophy . ( d ) postoperative autofluorescence image showing an apparent increase in the hypoautofluorescence due to unfolding of the retina a 31-year - old - male , a diagnosed case of stargardt disease [ fig . 1a and b ] on follow - up since 6 years presented with gradual diminution of vision in the right eye since 2 months . his best - corrected visual acuity ( bcva ) had dropped from 20/40 , n6 to 20/400 , n36 in the right eye while left eye was 20/40 , n6 as before . fundus examination revealed the presence of a thick erm with macular edema [ fig.1c ] . 1-month later macular thickness increased to 796 . no obvious vascular pathology was noted that could account for increased thickness . the rapid increase in the thickness of the retina could have been due to an attempt at spontaneous posterior vitreous detachment which did not occur . 1d ] and restoration of the foveal contour on sd - oct [ fig.2b ] and unchanged autofluorescence pattern [ fig.2c and d ] . at 6 months follow - up , bcva had improved to 20/120 n18 . ( a and b ) colour fundus picture of the postpole of right and left eyes of case 1 showing numerous flecks in the perifoveal area and posterior pole ( c ) colour fundus picture of the right eye 6 years later showing a thick epiretinal membrane at the macula . ( d ) postoperative colour fundus picture of the right eye showing absence of epiretinal membrane with retinal pigment epithelial atrophy at the macula ( a ) preoperative optical coherence tomography ( oct ) of case 1 showing increased macular thickness secondary to thick epiretinal membrane . ( c ) preoperative autofluorescence image showing hypoautofluorescence corresponding to central area of retinal pigment epithelial atrophy . a 24-year - old myopic male , a diagnosed case of stargardt disease [ fig . 3a ] on follow - up since 3 years with a bcva of 20/400 , n36 in both eyes , presented with sudden onset metamorphopsia in the left eye of 1-week duration . though , bcva was unchanged , fundus showed an erm causing traction on the macula with associated edema and subretinal hemorrhage [ fig . choroidal neovascular membrane was ruled out on fundus fluorescein angiography ( ffa ) [ fig . 4 ] . the ill defined leak seen on ffa could be due to traction on the capillaries , which is one of the causes for leakage in erms on ffa . on review 1-month sd - oct revealed thick erm with a taut posterior hyaloid and increased retinal thickness of 1198 microns [ fig . 25 g pars plana vitrectomy with erm removal was done . with an uneventful postoperative period , 3d and 5b ] and a slight increase in the hypoautofluorescence due to unfolding of the retina was noted [ figs . ( a ) colour fundus picture of right eye of case 2 patient at 3 years follow - up showing beaten metal appearance at the macula . ( b ) colour fundus picture of the left eye showing thick epiretinal membrane causing macular traction and associated with subretinal hemorrhage . ( c ) colour fundus picture of the left eye 1-month later showing further increase in the macular traction with resolution of subretinal hemorrhage . ( d ) postoperative colour fundus picture at 5 month follow - up showing traction relief at macula with retinal pigment epithelial atrophy colour photograph and fundus fluorescein angiography ( ffa ) images of second patient showing the epiretinal membrane with traction and hemorrhage . ffa shows an ill defined leak from distorted capillaries , temporal to the fovea and late staining in the foveal region . no choroidal neovascular membrane can be made out ( a ) preoperative optical coherence tomography picture showing increased macular thickness due to thick epiretinal membrane . ( b ) postoperative optical coherence tomography picture showing restoration of foveal contour with foveal thinning . ( c ) preoperative autofluorescence image showing an area of hypoautofluorescence corresponding to area of retinal pigment epithelium atrophy . ( d ) postoperative autofluorescence image showing an apparent increase in the hypoautofluorescence due to unfolding of the retina reports of spontaneous separation of idiopathic erms in the young as well as in a child with stargardt disease suggest observation rather than surgical intervention . though a compromised retinal structure hampers the expected results of surgical intervention , some visual improvement can be expected if the deterioration is mainly due to the erm . as both our patients were on regular follow - up , reduction in visual acuity corresponded to the development of a thick erm , which further progressed during the short period of observation . both on oct and intraoperatively case 1 had always had better vision than case 2 , and this patient had a significant improvement following surgery . subretinal fibrosis and hyperlipofuscinosis have been reported previously in these patients following blunt ocular trauma with resultant visual loss suggesting the need for protection from even minor ocular trauma . in our patients , there was no apparent surgical damage to the retinal pigment epithelium , which is depicted by the preoperative and postoperative autofluorescence images . in the second patient , an apparent increase in the area of hypoautofluorescence seen temporally could have been due to unfolding of the retina following release of traction caused by the erm . epiretinal membranes have been described in stargardt disease and observation rather than surgery has been one option in the past , with isolated reports suggesting limited improvement after surgery . our experience shows that visual improvement following surgery is possible in these eyes without compromise or worsening of the preexisting pathology .

epiretinal membranes ( erms ) in stargardt disease have been known to undergo spontaneous separation in children . results of surgical intervention in adult patients with stargardt disease have rarely been reported . a retrospective review of results of surgical intervention for erm causing visual impairment in two adult patients of stargardt disease was carried out . both patients developed erm in one eye during their follow - up period with the resultant drop in their preexisting visual acuity . postsurgery , restoration of foveal contour with some improvement in visual acuity was observed in both patients . no adverse effect of surgery was noted .

in spite of the efficacy of standard androgen deprivation therapy for metastasis tumors of prostate gland , almost all of the patients progress with their disease . in the last few years the prostate tumors progress although the androgen blockade is defined as castration - resistant prostate cancer despite obvious efforts for revealing the reasons for hormonal resistance after androgen deprivation , the treatment remains a challenge . the duration of remission after treatment of hormone - resistant tumors of prostate gland with secondary hormonal manipulation is short and there is no serious impact on survival . up to 1990 the results after chemotherapy for hormone - resistant tumors of prostate gland are disappointing . later a canadian researcher drew our attention to the potentialities of the combination mitoxantrone with prednisone where an improvement in pain and quality of life was indicated but there was no effect on the survival [ 3 , 4 ] . in 2004 two trials registered and prolonged survival after using docetaxel . the overall survival with docetaxel was 18,9 months against 16,5 months with mitoxantrone and prednisolone . there were registered improvements also in toxicity and quality of life . at this moment the treatment with docetaxel in combination with prednisone is accepted as standard of care for metastatic castration - resistant prostate tumors [ 5 , 6 ] . in a process of investigation there are some new chemotherapeutic agents like epothilones and satraplatin [ 7 , 8 ] . in standard chemotherapy used for treatment of diverse tumors , we usually use the maximum tolerated dose ( mdt ) as we intend to achieve better results . despite many clinical researches with different combinations of chemotherapeutics , the progress in effectiveness is very modest and there are many toxic effects . on the other hand prolonged intervals between the applications of the chemotherapeutics are factors which contribute to chemoresistance . in searching other possibilities for reducing the toxic effects of chemotherapy without decreasing its antitumor efficacy in the last few years intensive researches have been made on chemotherapy in low doses and in short intervals between the applications the so - called metronomic chemotherapy . preclinical research and small clinical research show serious possibilities for lowering the toxic effects without diminishing the efficacy [ 9 , 10 ] . usage of low doses chemotherapeutics with increased frequency suggests another method called insulin - potentiated therapy ( ipt ) or now called insulin - potentiated targeted low - dose therapy ( iptld ) , where standard schemes of chemotherapy are used in combination with intravenous insulin , 10 times lower doses of chemotherapeutics , and short intervals between the applications . this treatment has very low toxicity and our personal experience shows that efficiency is not deferring from the standard chemotherapy . according to our previous experience in implementation of iptld in different tumors including castration resistant prostate , tumors we conducted a research for new possibilities where our main target was to improve the quality of life [ 12 , 13 ] . this study is focusing on the potential of iptld in combination with hormone therapy for treatment of patients with castration - resistant prostate tumors . between april 2006 and may 2011 a total of 406 patients with diverse tumors were treated with iptld and 21 out of them were with prostate cancer . sixteen of them were with advanced prostate cancer ( stage iii - stage iv and nodal , bone , or visceral secondary ) and cynically apparent hormonal independence entered the study . they were divided into two groups : group a : 8 patients treated with epirubicin , vinblastine , and cyclophosphamide in combination with lhrh agonist ( goserelin depot 3,6 mg ) . in group b another 8 patients were treated with docetaxel in combination with lhrh agonist ( goserelin depot 3,6 mg ) . before the treatment all patients gave informed content . the main requirement for eligibility was objective and subjective data for progression of the disease after surgical castration , androgenic therapy , and androgen withdrawal . in table 1 pretreatment evaluation of the patients includes history of the disease , physical exam , karnofsky performance status ( kps ) , subjective condition evaluated with beretta , fbc , biochemistry including af , prostate - specific antigen ( psa ) , urine analysis , chest radiographs , ultrasound , bone scan , and ct . control lab exams include tumor markers after 6th ipt application and after every fourth ipt after that and control exams of bone scan and ct after the 10th application and after that on 3- or 6-month intervals from the beginning . every month patients complete berettas questionnaire for their subjective state and we note only sections a and b in table 4 . ( 0,4 ui / kg . ) in combination with cyclophosphamide ( 0,100,15 g / m)/epirubicin ( 3 mg / m ) ; vinblastine ( 0,5 mg / m ) i.v . in 8 patients . ( 0,4 ui / kg . ) in combination with docetaxel ( 3,6 mg / m ) i.v . in 8 patients . length of one scan treatment : 6 applications in every 5-day interval , then sustaining treatment in gradual increasing intervals ( four applications in 10 days , 2 , 3 , and more weeks ) . in the interval , have dexamethason , 20 mg ; cyclophosphamide , 50 mg p.o . ; doxycyclin , 100 mg ; legalon , 3 140 mg ; celebrex , 2 7,5 mg ; antioxidants , and ozone therapy . maintaining treatment consists of no more than 24 ipt applications . hormone therapysix application ( one per month ) with lhrh agonist ( goserelin depot 3 , 6 mg ) in every 28 days for six months . six application ( one per month ) with lhrh agonist ( goserelin depot 3 , 6 mg ) in every 28 days for six months . objective responseto treatment was assessed as per psa levels and bone scan results . a complete response required normal psa levels and normal bone scan . a partial response required a decrease of > 50% of the baseline value of psa and decrease in the measurable lesions seen on the bone scan and also the absence of signs of disease progression . stabilization of disease was defined as decrease of < 50% of the baseline levels of psa , an absence of increase of lesions seen on the bone scan . patients were considered to have disease progression if they showed an increase in 2 successive psa level measurements and/or appearance of new neoplastic lesions . to treatment was assessed as per psa levels and bone scan results . a complete response required normal psa levels and normal bone scan . a partial response required a decrease of > 50% of the baseline value of psa and decrease in the measurable lesions seen on the bone scan and also the absence of signs of disease progression . stabilization of disease was defined as decrease of < 50% of the baseline levels of psa , an absence of increase of lesions seen on the bone scan . patients were considered to have disease progression if they showed an increase in 2 successive psa level measurements and/or appearance of new neoplastic lesions . additional parametersfor evaluation the effect of the treatment includes mean remission duration and median overall survival . for evaluation the effect of the treatment the toxicitythe toxicity of treatment is recorded according to the criteria of world health organization ( who ) . the toxicity of treatment is recorded according to the criteria of world health organization ( who ) . the mean follow - up period was 7,6 months ( 2 to 23 months ) . after 6th ipt application 3 patients of group a and 4 patients of group b discontinued the treatment because of social reasons . in group common side effects include light weakness and sleepiness on the day of the procedure . two ( 16 ) patients complained of nausea and vomiting a few hours after the procedure but this disappears on the next day . blood transfusion was necessary before treatment in 3 and during it in 2 patients with low starting hemoglobin . the results after 6th ipt concerning psa criteria for both groups show partial effect in 8 of 16 p. ( 50% ) , stabilization in 4 out of 16 ( 25% ) , and progression in 4 out of 16 ( 25% ) . the results after 10th ipt show complete response in 3 of 9 ( 33% ) , partial response in 1 of 9 ( 11% ) , stabilization in 2 of 9 ( 22% ) and progression in 3 of 9 ( 33% ) . in table 2 are presented the results of treatment according to psa criteria after the end of the core 6 weekly course of ipt . in table 3 the mean values of psa in those who responded to the treatment decrease from 256,7 to 94,6 ng / ml in group a and from 2215,3 ng / ml to 956,2 ng / ml in group b after 6th ( course of treatment ) ipt . after the 10th course of treatment the values were , respectively , 36,3 nag / ml in group a and 4,9 nag / ml in group b. the mean duration of the remission in group a is 7,6 months ( range 318 months ) and for group b it is10 months ( range 318 months ) . the median survival for all treated patients is 11,7 months ( range 330 months ) . subjective improvement is observed in all patients in both groups , average from 24,2 points before treatment to 8,9 points after 6th iptld . more than 50% decrease in symptomatic index is observed in 7 ( 8) from group a and 3 ( 8) from group b. in figures 1 and 2 are presented the results from the subjective improvement in both groups after 6th ipt . the method of insulin potentiation therapy was empirically invented in 1930 from mexican doctor d. perez garsia , who was applying it successfully for the treatment of chronic and oncology diseases for 41 years . lately his practice is continued by his son and grandson who now gathers more and more popularity and the method is used in practice from increasing number of doctors ( more then 400 ) and clinics all around the world . the theoretical conception for the mechanisms of action of ipt is explained in two publications of ayre s. g. , d. perez garcia y bellon , and d. perez garcia , in 1986 and 2000 [ 10 , 16 ] . same authors in 1990 submitted in european journal of cancer one case from their practice demonstrating complete tumor regression of ductal breast carcinoma in 32-year woman explaining the mechanisms and method that they had use . in 2003 published in cancer chemotherapy and pharmacology the first clinical research investigating the effect of the combination of insulin and methotrexate in patients with breast cancer . basic role for the efficacy of the method plays the usage of hormone insulin in diabetics . despite of the diversity of the actions of the insulin in the human body many investigations were conducted and they show that besides its effect in lowering the blood sugar insulin has serious effect in the whole metabolism : increases the permeability of cell membrane , influences the metabolic processes in human body with the increase of the regenerating processes , facilitates the transport of intra and extra cellular liquids which helps the organism to eliminate the toxic products , have other endocrine effects : directly stimulates suprarenal gland to produce epinephrine and glucocorticoid hormones and stimulates acth secretion . various researches show that insulin hormone has a significant impact also on the tumor cells themselves . as a result of the current knowledge of the effect of insulin on biology of tumor cells increased permeability after the insulin effect on the cellular membrane results in increased intracellular quantity of antitumor agents . insulin influences the intracellular metabolism of the tumor cell , which leads to increase of the number of cells in phase s , where they are with highly sensitive to specific chemotherapeutics . the increased number of insulin receptors on the tumor cell , in comparison to the normal one , allows the before mentioned 2 factors to act predominantly . despite the serious achievements in the field of revealing the intimate mechanisms of the action of the insulin hormone in the human body we are still far away from getting answers to all our questions . future researches will probably reveal more details regarding the effective clinical usage of insulin . having in mind the serious problems of the treatment of hormone - resistant prostate tumors and taking into consideration previous experimental researches , we conducted a clinical research on the direct inhibitory effect of lhrh analog triptorelin acetate depot on the cellular proliferation . the results confirmed local inhibitory action from the experimental researches and showed that they can successfully be combined with chemotherapy [ 19 , 20 ] . in search of a new method for lowering the toxicity of the chemotherapy for treating oncological diseases , we began in 2006 to implement iptld combined with lhrh agonist gosrelin depot 3,6 mg . among the all treated patients 21 are with advanced prostate tumors , 16 are hormone - resistant , and those 16 are divided according to the used chemotherapeutics into 2 groups group a and group b. after the first 6 ipt applications overall ( groups a and b ) response to treatment on psa criteria shows partial effect and stabilization in 12 of 16 ( 75% ) patients . after the 10th iptld application or 3 months after starting treatment , complete response , partial response , and stabilization were observed in 4 of 9 ( 66.6% ) , while in 3 of 9 ( 33.3% ) was registered complete effect . symptomatic improvement of the treatment after the sixth iptld is observed in all patients in both groups . more than 50% improvement in symptomatic index was reported in 10 of 16 ( 62.5% ) for the same period . the mean duration of remission in patients with complete response in both groups was 17 ( range 1518 months ) months . in one patient treated with docetaxel , after a 15-month remission that progressed we used again iptld with cyclophosphamide , epirubicin , and vinblastin in combination with i lhrh agonist . after the sixth application the psa values decreased from 399,9 ng / ml to 35,0 ng / ml . despite the advanced stage of disease in patients treated by us , the treatment is well tolerated without any serious side effects . quality of life after the second iptld application is significantly improved , and this applies even to patients with treatment failure in terms of psa criteria . the research was carried out in a private medical centre and the financial expenses are not covered by the national health insurance institution . still the small number of treated patients and the short - term of the follow - up do not allow us to do a serious comparative analysis of the results of the treatment in both groups , as well as making definitive conclusions regarding the effectiveness of the treatment . the preliminary presented results give us reason to assume that extensive comparative researches are necessary for better proving the effectiveness of the method . our present experience with iptld ( in more than 400 treated patients ) with various tumors as well as the practical experience of the growing number of doctors practicing the method gives us a reason to assume that iptld method provides a real opportunity for resolving one of the most serious problems of toxicity associated with chemotherapy using maximum tolerated doses . a certain advantage of the method along with its effectiveness is the significantly improved quality of life of the treated patients . in spite of the small number of patients treated by us with castrate - resistant prostate tumor , the preliminary results are promising and this gives us hope and expectations for future serious researches on the potential of widespread clinical use of iptld .

purpose . to evaluate the results and quality of life of patients with resistant of castration - resistant tumors previously treated with insulin - potentiation therapy ( ipt ) combined with hormone therapy . materials and methods . sixteen patients with metastasis prostate tumors after bilateral castration , androgenic blockade , and progression of the disease were observed during the study . the patients were divided into two groups : group a consisting of 8 patients treated with low - dose chemotherapy epirubicin , vinblastine , and cyclophosphamide combined with lhrh agonist and group b consisting of another 8 patients treated with low - dose chemotherapy docetaxel combined with lhrh agonist . results . the overall ( groups a and b ) results concerning psa after the sixth ipt show partial effect in 8 out of 16 ( 50% ) patients , stabilization in 4 out of 16 ( 25% ) , and progression in 4 out of 16 ( 25% ) . the median survival for all treated patients is 11,7 months ( range 330 months ) . during the treatment no significant side effects were observed , and no lethal cases occurred . conclusion . in spite of the small number of the treated patients with castration - resistant prostate tumors , the preliminary results are promising and this gives us hope and expectations for future serious multicenter research over the possibilities for routine implementation of iptld .

pharmacists embedded in primary care medical practices , such as those modeled on the patient - centered medical home , are playing increasing roles in interdisciplinary primary healthcare teams.13 however , with limited pharmacist resources and high patient to pharmacist ratios , it is important to identify which patients would benefit most from consultation with a primary care pharmacist . we have previously completed a similar assessment of patient requirements for dietician services in primary care.4 while commonly used approaches to identify patients for pharmacist care include physician referrals , drug- or disease - specific programs ( eg , chronic disease management ) , and patients recently discharged from hospital , an alternative strategy is to focus on patients at increased risk of medication - related problems ( mrps).2,57 mrps are events or circumstances involving drug therapy that actually , or potentially , interfere with an optimum outcome for the patient.8,9 several studies have identified factors associated with an increased risk of mrps,1014 and short , self - administered surveys have been developed to help identify patients at risk of mrps.12,15 our objective was to determine the feasibility of using a locally adapted , self - administered , paper - based , patient survey , to identify patients at risk of mrps , in a convenience sample of patients at a single academic family medicine clinic . we conducted a cross - sectional pilot study based on a convenience sample of 100 completed surveys at the university of alberta hospital family medicine clinic in edmonton . at the time of the survey , seven family physicians , two family medicine resident doctors , two full time clinic nurses ( one dedicated to chronic disease management ) , and two academic clinical pharmacists ( each working ~0.2 full - time equivalent ) provided service to approximately 7,000 patients . all patients registered to be seen by one of the clinic physicians were eligible to participate in this survey , unless they were less than 18 years old , could not read english , or were not responsible for self - management of their medications . after registration at the reception desk , a medical office assistant asked eligible patients if they would be willing to participate . those who expressed interest were given an information letter and a copy of the survey as they were placed in an examination room , and asked to complete the survey while waiting to be seen by their physician . the university of alberta health research ethics board ( reb ) approved the study protocol . we used the previously validated , ten - item mrp questionnaire , developed by barenholtz levy , and the modified five - item questionnaire , developed by langford et al , to create an adapted ten - item questionnaire ( figure 1).12,15 to adapt the questionnaire for the primary care ambulatory population , several changes were made . the two most significant changes made were : 1 ) the focus of question 3 ( present in both surveys ) was changed from high risk medications to a chronic diseases focus , and 2 ) question 9 , does someone else bring any of your medications to your home for you ? was replaced ( as all patients attending the family medicine clinic were ambulatory ) with do you sometimes worry about the long - term effects of your medications ? to determine feasibility , we recorded patient - reported time to complete the questionnaire , and we solicited opinions from the medical office assistants and physicians about the impact of the questionnaire on clinic flow . to estimate the number of patients at high risk for an mrp , we calculated a summary risk score . responses , after collapsing question 3 into a single yes or no response . as done by others in the literature , we defined patients who answered yes to three or more questions as being at high risk for a mrp.11,15,16 to allow further comparisons , we did a sensitivity analysis , in which we examined the number of patients who were at risk , while restricting analysis to the five questions developed by langford et al.15 we conducted a cross - sectional pilot study based on a convenience sample of 100 completed surveys at the university of alberta hospital family medicine clinic in edmonton . at the time of the survey , seven family physicians , two family medicine resident doctors , two full time clinic nurses ( one dedicated to chronic disease management ) , and two academic clinical pharmacists ( each working ~0.2 full - time equivalent ) provided service to approximately 7,000 patients . all patients registered to be seen by one of the clinic physicians were eligible to participate in this survey , unless they were less than 18 years old , could not read english , or were not responsible for self - management of their medications . after registration at the reception desk , a medical office assistant asked eligible patients if they would be willing to participate . those who expressed interest were given an information letter and a copy of the survey as they were placed in an examination room , and asked to complete the survey while waiting to be seen by their physician . the university of alberta health research ethics board ( reb ) approved the study protocol . we used the previously validated , ten - item mrp questionnaire , developed by barenholtz levy , and the modified five - item questionnaire , developed by langford et al , to create an adapted ten - item questionnaire ( figure 1).12,15 to adapt the questionnaire for the primary care ambulatory population , several changes were made . the two most significant changes made were : 1 ) the focus of question 3 ( present in both surveys ) was changed from high risk medications to a chronic diseases focus , and 2 ) question 9 , does someone else bring any of your medications to your home for you ? was replaced ( as all patients attending the family medicine clinic were ambulatory ) with do you sometimes worry about the long - term effects of your medications ? to determine feasibility , we recorded patient - reported time to complete the questionnaire , and we solicited opinions from the medical office assistants and physicians about the impact of the questionnaire on clinic flow . to estimate the number of patients at high risk for an mrp , we calculated a summary risk score . responses , after collapsing question 3 into a single yes or no response . as done by others in the literature , we defined patients who answered yes to three or more questions as being at high risk for a mrp.11,15,16 to allow further comparisons , we did a sensitivity analysis , in which we examined the number of patients who were at risk , while restricting analysis to the five questions developed by langford et al.15 demographic characteristics , summary risk scores , and time to complete the questionnaire are presented in table 1 . from patient reports in answer to the questionnaire , the median time to complete the questionnaire was 2 minutes ( interquartile range : 1.53 minutes ) . there were no comments from the medical office assistants , or physicians approached during regular staff meetings , to suggest that the questionnaire was intrusive on the clinic s workflow . the questionnaire was not distributed by medical office assistants to consecutive patients , because research was not considered to be a priority in their responsibilities , and clinical tasks sometimes intervened . forty - three respondents had three or more risk factors identified by the full ten - point questionnaire , and 26 respondents met this criterion in the sensitivity analysis . in view of the limited data , and bearing in mind that this was a pilot study , no further mining of the data was undertaken . the findings of this pilot study suggest that distribution of a mrp questionnaire at the point of care is feasible in a busy family medicine clinic , and that there was a strong signal for potential mrps in the sample studied . however , there may have been a selection bias introduced by the office assistants , in terms of who they approached to participate , especially when they were busy . we have identified issues relating to the efficiency of this approach ( consecutive patients were not all approached ) , and we suggest that asynchronous methods to systematically screen larger numbers of patients , such as the use of electronic medical record data , should also be explored to supplement the direct approach . this anonymous pilot study , as approved by the reb , did not permit the pharmacists to act upon the mrps identified , whereas a chart survey would allow this . this study adds to existing literature that demonstrates in practice the clinical utility of a mrp screening tool , and demonstrates its potential to generate pharmacist referrals . while our overall estimate of patients at high risk for an mrp was double that obtained by langford et al15 ( 43% versus 18% , respectively ) , our sensitivity analysis , using only langford s questions , demonstrated more similar proportions ( 26% versus 18% ) . the difference is likely related to the change in focus of one question , from high risk medications to the presence of common chronic diseases . in view of the differences between the two studies , unfortunately , comparisons with the sample studied by barenholtz levy are not possible , as they did not report summary risk scores , nor use a cut - off to define patients as being at high risk.12 perhaps one of our study s biggest strengths was its use of medical office assistant staff to screen patients , rather than using research personnel or clinic pharmacists . from a practical standpoint , part - time clinic pharmacists are not available to screen all patients attending clinics . despite this strength , since our study was an uncontrolled , single site , cross sectional survey based on a convenience sample , the time taken to achieve 100 completed surveys , as well as our estimates of the proportions of patients at high risk of mrps , may be unreliable . framing the survey as research to the office assistants ( rather than quality improvement ) may have contributed to the prolonged recruitment period ; this was also contributed to by the deliberate arms length policy of the investigators , to test the feasibility of the intervention in the practice context . volunteer bias or preselection by medical office assistants could have led to overestimation of those at high risk . finally , as demonstrated by our sensitivity analysis , the reliability of this estimate is influenced by the number of questions included in the survey , and the arbitrary nature of the cut - off points to define high risk . future study is required to compare different methods of patient identification for primary care pharmacists , determine what threshold accurately defines high risk of an mrp , and , ultimately , the effect of pharmacist intervention on risk of mrps . in conclusion , our results suggest that screening patients at the point of care , using a self - administered mrp questionnaire , was feasible , and that a large proportion of those who complete the questionnaire will have multiple risk factors for mrps . this is similar to the opportunity found in our dietician study.4 however , other strategies , including electronic medical record screening or focusing on patients attending the chronic disease management nurse , may represent complementary methods to increase screening efficiency , and allow for more systematic screening of clinic patients for risk of mrps . our findings support the integration of a pharmacist into family medicine practices , as a member of the patient - centered medical home . future work will include further content and construct validation of the revised tool and its predictive ability for measures of health care utilization . additionally , we plan to compare the predictive accuracy and feasibility of an automated electronic medical record medication list screening approach for identifying patients at high risk of mrps .

background and objectivespharmacists working in primary care clinics are well positioned to help optimize medication management of community - dwelling patients who are at high risk of experiencing medication - related problems . however , it is often difficult to identify these patients . our objective was to test the feasibility of a self - administered patient survey , to facilitate identification of patients at high risk of medication - related problems in a family medicine clinic.methodswe conducted a cross - sectional , paper - based survey at the university of alberta hospital family medicine clinic in edmonton , alberta , which serves approximately 7,000 patients , with 25,000 consultations per year . adult patients attending the clinic were invited to complete a ten - item questionnaire , adapted from previously validated surveys , while waiting to be seen by the physician . outcomes of interest included : time to complete the questionnaire , staff feedback regarding impact on workflow , and the proportion of patients who reported three or more risk factors for medication - related problems.resultsthe questionnaire took less than 5 minutes to complete , according to the patient s report on the last page of the questionnaire . the median age ( and interquartile range ) of respondents was 57 ( 4569 ) years ; 59% were women ; 47% reported being in very good or excellent health ; 43 respondents of 100 had three or more risk factors , and met the definition for being at high risk of a medication - related problem.conclusionsdistribution of a self - administered questionnaire did not disrupt patients , or the clinic workflow , and identified an important proportion of patients at high risk of medication - related problems .

kmmell 's disease ( kd ) is rare form of post - traumatic delayed avascular necrosis of the vertebral body . to best of authors knowledge , there is no reported case of kd from india . it 's close radiological and histological resemblance to endemic tuberculosis of the vertebrae posses a diagnostic dilemma . we are reporting a case , whose clinical , radiological , and histological profile were compatible with kd and discuss the dilemma of diagnosing kd in indian context . a 60-year - old gentleman , a laborer by profession , presented with the complaints of severe low back ache of 3 days duration and low grade fever for the past 2 weeks . one year back , he had low backache of moderate intensity , precipitated by lifting and carrying heavy loads on his back , which resolved spontaneously . on clinical examination , there was focal tenderness over l4 - 5 vertebral levels with gross restriction of spinal movements and marked paraspinal spasm . routine hematological and biochemical parameters were normal except for high esr of 94 mm/1 h ( westergren ) . magnetic resonance imaging ( mri ) of lumbosacral spine [ figure 1 ] revealed collapse of l4 body with relatively preserved disc spaces . the collapsed l4 vertebral body was hypointense on t1w image and hyperintense on t2w image [ figure 1 ] . computed tomography ( ct ) of spine [ figures 2 and 3 ] revealed l4 vertebral body collapse . he underwent l4 laminectomy , l3 - 5 transpedicular fixation , and the biopsy of the l4 body . histopathology revealed thin irregular osteoporotic bony lamellae and fragments of pale ischemic bony bits [ figure 5a ] . the enclosed marrow spaces revealed fresh hemorrhage bordered by clusters of foamy histiocytes as response to fresh hemorrhage reflecting trauma [ figure 5b ] . there was abundant fibrin and early necrosis of the marrow without any osteoblastic or osteoclastic activity . there was no evidence of tuberculosis ( absence of granulomatous pathology or acid fast bacilli ) . the possibility of post - traumatic avascular necrosis ( kd ) was suggested . as tuberculosis mri t2w and t1w images show collapse of l4 vertebra and signal changes within the collapsed body ( hyperintense on t2w image and hypointense on t1w image ) , without pre or postvertebral collection / soft tissue and relatively preserved disc ct axial scan through l4 vertebra show fracture of left lamina sagittal reformatted ct shows collapse of vertebra with increased attenuation without evidence of bone destruction or erosion coronal reformatted ct shows collapse of vertebra and presence of air within the collapsed body ( kmmell 's sign ) histopatholgy reveal osteoporotic bony lamellae with preserved calcium lines contiguous with fragmented , ischemic bone with loss of calcium ( asterix , a ) closely apposing ischemic fragments , densely eosinophillic necrotic bone with remnants of osteocytes are seen . marrow spaces showed fresh hemorrhage walled in by foamy histiocytes ( b ) reflecting reparative process . [ a : he16 , b : he240 ] on subsequent reviewing of mri , radiological features were not favoring tuberculosis , as the disc space was well preserved . there was no pre or paravertebral collection of inflammatory granulation tissue and the collapsed vertebral body did not have abnormal enhancement or signal changes . review of the ct scan revealed l4 vertebral body collapse with cortical breach , linear fracture of lamina on the left side , and presence of intravertebral vacuum cleft on radiograph [ kmmell 's sign a retrospective observation ( figure 4 ) ] . clinical history was suggestive of mild trauma and delayed acute presentation , and hence , diagnosis of kd was made and att was stopped . serum ada performed at 3 months of follow up , which was normal . at 12 months kd is a rare form of delayed post - traumatic osteonecrosis , typically precipitated by mild trauma to the lower thoracic and lumbar spine . the initial pain following trauma resolves after a few days and reappear some months later . though first described in 1895 by hermann kmmell , this disease is uncommon and occasionally reported in english literature , possibly because of nonspecific symptomatology , difficulty in recalling trivial trauma , spontaneous resolution of initial pain , and absence of specific diagnostic tests . there is neither a consensus on the degree of initial trauma precipitating the event nor on the duration and/or degree of symptoms during the asymptomatic period . the term kmmell 's disease has been inappropriately applied to vertebral collapses with vacuum phenomenon , resulting in great confusion about the exact nature of cases reported as reviewed the available english literature on kd since the 1950s and authors felt that only five cases meet the criteria set out by kmmell . most of reported cases are isolated case reports affecting the elderly and more frequent in males . osteoporosis , chronic corticosteroid use , and local radiotherapy are the associated risk factor for this disorder . repetitive spinal loading in hyperflexion has been described as triggering factor for young onset kd . diagnosis usually requires exclusion of other causes of vertebral body collapse and osteonecrosis , as various disorders can cause vertebral body collapse beside trauma , which includes neoplasm , infection ( especially tuberculosis ) , metabolic disorders , radiation , and osteoporosis . there is no diagnostic imaging for the disease and the diagnosis remains a diagnosis of exclusion ; however , careful review of imaging and exclusion of other common cause of vertebral body collapse on radiology is vital to avoid missing the diagnosis . an intravertebral vacuum cleft on radiograph , referred to as kmmell sign , is highly suggestive of ischemic necrosis and corresponds to the one of the most characteristic features of kd is normal radiological study immediately following trauma and subsequent development of rarefaction and loss of bone tissue leading to collapse of the vertebral body after a delay of weeks or months . probably , the ideal investigation is serial imaging . it is suggested that the initial trigger of trivial trauma results in subtle disruption in the bony spongiosa with microhemorrhages . the etiological contribution of vascular insufficiency is supported by finding of delayed vertebral body collapse with normal appearance on routine ct imaging but with an abnormal bone scan . in our case , though there was no histological evidence of tuberculosis , however , the diagnostic possibility of tuberculosis could not be excluded , especially in an endemic area like india and in the presence of systemic symptoms of fever and raised esr . histopathological review revealed osteoporotic bony lamellae , a described risk factor for kmmell 's disease in addition to the presence of necrosis , and histiocytes . our patient was not investigated after initial trauma and we did not have the opportunity to establish the delayed radiological vertebral body collapse . however , review of the initial radiological imaging of l4 body revealed cortical breach and fracture line extending to involve whole body with presence of kmmell 's sign with conspicuous absence of features of tuberculosis . reconsideration and review of the clinicoradiological and histopathology is crucial to avoid under diagnosing the kd . in one of the published cases of kd , authors could diagnose only after clinical and histopathological review , who had two biopsies at different center and even received radiation for suspected malignancy . it is really difficult not to favor the diagnosis of tuberculosis , in endemic countries like india . it is therefore possible that kd is probably overlooked / under - reported rather than nonexistent . a high degree of clinical suspicion with careful review of imaging and histopathology and clinical correlation may aid in diagnosis and avoid unnecessary work up and prolonged treatment .

kmmell 's disease is a rare form of vertebral body osteonecrosis , which develops as a delayed post - traumatic event . it is infrequently reported in literature and to the best of our knowledge , has not been reported from india . we describe the clinical , radiological , and pathological features of a case occurring in a 60-year - old man and relevant brief review of the literature of this rare disease . its close resemblance to more commonly occurring bony tuberculosis poses a diagnostic dilemma particularly in developing country like india , where tuberculosis is endemic . awareness of this entity , though rare , is essential to avoid unnecessary diagnostic work up and treatment .

in this commentary we discuss the study of scannapieco and colleagues published in a recent issue of critical care . ventilator - associated pneumonia ( vap ) frequently occurs in icus , with reported incidences ranging from 9% to 27% . it is a leading cause of morbidity and , possibly , of mortality . as a result , colonization of the upper respiratory tract generally precedes the occurrence of vap , most probably because of a reduced capacity to clear pathogens and/or an increased adherence of micro - organisms to the respiratory tract . prevention of oropharyngeal colonization has been achieved with topically applied non - absorbable antibiotics ( referred to as selective oropharyngeal decontamination with antibiotics ( ab - sod ) ) or with topically applied chlorhexidine gluconate ( chx - sod ) . ab - sod was associated with reduced incidences of vap in various studies [ 4 - 6 ] , and recently also with a better 28-day survival in a large dutch multi - center study . in that study , ab - sod was equally effective in improving patient outcome as selective decontamination of the digestive tract ( sdd ) , which combines ab - sod with intestinal decontamination and 4 days of intravenous cefotaxim . the occurrence of resistance as a result of ab - sod or sdd , however , remains of concern , especially in countries with endemic levels of antimicrobial - resistant bacteria ( amrb ) . therefore , simply replacing antibiotics with antiseptics for oral decontamination might offer an effective and safe measure for icu patients , even in settings with high levels of amrb . indeed , chx - sod appeared effective in reducing vap incidence in several studies [ 8 - 13 ] . however , the regimens used were not always carefully described and concentrations and dosing frequencies varied from 0.12% chx twice daily to 2% chx four times a day . in addition , patient populations varied widely : from mixed icu populations to surgical icu patients and patients undergoing cardiac surgery . furthermore , nasal application of chx was used in one study and chx was combined with colistine in another , and in one study effects were compared to historic controls . moreover , all individual studies published so far have been underpowered to demonstrate effects of chx - sod on patient survival . in a recently published systematic review and meta - analysis , chx - sod was associated with a significant reduction in vap incidence of 44% , although the studies were very heterogeneous , which precludes firm conclusions about its protective effects . no reductions in overall mortality , duration of mechanical ventilation or length of stay could be demonstrated . in their article , scannapieco and colleagues aimed to determine the optimal frequency of chx - sod to prevent vap in trauma icu patients . the study contains a control group ( 49 patients ) and two intervention groups receiving chx 0.12% either once ( 47 patients ) or twice daily ( 50 patients ) . they conclude that the number of staphylococcus aureus in dental plaque was reduced in both intervention groups , but no significant reductions were observed in the total number of respiratory pathogens or incidence of vap . estimated reductions in colonization were 25% and 30% in the ' twice - daily ' and ' once - daily ' groups , respectively . the odds ratio for developing vap was 0.54 ( 95% confidence interval 0.23 to 1.25 ) for patients receiving chx - sod , which is remarkably similar to the pooled estimate from the most recent meta - analysis . although this may suggest a beneficial effect of chx - sod , it can not be demonstrated by a study of this sample size . in summary , the evidence that both ab - sod and chx - sod reduce vap incidence in icu patients is accumulating . the optimal frequency and concentration for chx - sod remains to be demonstrated . from scannapieco and colleagues ' study we can conclude that twice daily is not necessarily better than once daily , but maybe a four times daily regimen with 2% instead of 0.12% chx does make a difference . what we need now are well - designed and adequately powered studies to evaluate the effects of these measures on length of icu stay and survival . if these effects were demonstrated , chx - sod would offer a very cheap and ( ecologically ) safe infection prevention measure in patient populations increasingly suffering from infections caused by amrb . ab : antibiotics ; amrb : antimicrobial - resistant bacteria ; chx : chlorhexidine ; sdd : selective decontamination of the digestive tract ; sod : selective oropharyngeal decontamination ; vap : ventilator - associated pneumonia .

ventilator - associated pneumonia ( vap ) is a common cause of morbidity , antibiotic use , increased length of stay and , possibly , increased mortality in icu patients . colonization of the oropharyngeal cavity with potentially pathogenic micro - organisms is instrumental in the pathogenesis of vap , and selective oropharyngeal decontamination ( sod ) with antibiotics ( ab - sod ) or antiseptics , such as chlorhexidine gluconate ( chx - sod ) , has been associated with reduced incidences of vap . in a recent issue of critical care scannapieco and colleagues investigated differences in oropharyngeal colonization between mechanically ventilated patients receiving oropharyngeal decontamination with 0.12% chx - sod either once or twice daily compared to placebo . chx - sod was associated with a reduction in staphylococcus aureus colonization , but the study was underpowered to demonstrate a reduction in vap incidence . we urgently need well - designed and adequately powered studies to evaluate the potential benefits of chx - sod on patient outcome in icus .

these include the presence of at least two of the following : clinical and/or biochemical features of hyperandrogenism , menstrual dysfunction , and the appearance of polycystic ovaries on ultrasound , once other endocrine conditions have been excluded . other criteria that can be used include those from the national institutes of health ( nih ) and the androgen excess society ( aes ) . pcos has estimated prevalence of over 10% in women of childbearing age . besides being associated with infertility , pcos is also associated with a higher incidence of type 2 diabetes mellitus ( t2d ) , endometrial carcinoma , and cardiovascular disease including stroke and coronary heart disease . the exact etiology of pcos is unknown and probably represents a complex interaction between environmental and genetic factors . obesity in women of childbearing age is associated with anovulation , infertility , pregnancy loss , pregnancy - associated complications such as preeclampsia and gestational diabetes , and postpartum complications including hemorrhage as well as higher rates of infant mortality and congenital defects [ 3 , 4 ] . obesity in patients with pcos is also associated with delayed or failed response to fertility treatments including clomiphene citrate , gonadotropins , and assisted insemination [ 5 , 6 ] . the british fertility society advises that fertility treatment should be deferred until women have a body mass index ( bmi ) of less than 35 or bmi under 30 if they are below 37 years of age . metformin and nonsurgical weight loss measures have been advocated as first - line management for pcos [ 8 , 9 ] . it has been suggested that even a modest loss of up to 5% of the initial body weight can result in spontaneous ovulation , restoration of menstrual cycle regularity , and pregnancy in obese women with pcos [ 1012 ] . bariatric surgery is the most durable and effective treatment for morbid obesity and also results in the improvement of the metabolic syndrome . with the safety of the laparoscopic approach and improved understanding of the metabolic changes occurring in bariatric patients postoperatively , morbidly obese women with infertility secondary to pcos have resorted to bariatric surgery . historically , epidemiological studies have suggested that the rapid weight loss in the first year or two after bariatric surgery may increase women 's chance of conception . while the incidence of pcos decreases significantly after surgery , there are very few studies assessing fertility before and after bariatric operations . at present , there is no consensus on the role of such surgery in the management of infertility and whether surgery can also be beneficial in women who have a bmi of under 40 kg / m . in this article , we systematically review the published literature to assess the effects of bariatric surgery on fertility in women with pcos . the prisma statement for reporting systematic reviews and meta - analyses of studies that evaluate health care interventions : explanation and elaboration was utilized as a framework for this systematic review . all manuscripts assessing the quantitative effect of gastric bypass , gastric banding , sleeve gastrectomy , and gastric plication on infertility in females with pcos published between 1 january 1974 and 20 march 2015 were considered eligible for inclusion in this systematic review . search databases , pubmed , embase from 1974 to 20 march 2015 , and medline and medline non - indexed items , were searched using the following keywords : polycystic ovary syndrome , infertility , bariatric surgery , gastric bypass , laparoscopic , roux - en - y , gastric band , sleeve gastrectomy , and gastric plication . manuscripts lacking quantitative data , those lacking in relevance to the study question , and those relating to male fertility were excluded . results and data were extracted following analysis and critical review of the results section of original manuscripts . this included sample numbers , age , body mass index , and basic demographics . surgical procedure and technique . this list included roux - en - y gastric bypass ( rygb ) , gastric band ( gb ) , gastric plication ( gp ) , or sleeve gastrectomy ( sg ) , open or laparoscopic . comparisons . outcomes were conception rate , pregnancy , biochemical markers of fertility and pcos , and menstrual regularity . study design . the type of study and level of evidence were recorded . each study was individually assessed for risk of bias giving particular attention to funding sources , limitations of study , and conflicts of interest declared in the discussion section . as the literature primarily is composed of epidemiological studies , the principle summary measure is sample conception rates before and following surgery in addition to / or other biochemical markers of fertility and pcos within the same sample . results of studies have been summarized ; however , quantitative data have not been combined in analysis . the 6 manuscripts that were included in the analysis and their results are summarized in table 1 . . demonstrated that , after laparoscopic rygb ( 75 cm roux limb ) , weight loss was associated with amelioration of pcos - associated symptoms including resolution of menstrual abnormalities in all patients and resolution of hirsutism in 52% of patients . five patients who were unable to conceive preoperatively were able to conceive without the use of hormones postoperatively although the time interval after surgery is not mentioned in this paper . in a study by jamal et al . , 20 patients with pcos were followed up after rygb ( 75 cm roux limb and 30 cm biliopancreatic limb ) for a mean postoperative follow - up of 46.7 months . preoperatively , 50% of the patients with pcos were infertile , 85% had menstrual dysfunction , and 70% had hirsutism . following surgery - induced weight loss , menstrual irregularities were corrected with return of regular cycles in 82% of patients without the need for hormonal treatment . hirsutism completely resolved in 29% and 77.8% of those with t2d that had complete remission . of the 10 patients who did not conceive before surgery , 4 no longer desired pregnancy with the remaining 6 patients becoming pregnant within 3 years of surgery ( 5 of whom conceived without any hormonal treatment ) . series of 5 patients who underwent ivf after bariatric surgery , two women with infertility secondary to pcos and male infertility underwent rygb and gastric banding , respectively . both conceived postoperatively following in vitro fertilization / intracytoplasmic sperm insemination and had uncomplicated deliveries . this paper suggests that although ivf appears to be safe after bariatric surgery , ovarian hyperstimulation may present with features similar to complications after weight loss surgery ( especially internal herniation after rygb ) and a high index of suspicion is thus required . talebpour reported that gastric plication and gastric bypass appear to have a positive effect on fertility in an abstract presented at ifso 2011 . 10 women ( 71% ) out of 14 who were infertile preoperatively became pregnant one year postoperatively . out of 87 premenopausal patients who had irregular menstrual cycles preoperatively , 70 ( 80% ) had regular cycles by the end of the first year postoperatively . this work , only published in abstract form , does not compare the effects of the two operations . in another abstract presented by george and azeez at ifso 2013 , 156 female patients between the ages of 20 and 50 underwent laparoscopic sleeve gastrectomy ( sg ) and were followed up 6-monthly . postoperatively , hirsutism resolved in 77 out of 96 patients ( 80% ) and 54 out of 67 showed resolution of radiological evidence of pcos . 132 patients had menstrual irregularities before surgery but all of these returned to a normal menstrual pattern after sg . four out of the 11 patients who were unsuccessfully treated for infertility preoperatively became pregnant postoperatively . this abstract also showed that urinary stress incontinence resolved or substantially improved in over 40% of patients . unfortunately , this work was only published as an abstract and there is no information regarding the mean follow - up period , the preoperative bmis , and the nature of the study . dixon and o'brien found that 2 infertile patients in their cohort became pregnant after laparoscopic gastric banding . unfortunately , the paper does not make the denominator clear and it is not possible to ascertain how many of the 28 women with primary or secondary infertility were followed up 1 year postoperatively . the definition of infertility , the age of the patients , and the operations were different . controls were not present in the considered papers with patients being control of themselves before and after bariatric surgery . it was difficult to carry out analysis for bias in studies that were only presented as conference abstracts . another bias deals with one of the analyzed studies that considered in vitro fertilization ( often performed for the male factor ) , while the others considered spontaneous pregnancy . women of childbearing age form a significant percentage of patients being referred for and undergoing bariatric surgery . a review of admission data from more than 1,000 us hospitals between 1998 and 2005 revealed that almost half of all patients undergoing inpatient surgical weight loss procedures were women between the ages of 18 and 45 . a recent meta - analysis has shown that pcos decreases significantly after bariatric surgery from 45.6% preoperatively to 6.8% at 1 year postoperatively . the reproductive health of female participants was investigated as part of the longitudinal assessment of bariatric surgery ( labs-2 ) study with a self - administered survey within 30 days preoperatively . 42% of women who tried to become pregnant preoperatively in this cohort of patients experienced infertility ( defined as 12 months of regular intercourse with a man without contraception but no resulting pregnancy ) . 65% of these patients however had at least 1 pregnancy after experiencing a period of infertility . a high rate of stillbirths was self - reported by these women with the rate being twice that expected in the usa ( 13.2 versus 6.2 per 1000 live births ) . future pregnancies ( in the postoperative period ) were an important consideration to 30% of patients who were aged 1844 and who did not report natural / surgical menopause , hysterectomy , endometrial ablation , or sterilization ( personal or partner ) . this study revealed that women who were obese by the age of 18 were more likely to report pcos ( 14.4% versus 5% ) and infertility ( 56% versus 25% ) and less likely to have ever been pregnant ( 75% versus 92% ) , compared with women whose obesity started after the age of 30 . obesity at a young age may be considered an indication for bariatric surgery in effort to prevent infertility developing in later life . the labs-2 study is yet to report the postoperative reproductive health results in this cohort of women . pcos in obese patients primarily manifests itself with irregular or infrequent menstrual bleeding / amenorrhea , hirsutism , and infertility . it is thought that a 5% weight loss can result in resolution of obesity - related anovulation ; however , there is little evidence that this is sufficient in morbidly obese patients . in a retrospective survey , 50% of women ( 98 patients ) aged 40 years or younger with intact uterus and ovaries had anovulatory cycles , defined as cycles of > 35 days longer , prior to bariatric surgery . of these 98 patients , 70 patients ( 71% ) patients who regained ovulation postoperatively had statistically significant greater weight loss compared to those who remained anovulatory . patients who had normal cycles preoperatively still had normal menstrual cycles postoperatively despite the weight loss . in a prospective study of 14 females with pcos , amelioration in clinical symptoms was associated with significant improvements in testosterone , fasting glucose , cholesterol , insulin , and triglyceride levels at 6 and 12 months after rygb when compared to baseline . improvements in biomarkers , hirsutism , and regularity of the menstrual cycles did not correlate with the degree of weight change in this study . . also showed similar improvements in total and free testosterone and amelioration of insulin resistance estimated in a prospective study of 12 premenopausal women with pcos who underwent either rygb or biliopancreatic diversion . traditionally , bariatric surgery has been reserved for patients with a body mass index ( bmi ) > 40 kg / m or with bmi > 35 kg / m and one or more significant comorbid conditions , when nonsurgical methods of weight loss have failed . recently , the national institute for health and care excellence ( nice ) in the uk suggested lowering the bmi down to 30 infertility due to anovulation and pcos amongst morbidly obese women could potentially be viewed as an additional indication for bariatric surgery . although most studies show amelioration of pcos postoperatively , to date , the number of studies showing improved fertility is small and they mainly consist of retrospective analysis of small cohorts of patients . as females of childbearing age make up a large percentage of patients undergoing surgery , more research is required in this area of metabolic and bariatric surgery to enable clinicians to advise these women regarding their reproductive health and fertility after surgery . careful follow - up of these patients is required , as pregnancy is usually not advised in the first 1218 months postoperatively . bariatric surgery results in improvement of menstrual irregularities and hirsutism and amelioration of the metabolic profile . however , at this stage , it is difficult to recommend the lowering of bmi criteria for patients with primary infertility and pcos and larger studies are required to confirm the effects of bariatric surgery on fertility and to determine whether different bariatric operations have different results compared to nonsurgical methods of weight loss . polycystic ovary syndrome ( pcos ) is the most frequent cause of female infertility.pcos , hirsutism , and menstrual irregularities improve after bariatric surgery.the evidence for improvement in fertility after bariatric surgery is still limited.more studies are required to understand which operation ( if any ) would be best for this cohort of young infertile women . more studies are required to understand which operation ( if any ) would be best for this cohort of young infertile women .

background . polycystic ovary syndrome ( pcos ) is the commonest cause of female infertility . visceral obesity and insulin resistance are key pathophysiological mechanisms behind pcos . women suffering from this syndrome and infertility often seek bariatric surgery hoping that they would be able to conceive postoperatively . objective . at present , there is no consensus on the role of bariatric surgery in the management of pcos - associated infertility within the medical community , making it difficult to give specific advice to these women , so a review of the literature was necessary . results . a detailed review of the literature was performed . only 6 manuscripts were relevant and contained quantitative data . they demonstrated that bariatric surgery results in postoperative conception rates varying from 33% to 100% . surgery is also associated with amelioration of menstrual irregularities , hormonal abnormalities , and hirsutism that are associated with pcos . these studies were retrospective and only had a small number of participants with infertility . conclusions . bariatric surgery has been shown to conclusively improve life expectancy , quality of life , and comorbidities like type 2 diabetes and obstructive sleep apnea . however , further research is required to identify whether weight loss surgery results in significant improvement in fertility of women with pcos and to investigate which operation has the best results .

human parvovirus b19 is a dna virus most known for causing erythema infectiosum in children , and polyarthropathy or transient aplastic crisis in adults . we describe a young patient who presented with splenic infarcts as a rare complication of b19 infection . a 33-year old previously healthy man was admitted to our hospital because of a 5-day history of fever and headache . endocarditis was ruled out , whereas serologic testing for b19 was indicative of acute infection . to our knowledge , three cases of thromboembolism in the setting of b19 infection have been reported up to now , including one occurence of splenic infarction . human parvovirus b19 is most known for causing erythema infectiosum in children , transient red cell aplasia which can be significant in those with hemolytic anemia or polyarthropathy in adults , . a 33-year old man was admitted to our hospital because of a 5-day history of continuous fever up to 39 c , and diffuse headache . he was unmarried and lived with his parents in thirasia island near santorini , working as a builder , was sexually active and consistently used condoms . he did not smoke , use illicit drugs , or misuse alcohol , had no pets or exposures to animals , and had never traveled abroad . the man reported no recent tick or mosquito bites , no ingestion of unpasteurized milk or milk products , and no sick contacts and had received all routine childhood vaccinations , had no known medical problems , and did not take any medications . his family history was negative for tuberculosis , cancer or autoimmune disease . on physical examination , the patient appeared well and was in no acute distress ; he was alert and communicative . his temperature was 39 c , heart rate 73 beats per minute , blood pressure 120/80 mmhg , respiratory rate 16 breaths per minute , and oxygen saturation 99% while breathing ambient air . his abdomen was soft , with mild tenderness in the left upper quadrant on deep palpation . there was no hepatomegaly or splenomegaly , and no cervical , axillary , or femoral lymphadenopathy . laboratory analyses revealed normocytic , normochromic anemia ( hemoglobin level , 12 g per deciliter ) , with a low reticulocyte count ( 0.3% ) , leukopenia ( white - cell count , 3420 per cubic millimeter , with 68% neutrophils , 25% lymphocytes , 7% monocytes , and no eosinophils or band forms ) , thrombocytopenia ( platelet count , 87,000 per cubic millimeter ) , and elevated levels of hepatic aminotransferases ( aspartate transaminase 138 u per liter , alanine transaminase 111 u per liter ) , lactate dehydrogenase ( 1383 u per liter ) , creatine phosphokinase ( 1019 u per liter ) , and c - reactive protein ( 112 mg per liter ) . clotting times ( dilute prothrombin time and activated partial - thromboplastin time ) were not prolonged . an abdominal ultrasound examination revealed a mildly enlarged spleen , with two peripheral wedge - shaped hypoechoic lesions , which were characteristic of splenic infarcts . 1 ) , obtained after the administration of intravenous and oral contrast material , confirmed the results of ultrasonography , demonstrating splenomegaly ( with the spleen measuring 15.4 cm in the craniocaudal dimension ) , and two wedge - shaped areas of hypoenhancement in the periphery of the spleen , features that were consistent with splenic infarcts . no other organ abnormality was observed . hepatitis a immunoglobulin m ( igm ) antibody , hepatitis b surface antigen and core igm antibody , a polymerase chain reaction ( pcr ) assay for hepatitis c viral rna , and serologic tests for cytomegalovirus ( cmv ) , herpes simplex virus , coxsackie a virus , echovirus , human immunodeficiency virus , rickettsia species , coxiella burnetii , brucella melitensis , and leishmania were negative , as was real - time reverse transcriptase pcr for h1n1 influenza a virus . barr virus ( ebv ) , coxsackie b virus , and toxoplasma gondii showed evidence of past infection . parvovirus b19 igm antibodies were positive , while parvovirus b19 immunoglobulin g ( igg ) antibodies were negative , indicating an acute infection . the patient received symptomatic treatment with antipyretic and analgesic agents . over the following few days whether he developed parvovirus b19-specific igg antibodies during the convalescent phase remained unknown , because the relevant test could not be performed in his island . splenic infarction occurs as a result of the occlusion of the splenic artery or its branches due to thrombosis or embolism . the most common etiologies are hematologic diseases , including myeloid disorders , lymphomas , hemoglobinopathies , and hypercoagulable states ; cardioembolic events ; viral infections ; and trauma , . the majority of patients present with pain in the left upper quadrant or left flank or both . however , as in this case , a surprising percentage of patients have no symptoms localized to the splenic area . the most frequent laboratory abnormality is elevated lactate dehydrogenase levels , as seen in our patient . a major concern , when evaluating a patient with fever accompanied by splenomegaly and splenic infarcts , is bacterial endocarditis . this diagnosis was ruled out by sterile blood cultures and normal transesophageal echocardiography as well as recovery without antimicrobials . additionally , the acuity of our patient 's illness , and his rapid and complete recovery argued against hematologic malignancies , being most consistent with a viral infection . the viral infections that have been associated with splenic infarcts are infectious mononucleosis , and acute cmv infection . our patient did not present with any of the classic syndromes associated with pvb19 infection . virus - induced cytotoxicity results in cessation of red cell production . in individuals without hemolytic anemia , this transient arrest of red cell production will lead to only a minimal drop in hemoglobin levels but in patients with increased destruction of red cells , who depend on continual rapid production of erythrocytes ( hemoglobinopathies or hemolytic anemias ) , this suppression of erythropoiesis can cause a transient aplastic crisis with severe anemia , . a wide range of unusual clinical manifestations have been reported in association with pvb19 infection , including vasculitis , myocarditis , glomerulonephritis , peripheral neuropathy , meningitis and encephalitis , immune thrombocytopenia , and hemophagocytic syndrome . three cases of thromboembolism have also been described , to our knowledge ; a man with a splenic infarct , a woman with multiple pulmonary emboli , and a woman with right pulmonary arterial and superior mesenteric venous thromboses . similar to our case , all these occurrences involved young previously healthy patients who recovered fully . in regards to pathogenesis , they were attributed to the development of a transient antiphospholipid antibody syndrome . consequently , other unknown mechanisms also account for the thromboembolic phenomena observed in the setting of pvb19 infection . pvb19 infection should be considered in the differential diagnosis of fever accompanied by splenic infarcts in immunocompetent young adults . clinicians must be aware of pvb19 propensity to induce thromboembolic events , regardless of the presence of antiphospholipid antibodies .

introductionhuman parvovirus b19 is a dna virus most known for causing erythema infectiosum in children , and polyarthropathy or transient aplastic crisis in adults . however , various unusual clinical manifestations have also been reported in association with it . we describe a young patient who presented with splenic infarcts as a rare complication of b19 infection.case reporta 33-year old previously healthy man was admitted to our hospital because of a 5-day history of fever and headache . imaging studies revaled two splenic infarcts . endocarditis was ruled out , whereas serologic testing for b19 was indicative of acute infection.discussionto our knowledge , three cases of thromboembolism in the setting of b19 infection have been reported up to now , including one occurence of splenic infarction . these events were attributed to the development of a transient antiphospholipid antibody syndrome . in contrast , our patient did not have elevated titers of antiphospholipid antibodies.conclusionssplenic infarcts can be an atypical presentation of b19 infection . parvovirus b19 may induce thromboembolic events , even in the absence of antiphospholipid antibodies .

the genus acanthotrema ( digenea : heterophyidae ) are intestinal parasites of fish - eating birds and mammals . acanthotrema acanthotrema , the type species , was originally described from a bird ( sterna maxima ) in 1928 in brazil , and other species were subsequently documented . currently , 8 species are included in this genus ; a. acanthotrema , a. armata , a. cursitans , a. felis , a. hancocki , a. martini , a. tanayensis , and a. tridactyla . the newest species is a. felis , which was described in 2003 from the small intestine of stray cats in the republic of korea . various brackish water fish have been reported to be the second intermediate host for acanthotrema species including the mud sucker gillichthys mirabilis and the southern california killifish for a. hancocki and the goby acanthogobius flavimanus for a. felis . for example , a. acanthotrema has been reported from brazil , a. hancocki , a. martini , and a. cursitans from the united states , a. tridactyla from egypt and kuwait , a. armata from spain , a. tanayensis from the philippines , and a. felis from korea . the definitive hosts for a. acanthotrema , a. armata , a. hancocki , and a. martini are birds , including sea gulls , and those of a. felis , a. cursitans , and a. tanayensis are mammals , particularly cats . , with the exception of a. felis . a 70-year - old man residing in muan - gun , korea , expelled a single adult worm of a. felis after praziquantel treatment , together with 148 heterophyes nocens , 65 gymnophalloides seoi , 30 stictodora fuscata , 1 stictodora lari , and 1 stellantchasmus falcatus . we describe here 4 additional human cases naturally infected with a. felis in southern coastal areas of korea . during surveys of human intestinal flukes in shinan - gun ( 1990 ) , muan - gun ( 1995 ) , and yeongam - gun ( 2003 ) , jeollanam - do , korea , small trematode egg ( ste ) positive cases in kato - katz fecal smears were subjected to adult fluke collection after anthelmintic treatment and purging . briefly , praziquantel at a dose of 10 - 15 mg / kg was orally administered and an hour later the patient was given 30 g of magnesium sulfate . all the diarrheic stools were collected from each case for 4 - 5 hr and examined under a stereomicroscope . ten specimens of a. felis were recovered from 4 patients ( 1 - 7 per person , average 2.5 worms ) ( table 1 ) . all 4 patients were commonly infected with other heterophyid flukes including h. nocens , pygidiopsis summa , s. fuscata , and heterophyopsis continua . the number of specimens of the aforementioned fluke species was much higher than that of a. felis ( table 1 ) . the patients were suffering from vague abdominal discomfort and indigestion , sometimes accompanied by frequent stool , lethargy , and anorexia . however , it was considered that these symptoms were not necessarily due to a. felis infection . the patients had habitually eaten raw flesh of brackish water fish , such as the mullet and goby . the morphological characteristics and measurements ( m ) of the recovered a. felis adults are as follows ( table 2 ) . the worms were pear - shaped and were covered with fine tegumental spines , except near the posterior extremity ( fig . their average body size was 472 in length ( range , 405 - 510 ) and 269 ( 230 - 300 ) in width . the ventral sucker was embedded in the ventrogenital sac which had a distinct membranous covering . the ventrogenital sac was located in the median line , usually at the anterior end of the middle third of the body . inside the ventrogenital sac , 3 sclerites were observed ; 1 was short and thumb - like , 1 was long and blunt - ended , and 1 was long and broadly tipped ( fig . the gonotyl was u - shaped , unspined , located right to the sclerite complex , and was included in the ventrogenital sac . two testes were ovoid , posterolateral to the ovary , and a little obliquely side - by - side , and were located at the posterior half of the body . the bipartite seminal vesicle was connected to the genital sinus and then to the genital opening in the ventrogenital sac . vitellaria distributed from the post - testicular level to the posterior extremity , forming 2 large groups of follicles . the uterus formed a transverse loop immediately in front of the ovary , and nearly filled up the posterior half of the body . the morphological characteristics and measurements ( m ) of the recovered a. felis adults are as follows ( table 2 ) . the worms were pear - shaped and were covered with fine tegumental spines , except near the posterior extremity ( fig . their average body size was 472 in length ( range , 405 - 510 ) and 269 ( 230 - 300 ) in width . the ventral sucker was embedded in the ventrogenital sac which had a distinct membranous covering . the ventrogenital sac was located in the median line , usually at the anterior end of the middle third of the body . inside the ventrogenital sac , 3 sclerites were observed ; 1 was short and thumb - like , 1 was long and blunt - ended , and 1 was long and broadly tipped ( fig . the gonotyl was u - shaped , unspined , located right to the sclerite complex , and was included in the ventrogenital sac . two testes were ovoid , posterolateral to the ovary , and a little obliquely side - by - side , and were located at the posterior half of the body . the bipartite seminal vesicle was connected to the genital sinus and then to the genital opening in the ventrogenital sac . vitellaria distributed from the post - testicular level to the posterior extremity , forming 2 large groups of follicles . the uterus formed a transverse loop immediately in front of the ovary , and nearly filled up the posterior half of the body . since the first discovery of a. felis in korea , its presence has been documented several times in the country . adult flukes were reported from feral cats caught near pusan and shinan - gun and from a human in muan - gun . the present study adds shinan - gun and yeongam - gun as new localities for human infections . metacercariae of a. felis were discovered in 7 ( 35.0% ) of 20 a. flavimanus in haenam - gun , and in 31 ( 91.2% ) of 34 a. flavimanus in muan - gun , with the total number of metacercariae being 116 ( 17 per fish ) and 7,292 ( 235 per fish ) , respectively . among the 8 species of acanthotrema , only a. felis is so far known to distribute in korea , and is the only species infecting humans around the world , based on a previous report and the present study . however , humans seem to be a less susceptible host than birds or mammals , and the pathogenicity of acanthotrema worms to humans is unknown . in the 5 known human cases , the number of worms recovered per individual was usually 1 with the present exception of 1 patient who expelled 7 worms . this low number of infected worms probably would not cause any harmful effect to humans . humans may have innate resistance against this fluke , although this issue needs further clarification . however , acanthotrema has fewer than 12 spines or sclerotizations , and stictodora has more than 12 spines on the ventral sucker or ventrogenital sac . it was not difficult to assign our specimens to acanthotrema because they typically had 3 sclerotizations in the ventrogenital sac . other morphological characters included a submedian ovary , a bipartite seminal vesicle , a short prepharynx , absence of spines on the gonotyl , and the presence of 3 sclerites on the ventrogenital sac . a. tanayensis has 4 sclerites which are unarmed and the sclerites of a. acanthotrema consist of 2 armed pieces and a third unarmed piece . a. armata has 2 armed sclerites , and a. cursitans has 2 unarmed , branched sclerites . the present specimens displayed 3 sclerites with spines on the tip , differing from a. acanthotrema and a. tridactyla , which have 2 - 3 clerites with minute spines on the tip . the shape of the sclerites in our specimens was slightly different from that in the original report of this species . the sclerites in the original report consisted of 1 short , thumb - like piece and 2 long and pointed ones . however , in our specimens , the sclerites consisted of 1 short and thumb - like piece , 1 long and broadly tipped piece , and 1 long and blunt - ended piece . the present study is the second report on human a. felis infection following the report of cho et al . . this fluke should be included among the list of human - infecting foodborne intestinal flukes in korea and asia . further studies are required to clarify the geographical distribution and host - parasite relationships of a. felis infection .

acanthotrema felis is an intestinal trematode of cats originally reported from the republic of korea . only 1 human case infected with a single adult worm has been previously recorded . in the present study , we report 4 human cases infected with a total of 10 worms recovered after anthelmintic treatment and purging . all 4 patients reside in coastal areas of jeollanam - do , korea , and have consumed brackish water fish including the gobies , acanthogobius flavimanus . the worms averaged 0.47 mm in length and 0.27 mm in width , and had 3 sclerites on the ventrogenital sac ; 1 was short and thumb - like , another was long and blunt - ended , and the 3rd was long and broad - tipped . they were identified as a. felis sohn , han , & chai , 2003 . surveys on coastal areas to detect further human cases infected with a. felis are required .

nosocomial infections cause prolonged duration of hospitalization and increased treatment costs with high morbidity and mortality . acinetobacter species have become the most common etiology of hospital acquired infections due to the present frequent use of wide spectrum antibiotics ( 1 ) . acinetobacter species cause pneumonia , skin and wound infections , bacteremia , and urinary tract infections while they have shown resistance to various antibiotics ( 2 ) . carbapenems , sulbactam and colistin seem to be the most effective antibiotics for treatment ( 3 ) . sulbactam shows high efficacy against acinetobacter species in vitro and in vivo ( 4 - 6 ) . previously in turkey , sulbactam could not be found alone ; therefore sefoperazone and sulbactam combination was widely used for the treatment of multi - resistant acinetobacter species . today , however , commercially available sulbactam antibiotics are being used thus , the determination of the minimum inhibitory concentration ( mic ) of sulbactam is necessary . the aim of this study was to determine the susceptibility rate of genotypically different a. baumannii species , isolated from various clinical samples of patients with hospital acquired infections , to sulbactam , amikacin , netilmicin , meropenem , tigecycline and colistin . the isolates are defined as the agent of hospital acquired infections , if they emerged after 72 hours of hospitalization . , 300 a. baumannii were isolated from various clinical samples and detected by the vitek 2 compact system ( biomerieux , france ) at the microbiology laboratory of our hospital between january 2010 and march 2012 . the isolates were stored at -80c in brain heart infusion broth including 10% glycerol until susceptibility tests and genotypic evaluations were completed . according to the instructions of the manufacturer , these isolates were subjected to pcr analysis using the appropriate diversilab dna fingerprinting kit ( biomerieux , france ) . an analysis of the bands was completed using the diversilab software 3.3 gel images , while percentage similarity rates and dendrogram reports were formed for each dna sample . the samples were classified as different ( similarity < 95% and > 2 band difference ) , similar ( similarity in between 95 - 97% ) , and indistinguishable ( similarity > 97% , 1 - 2 band difference ) ( 7 ) . the susceptibilities of genotypically different isolates ( similarity < 95% and > 2 band difference ) , to sulbactam , amikacin , netilmicin , meropenem , tigecycline and colistin were tested . antibiotic susceptibilities were studied using the e - test method for sulbactam and tigecycline ( biomerieux , france ) , while the bouillon broth microdilution method was used for meropenem ( tumekip , turkey ) , colistin , amikacin and netilmicin ( sigma aldrich , usa ) . for the e - test method , bacterial suspensions with turbidity equivalent to 0.5 mcfarland standard were prepared and spread onto mueller hinton agar ( mha ) medium ( himedia , mumbai , india ) , followed by the addition of e - test strips . after the plates were incubated at 37c for 24 hours , the e - test mic value was read as the concentration at which the growth on the plate intersected the e - test strip . according to the food and drug administration ( fda ) criteria ; if the e - test mic value of tigecycline was 8 g / ml , it should be accepted as resistant , if the value was 4 - 6 g / ml , it should be accepted as intermediate , and if the value was 2 g / ml it should be accepted as susceptible ( 8) . the cut - off value for sulbactam alone was not defined for a. baumannii therefore , we determined the cut - off value of sulbactam considering the values of ampicillin / sulbactam for a. baumannii isolates in the clsi document . accordingly , if the value was 4 g / ml , it was accepted as susceptible , if the value was 8 g / ml it was accepted as intermediate and if the value was 16 g / ml it was accepted as resistant . for the bouillon broth micro dilution method , cation - adjusted mueller - hinton broth ( bbl , becton dickinson , usa ) was used according to clsi recommendations ( 9 ) . serial dilutions of each of the four antibiotics ( 32 - 0.062 g / ml ) were prepared . a. baumannii isolates were suspended to 0.5 mcfarland turbidity and added to micro dilution plates with 96 wells . escherichia coli ( atcc 25922 ) and pseudomonas aeruginosa ( atcc 27853 ) strains were used as quality control strains for all mic determinations . the lowest antimicrobial drug concentration at which there was no growth was considered as the minimum inhibitory concentration ( mic ) . the isolates are defined as the agent of hospital acquired infections , if they emerged after 72 hours of hospitalization . , 300 a. baumannii were isolated from various clinical samples and detected by the vitek 2 compact system ( biomerieux , france ) at the microbiology laboratory of our hospital between january 2010 and march 2012 . the isolates were stored at -80c in brain heart infusion broth including 10% glycerol until susceptibility tests and genotypic evaluations were completed . according to the instructions of the manufacturer , these isolates were subjected to pcr analysis using the appropriate diversilab dna fingerprinting kit ( biomerieux , france ) . an analysis of the bands was completed using the diversilab software 3.3 gel images , while percentage similarity rates and dendrogram reports were formed for each dna sample . the samples were classified as different ( similarity < 95% and > 2 band difference ) , similar ( similarity in between 95 - 97% ) , and indistinguishable ( similarity > 97% , 1 - 2 band difference ) ( 7 ) . the susceptibilities of genotypically different isolates ( similarity < 95% and > 2 band difference ) , to sulbactam , amikacin , netilmicin , meropenem , tigecycline and colistin were tested . antibiotic susceptibilities were studied using the e - test method for sulbactam and tigecycline ( biomerieux , france ) , while the bouillon broth microdilution method was used for meropenem ( tumekip , turkey ) , colistin , amikacin and netilmicin ( sigma aldrich , usa ) . for the e - test method , bacterial suspensions with turbidity equivalent to 0.5 mcfarland standard were prepared and spread onto mueller hinton agar ( mha ) medium ( himedia , mumbai , india ) , followed by the addition of e - test strips . after the plates were incubated at 37c for 24 hours , the e - test mic value was read as the concentration at which the growth on the plate intersected the e - test strip . according to the food and drug administration ( fda ) criteria ; if the e - test mic value of tigecycline was 8 g / ml , it should be accepted as resistant , if the value was 4 - 6 g / ml , it should be accepted as intermediate , and if the value was 2 g / ml it should be accepted as susceptible ( 8) . the cut - off value for sulbactam alone was not defined for a. baumannii isolates in the clinical and laboratory standards institute ( clsi ) document . therefore , we determined the cut - off value of sulbactam considering the values of ampicillin / sulbactam for a. baumannii isolates in the clsi document . accordingly , if the value was 4 g / ml , it was accepted as susceptible , if the value was 8 g / ml it was accepted as intermediate and if the value was 16 g / ml it was accepted as resistant . for the bouillon broth micro dilution method , cation - adjusted mueller - hinton broth ( bbl , becton dickinson , usa ) was used according to clsi recommendations ( 9 ) . serial dilutions of each of the four antibiotics ( 32 - 0.062 g / ml ) were prepared . a. baumannii isolates were suspended to 0.5 mcfarland turbidity and added to micro dilution plates with 96 wells . escherichia coli ( atcc 25922 ) and pseudomonas aeruginosa ( atcc 27853 ) strains were used as quality control strains for all mic determinations . the lowest antimicrobial drug concentration at which there was no growth was considered as the minimum inhibitory concentration ( mic ) . among the 300 a. baumannii isolates the susceptibilities of 30 isolates were determined as genotypically different ( figure 1 ) . twenty - one ( 70% ) of the 30 genotypically different a. baumannii isolates included in the study were isolated from tracheal aspirates , one ( 3.3% ) from blood , four ( 13.3% ) from the catheter , one ( 3.3% ) from a wound infection , two ( 6.6% ) from urine samples , and one ( 3.3% ) from peritoneal fluid samples . the susceptibility results of 27 carbapenem resistant isolate ( including the one with intermediate susceptibility ) to antibiotics other than carbapenem are shown in table 2 . the sensitivity rates for netilmicin , tigecycline , sulbactam , amikacin , and meropenem were 66.6% , 50% , 36.6% , 30% and 10% , respectively . the rates determined for carbapenem resistant isolates were 66.6% , 51.8% , 33.3% , and 25.9% for netilmicin , tigecycline , sulbactam , and amikacin , respectively . the mic50 values for netilmicin , tigecycline , sulbactam , amikacin , meropenem and colistin were 4 g / ml , 3 g / ml , 8 g / ml , 128 g / ml , 64 g / ml , and 1 g / ml , respectively , while the mic90 values were 512 g / ml , 8 g / ml , 12 g / ml , 1024 g / ml , 128 g / ml , and 1 g / ml , respectively . a. baumannii has become an important health problem due to the fact that it is a pathogen that causes hospital acquired epidemics and due to treatment failures caused by multiple antibiotic resistances ( 2 , 10 ) . the same bacteria obtained from different patients in a clinic shows that there is cross contamination between the patients originating from the same source . in such a case , the source of the microorganism causing the hospital acquired infection must be investigated . through determination of the clonal relationship between the isolates , the source of the infection , the porter , and the way of spread can be revealed , and proper prevention methods can be chosen . the aim of genotyping studies is to determine whether there is an epidemiological relationship between isolated and genotyped strains and to grade this relationship if one exists . rep - pcr is performed with primers specific to a repeated sequence in the genome and can be used in phylogenetic relationship studies for providing reliable results and repeatability ( 11 ) . in a study by caretto et al . , genotyping of acinetobacter isolates was carried out and they emphasized that rep - pcr is an expensive but a rapidly performed and repeatable method ( 12 ) . therefore in this study rep - pcr method ( diversilab , biomrieux , france ) was used for genotyping the isolates as it is a rapid and easy to use method . only genotypically different isolates were evaluated for antimicrobial susceptibilities as the genotypically related isolates could give the same susceptibility patterns . therefore , in the current study the antimicrobial susceptibilities were evaluated for only 30 isolates that were genotypically different and not for the 300 isolates that were collected in a two year period . monotherapy with sulbactam is not recommended for severe acinetobacter infections . however , wood et al . , reported that the use of sulbactam for the treatment of 14 patients with ventilator associated pneumonia caused by multi drug resistant acinetobacter was successful and that there was no difference between 63 patients treated with sulbactam or imipenem in terms of clinical results ( 13 ) . in the study of rodriguez - hernandez et al . , which was carried out on 150 a. baumannii isolates , it was indicated that sulbactam could be a good treatment option for multi resistant a. baumannii infections ( 6 ) . they tested ampicillin , piperacillin , ticarcillin , and beta lactamase inhibitors , such as clavulanic acid , sulbactam , and tazobactam , alone or in combination . it was emphasized that beta lactamase inhibitors , with the exception of sulbactam , had no effect and ampicillin sulbactam was the most effective combination . applied intravenous treatment of sulbactam to 18 patients and intravenous treatment of ampicillin sulbactam to 24 patients out of 42 patients in which multidrug - resistant acinetobacter was isolated . they found that the effect of ampicillin - sulbactam combination is deriven by sulbactam and this combination does not produce a synergistic effect ( 14 ) . furthermore , in an experimental model of pneumonia in which sulbactam sensitive a. baumannii was used , sulbactam was found as effective as imipenem ( 6 , 15 ) . in the current study , we found sulbactam susceptibility in a lower ratio , discordant from the literature . the sensitivity ratio of sulbactam in vitro was found to be 36.6% in all acinetobacter isolates and 34.6% in carbapenem resistant isolates . this ratio was determined when the mic value of sulbactam was accepted as sensitive if it was 4 g / ml . if the mic value 8 g / ml was accepted as sensitive , the sensitivity ratio would be 73.3% . furthermore , the low sulbactam susceptibility of acinetobacter isolates in the current study may be due to selecting clones different from each other and that the isolates were multidrug resistant . although the carbapenems are the most effective antibiotics for the acinetobacter species , recently in studies from turkey and worldwide , it was reported that acinetobacter isolates resistant to carbepenems were isolated with an increasing frequency ( 2 , 16 - 21 ) . there are studies from turkey demonstrating that there is an increasing resistance to imipenem ( 50 - 84% ) and meropenem ( 63 - 80.3% ) ( 18 - 21 ) . in the current study , there was 86.6% resistance to meropenem and3.3% of the isolates were intermediate . the high levels of carbapenem resistance may be due to the multidrug resistance of the isolates and the prolonged duration of empirical treatment with carbapenem at our studied hospital . this study is the first study from turkey where the mic value of sulbactam in acinetobacter was determined alone in vitro . further clinical studies are needed to determine the efficacy of sulbactam on a. baumannii . in the current study , the mic value of sulbactam 4 g / ml was accepted as susceptible , the value of 8 g / ml was accepted as intermediate and the value of 16 g / ml was accepted as resistant . if we had determine the cut - off mic value two folds higher it would have considerably affected the susceptibility results . therefore , it is important to determine the resistance profile for hospital settings in order to choose the proper antibiotic for empirical treatment .

background : the treatment of acinetobacter baumannii infections is difficult . carbapenems , sulbactam , and colistin are the most effective antibiotics.objectives:the aim of this study was to evaluate the susceptibilities of genotypically different a. baumannii isolates to sulbactam , amikacin , netilmicin , meropenem , tigecycline and colistin.patients and methods : isolates from various clinical samples of patients with hospital - acquired infections that were identified by the vitek 2 compact system in our hospital s microbiology laboratory between january 2010 and march 2012 were included in the study . to determine genetic relatedness of the isolates , the rep - pcr method was used . the broth microdilution method was used for amikacin , netilmicin , meropenem and colistin , while e - test was used for sulbactam and tigecycline.results:among the 300 isolates , 30 were found to be genotypically different and were evaluated in terms of their antimicrobial susceptibilities . all isolates were susceptible to colistin . the susceptibility rates were 66.6% , 50% , 36.6% , 30% , and 10% for netilmicin , tigecycline , sulbactam , amikacin , and meropenem , respectively . for carbapenem resistant isolates , the susceptibility rates were 66.6% , 51.8% , 33.3% , and 25.9% for netilmicin , tigecycline , sulbactam , and amikacin , respectively . the sulbactam minimum inhibitory concentration ( mic ) 50 and mic 90 were 8 g / ml and 12 g / ml , respectively.conclusions:in this study , it was concluded that determining the cut - off value for mic breakpoints for sulbactam alonehas a critical impact on the susceptibility results .

the mhc is the region with the most disease associations in the human genome , but by far the greatest number and strongest associations are with autoimmune diseases . in contrast , the chicken mhc has many associations with resistance and susceptibility to infectious disease ( reviewed in kaufman 2008 ) . the chance discovery that a single classical class i and a single classical class ii molecule are expressed at high levels in common chicken mhc haplotypes led us to propose the hypothesis that the bf - bl region of the chicken is a minimal essential mhc . by this , we meant the region responsible for the major histocompatibility reactions that contains single dominantly expressed classical class i and ii molecules whose properties determine the immune response ( kaufman et al . , we also found that there were actually two class i genes with at least one tap gene in between , which led us to propose that co - evolution with the antigen - presenting genes was responsible for the dominantly expressed class i gene ( kaufman et al . ; koch et al . 2007 ; salomonsen et al . 2003 ; shaw et al . 2007 ; walker et al . 2011 ; wallny et al . 2006 ) ; including the fact that many if not most non - mammalian vertebrates ( as well as at least one marsupial , belov et al . 2007 ) have these salient features of the chicken mhc , including the strong associations of the mhc with infectious disease ( grimholt et al . however , there is some confusion in the literature over whether the chicken mhc really is small and simple or is actually large and complicated . many investigators , including ourselves , have characterised genes located on the chicken mhc microchromosome that in other animals are found in regions of the mhc , adjacent to the mhc , or in mhc paralogous regions ( afanassieff et al . 2001 ; briles et al . 1993 ; guillemot et al . 1988 ; kaufman et al . 1994a , b , 1996 , 2005 ; rogers et al . 2005 ; rogers and kaufman 2008 ; ruby et al . 2005 ; salomonsen et al . this has led to an alternative view that the chicken mhc is in fact very large and complex , containing many genes located in at least two separate regions , called mhc - b and mhc - y ( afanassieff et al . we have concluded that the difference in these views lies in emphasis ( kaufman et al . 1995 , 1999a ) defines an mhc based on function , a region of the genome containing the classical polymorphic class i and class ii genes responsible for strong histocompatibility reactions ( that is , a major histocompatibility complex ) . the alternative view defines the mhc as a genomic region , with any gene ever found in or adjacent to an mhc becoming part of that mhc , and with the term itself becoming a genetic acronym rather than a functional definition . in our view , the minimal essential mhc is the selected and conserved unit of classical mhc molecules ( which we call " the classical mhc " and which has also been dubbed " the core mhc " , shiina et al . 2007 ) , around which other genes come and go as part of the mhc syntenic region ( which has also been referred to as " the extended mhc " ) . whichever way one wishes to view the chicken mhc , there is no doubt that there is a small and simple region ( the bf - bl region ) which contains the essential polymorphic components of antigen presentation by classical mhc molecules , and that this close proximity of classical mhc genes with the polymorphic genes that process and load their peptides is a feature of many , if not most , non - mammalian vertebrates . it therefore remains interesting and important to test to what extent the single dominantly expressed mhc molecules of the chicken mhc can explain the strong genetic associations with disease resistance and vaccine responses . we have determined simple peptide motifs for certain chicken class i molecules based on the peptides eluted from chicken class i molecules isolated from spleen and blood cells ( kaufman et al . there are strong mhc associations for resistance and susceptibility to the pathogens rous sarcoma virus ( rsv ) and marek s disease virus ( mdv ) , and we found many more peptides predicted to bind class i molecules from resistant than from susceptible chicken lines . we used cell binding and assembly assays to determine which of the peptides predicted by the simple motifs would actually bind the class i molecules . for rsv , we found that vaccination with a single identified peptide would eliminate disease ( hofmann et al . since then , the utility of these motifs has been confirmed by other researchers for the pathogen avian influenza virus ( hou et al . gumburo disease is caused by an immunosuppressive birnavirus called infectious bursal disease virus ( ibdv ) that infects and kills cells of the b lineage in chickens ( reviewed in ingrao et al . chicks up to 2 weeks old frequently survive , although the bursa is ravaged , the b cell system is depleted , and thereafter birds do not respond well to vaccines . there follows a period of susceptibility , peaking at 34 weeks of age , during which a virulent virus will kill birds . with time , an increasing resistance to mortality develops , although the birds may still suffer immunosuppression and morbidity , evidenced by weight loss or reduction in egg - laying . the disease is controlled mainly by vaccination in ovo , vaccination of chicks or vaccination of laying hens , usually with an attenuated live viral vaccine and often followed by an inactivated vaccine , both of which raise antibodies mostly to the major capsid protein , viral protein 2 ( vp2 ) . however , both antigenic variation and increases in virulence of the virus have been reported , so gumboro disease is still an economic concern for the poultry industry , and various approaches to vaccines continue to be developed . an early experimental vaccine called fp - ibd1 was created with the vp2 as a fusion protein with -galactosidase in a fowlpox virus vector ( bayliss et al . this vaccine protects some experimental chicken lines but not others ( shaw and davison 2000 ) . in particular , line 61 ( homozygous for mhc haplotype b2 ) and line c - b12 ( homozygous for b12 ) were shown to be protected by fp - ibd1 , while line 15i ( homozygous for b15 ) was not . the protection in line 61 was partially conferred by the fowlpox virus vector alone , while the protection in line c - b12 was not . in both cases , the protection was found not to be due to antibodies , which suggested the involvement of t cells recognising mhc molecules ( shaw and davison 2000 ) . in this report , we extend this work to examine the contribution of the chicken mhc to protection by the vaccine fp - ibd1 . the histories of the chicken lines 61 ( b2 ) , c - b4 ( b4 ) , c - b12 ( b12 ) and 15i ( b15 ) , bred and maintained under specific pathogen - free conditions at the institute for animal health ( compton , uk ) , have been described ( jacob et al . chicks were transferred at 1 day of age to isolation accommodation equipped with high - efficiency particle air filters . all procedures were carried out in accordance the united kingdom animals ( scientific procedures ) act and were subject to local ethical review . f1 crosses were produced between c - b12 and 15i birds and between 61 and 15i birds . typing by microsatellite analysis ( khatib et al . 1993 ) yielded 35 mhc heterozygous and 25 mhc homozygous progeny from the 15i x ( 61 15i ) backcross , and 24 mhc heterozygous and 25 mhc homozygous progeny from the 15i ( c - b12 15i ) backcross . together with 30 birds of each parental line , all chicks were vaccinated with fp - ibd1 and challenged with f52/70 as described below . the fowlpox virus recombinant vaccine fp - ibd1 encodes the vp2 sequence of the virulent ibvd virus f52/70 as a -galactosidase fusion protein ( bayliss et al . 1991 ) and was produced by tissue culture passage on chick embryo fibroblasts as previously described ( shaw and davidson 2000 ) . chicks were vaccinated at 1 and 3 weeks of age with 10 plaque - forming units fp - ibd1 in a total volume of 50 l . the vaccine was dropped onto a section of the wing web , and the skin punctured 30 times with a 25-gauge needle . local inflammation was evident at the site of scarification within 2 days and resolved shortly after . ibdv f52/70 was propagated in vivo , and the active concentration of virus was assayed to determine egg infectious dose ( eid ) as previously described ( butter et al . chicks were challenged with 10 eid50 ibdv f52/70 in a total volume of 100 l by the intranasal route , 10 days after the second vaccination with either peptide ( see below ) or fp - ibd1 ( see above ) . as in wallny ( 2006 ) , peptides were predicted from vp2 sequence derived from f52/70 ( accession number d00869.2 ) by using the gcg program findpatterns ( anonymous 1991 ) and relaxed motifs for the dominantly expressed class i molecules of two chicken haplotypes . these relaxed motifs allow both octamers and nonamers with all residues found in anchor positions by pool sequencing ( wallny et al . the motifs are denoted for findpatterns by the notation xxx(x , xx)(v , i)xx(v , l , i ) for b12 and xrxxxx(x , xx)y for b15 , in which x denotes any amino acid , single capital letters refer to amino acids in the iupac single letter code and parentheses enclose alternatives , so ( x , xx ) means either one or two amino acids , and ( v , l , i ) means either valine , leucine or isoleucine . peptides were synthesized by fluorenylmethoxycarbonyl chemistry , purified by hplc , and resuspended at 1 mg / ml in pbs ( with up to 1 % dmso ) . as in wallny ( 2006 ) , chicken peripheral blood lymphocytes without thrombocytes were cultured ( 10 cells per ml at 40 c , 5 % co2 ) overnight in dulbecco s modified minimal eagle s medium with 0.5 mg / ml bovine serum albumin ( bsa ) and with or without 1 mm synthetic peptides ( in triplicate ) . then , cells were washed ( phosphate - buffered saline , 0.5 % bsa , 0.1 % nan3 ) , and flow cytometric analysis using a monoclonal antibody against chicken 2-microglobulin ( f21 - 21 , skjdt et al . 1986 ) followed by fitc - conjugated goat anti - mouse igg ( silenus , paris ) was performed , gating on small lymphocytes . as in koch et al . ( 2007 ) , inclusion bodies from 2 m cdna sequence ( encoding the mature protein without a signal sequence but beginning with a start codon for methionine ) and for bf2 * 1201 cdna ( residues 1270 of the mature protein without a signal sequence but with a codon for met added at the beginning ) in pet22b(+ ) were isolated from bl21 ( de3 ) plyss rosetta cells , and dissolved in 8 m urea , 10 mm 2-mercaptoethanol , 100 mm nah2po4 , 10 mm triscl , ph 8 . small - scale assembly assays were carried out at 4 c in 1 ml refold buffer [ 100 mm triscl ph 8.2 , 400 mm arginine , 0.5 mm oxidized glutathione , 5 mm reduced glutathione , 2 mm ethylenediamine tetraacetic acid , 0.1 mm 4-(2 aminoethyl ) benzenesulfonyl fluoride ( pefablock , sigma ) ] . the peptide , 2 m and heavy chain were added slowly with vigorous stirring in a molar ratio of 10:2:1 ( usually around 1:24:31 g in 1 ml ) . after 1830 h , fplc size exclusion chromatography ( akta , pharmacia ) with a hiload 26/60 superdex 75 column ( pharmacia ) in 0.15 m nacl , 0.1 m triscl ph 8.2 was used to separate aggregates , monomers , heavy chains , 2 m , peptides and smaller molecules . as in hofmann et al . ( 2003 ) , 18 mg l--phosphatidylcholine , 2 mg l--phosphatidyl - dl - glycerol and 5 mg cholesterol ( all components from sigma ) were suspended in 5 ml chloroform and rotary evaporated under reduced pressure until a thin lipid film formed on the flask wall . three milligrams of peptide was dissolved in 1 ml pbs containing 0.4 mg quil a and 1 mm 2-mercaptoethanol . this solution was added to the dried lipids and slowly shaken until the lipids were re - suspended and then equilibrated 30 min at room temperature . c - b12 chicks were immunised at 1 and 3 weeks of age with 100 g peptide in 0.2 ml of liposomes per injection , i.m . into the pectoral muscle . some other chicks were not vaccinated , some others were vaccinated with fp - ibd1 and most chicks were challenged with f52/70 as described above . five days post - infection , the birds were sacrificed , the bursa dissected , rna extracted by standard techniques ( staines et al . bursal / body weight ratio , bursal damage score , peak blood viremia and bursal viral load were used as post - mortem measures of pathology . briefly , a histological scoring system ranging from 0 , representing normal bursal architecture , to 5 , representing complete loss of bursal architecture , was used to assess bursal histopathology . all slides were encoded and the scoring carried out blind , with the identity of the samples unknown . quantitative rt - pcr was performed to determine relative amounts of vp2 gene transcripts in blood and bursal samples using the primer - probe combinations previously described ( moody et al . 2000 ) and the results normalised against the expression of 28s rna , determined in a duplex reaction with vp2 , and presented as log2ro ( butter et al . given that fp - ibd1 elicits very different levels of protection in different strains of chicken , we examined whether the mhc is a major genetic locus in this response . using the high responder lines ( line 61 homozygous for mhc haplotype b2 , c - b12 homozygous for b12 ) and a low responder line ( 15i homozygous for b15 ) , we first bred f1 chickens ( 61 15i heterozygous for b2/b15 , and c - b12 15i heterozygous for b12/b15 ) , then bred two backcross families , immunised with fp - ibd1 , challenged with the virulent ibdv strain f52/70 , and measured damage to the bursa of fabricius . in the first comparison ( fig . 1 ) , the majority of the chickens of the low responder parental line 15i ( b15/b15 ) had a high bursal damage score , while nearly all of the chickens of the high responder parental line 61 ( b2/b2 ) had little or no bursal damage . from the backcross 15i f1 ( 61 15i ) family , about one quarter of the birds had a high bursal damage score , and the rest had little or no damage . after typing these chickens , we found that nearly all of the b15/b2 heterozygotes had little or no damage , but that half of the b15/b15 homozygotes had little damage and the other half had a high bursal damage score . since genes outside of the mhc should have segregated randomly , this shows that a single b2 haplotype in a b2/b15 heterozygote chicken confers protection , which was as much as two b2 haplotypes in a b2/b2 homozygote chicken . however , there must be another gene outside of the mhc which confers responsiveness as well since half of the b15/b15 homozygote chickens were protected . thus , there are two genetic loci that confer the responsiveness found in line 61 chickens.fig . 1backcross mapping of protection conferred by fp - ibd1 vaccination to bursal damage by ibdv strain f52/70 challenge in two families derived from line 61 ( mhc haplotype b2 ) , cb-12 ( b12 ) and 15i ( b15 ) . the graphs represent bursal damages score , with 0 as no perceptible damage and 5 as extremely damaged ( x - axis ) against percentage birds with that score ( y - axis ) . upper two panels are birds from the parental lines 15i and 61 , and birds from the backcross family 15i f1(15i 61 ) segregated into the homozygotes and heterozygotes . lower two panels are birds from the parental lines 15i and c - b12 , and birds from the backcross family 15i f1(15i c - b12 ) segregated into the homozygotes and heterozygotes backcross mapping of protection conferred by fp - ibd1 vaccination to bursal damage by ibdv strain f52/70 challenge in two families derived from line 61 ( mhc haplotype b2 ) , cb-12 ( b12 ) and 15i ( b15 ) . the graphs represent bursal damages score , with 0 as no perceptible damage and 5 as extremely damaged ( x - axis ) against percentage birds with that score ( y - axis ) . upper two panels are birds from the parental lines 15i and 61 , and birds from the backcross family 15i f1(15i 61 ) segregated into the homozygotes and heterozygotes . lower two panels are birds from the parental lines 15i and c - b12 , and birds from the backcross family 15i f1(15i c - b12 ) segregated into the homozygotes and heterozygotes in the second comparison ( fig . 1 ) , the majority of the chickens of the low responder parental line 15i ( b15/b15 ) had a high bursal damage score , while the majority of the high responder parental line c - b12 ( b12/b12 ) had little or no bursal damage . from the backcross 15i f1(c - b12 15i ) family , about half of the birds had a high bursal damage score , while half had less damage . after typing these chickens , we found that most of the b15/b15 homozygote chickens had high bursal damage score , but most of the b12/b15 heterozygote chickens had a moderate bursal damage score . since genes outside of the mhc should have segregated randomly , this shows that a single b12 haplotype in a b12/b15 heterozygote chicken confers about half as much protection as two b12 haplotypes in a b12/b12 homozygote chicken . thus , there is one major genetic locus that confers responsiveness found in line c - b12 chickens which is the mhc , but two copies of the c - b12 haplotype are necessary for the highest level of protection under these circumstances . given that the fp - ibd1 vaccine contains ibdv vp2 protein as a fusion protein with -galactosidase and that the mechanism of protection does not involve antibodies , it seemed most likely that the response was directed against peptides presented by class i molecules . having determined peptide motifs for the class i molecules of the b12 and b15 haplotypes ( bf2 * 1201 and the bf2 * 1501 ) , we used these motifs to predict peptides from the vp2 protein sequence that might bind these class i molecules . we found 12 peptides that fit the b12 peptide motif , but only 2 peptides that fit the b15 peptide motif ( fig . 2cell binding and assembly analysis of vp2 peptides predicted to bind b12 and b15 class i molecules . left hand panel , mean fluorescence intensities measuring the increase of f21 - 21 binding to cells after culture overnight with 1 mm peptide compared to culture overnight without added peptide . right hand panel , ratio of the area of peaks corresponding to assembled monomer compared to 2 m from size exclusion chromatography of renaturation assays with indicated peptides cell binding and assembly analysis of vp2 peptides predicted to bind b12 and b15 class i molecules . left hand panel , mean fluorescence intensities measuring the increase of f21 - 21 binding to cells after culture overnight with 1 mm peptide compared to culture overnight without added peptide . right hand panel , ratio of the area of peaks corresponding to assembled monomer compared to 2 m from size exclusion chromatography of renaturation assays with indicated peptides from work on mammals ( and our own work with these same chicken class i molecules in a previous study ) , generally only a fraction of peptides that fit the simple kind of motif used here actually bind the class i molecule with measurable affinity . so , we tested the binding of synthetic peptides to chicken peripheral blood lymphocytes with an assay we have used previously ( wallny et al . 2006 ) , a flow cytometry - based class i stabilisation assay analogous in procedure to the rma - s and t2 stabilization assays using mammalian cells with defective tap genes . as assessed by the fluorescence in comparison with no peptide controls , neither of the two b15 peptides predicted bound to b15 cells , whereas many of the predicted b12 peptides did bind ( fig . in addition , we tested the b12 peptides by an assembly assay , in which b12 heavy chain and 2 m were renatured in the presence of peptide , and the material analysed by size exclusion chromatography , as illustrated in fig . 3 . since the amount of 2 m in the assay was far in excess , ratio of the areas under the peaks of monomer was taken as a measure of the success of the assembly of heavy chain and 2 m dependent on peptide . ten of the 12 predicted b12 peptides supported assembly very well , in agreement with the stabilisation assay ( fig . class i heavy chain was renatured with 2 m and peptide ibdv-8 , the components separated by size exclusion chromatography , and the areas under the peaks corresponding to reassembled monomer and free 2 m ( labelled and indicated by dashed boxes ) determined an example of an assembly assay . class i heavy chain was renatured with 2 m and peptide ibdv-8 , the components separated by size exclusion chromatography , and the areas under the peaks corresponding to reassembled monomer and free 2 m ( labelled and indicated by dashed boxes ) determined having found many peptides from the vaccine fp - ibd1 that bind bf2 * 1201 , the dominantly expressed class i molecule from the b12 haplotype , we wanted to test whether immunisation of c - b12 chickens with one of them would protect against challenge by ibdv . we had previously developed a peptide immunisation protocol that protected against tumour progression after infection of chickens by rous sarcoma virus . we used the same protocol of intramuscular injection of liposomes loaded with quil a and various peptides , in comparison with no immunisation and with fp - ibd1 immunisation . three days after challenge with ibdv strain f52/70 , we measured bursal / body weight ratio , bursal damage score , peak blood viremia and bursal viral load ( figs . 4means of bursal / body weight ratio ( 1,000 ; upper panel ) , bursal damage score ( second panel ) , peak blood viremia ( log2ro ; third panel ) and bursal viral load ( log2ro ; lower panel ) in line c - b12 birds with no challenge , or after ibdv f52/70 challenge following vaccination with nothing , fp - ibd1 or different peptides . as an indication of variability means are shown with their standard errors ( though the data do not conform to the strict requirements of parametric analysis)fig . 5percentages of line c - b12 birds with different bursal damage scores after no challenge , or after ibdv f52/70 challenge following vaccination with nothing , fp - ibd1 or different peptides means of bursal / body weight ratio ( 1,000 ; upper panel ) , bursal damage score ( second panel ) , peak blood viremia ( log2ro ; third panel ) and bursal viral load ( log2ro ; lower panel ) in line c - b12 birds with no challenge , or after ibdv f52/70 challenge following vaccination with nothing , fp - ibd1 or different peptides . as an indication of variability means are shown with their standard errors ( though the data do not conform to the strict requirements of parametric analysis ) percentages of line c - b12 birds with different bursal damage scores after no challenge , or after ibdv f52/70 challenge following vaccination with nothing , fp - ibd1 or different peptides as expected , without the challenge infection , chickens had a normal bursa to body weight ratio , a low bursal damage score and no virus in the blood or bursa . all four measures rose after infection without vaccination , as well as after vaccination with two peptides , the control peptide from rsv ( rsv6 ) and one of the peptides from fp - ibd1 ( ibdv12 ) . in contrast , protection was evident by all measures after vaccination with fp - ibd1 and even more so after vaccination with another peptide from fp - ibd1 ( ibdv8 ) . thus , immunisation with a peptide from ibdv that binds the dominantly expressed class i molecule bf2 * 1201 will protect from challenge with a virulent ibdv ( figs . 4 and 5 ) . given that fp - ibd1 elicits very different levels of protection in different strains of chicken , we examined whether the mhc is a major genetic locus in this response . using the high responder lines ( line 61 homozygous for mhc haplotype b2 , c - b12 homozygous for b12 ) and a low responder line ( 15i homozygous for b15 ) , we first bred f1 chickens ( 61 15i heterozygous for b2/b15 , and c - b12 15i heterozygous for b12/b15 ) , then bred two backcross families , immunised with fp - ibd1 , challenged with the virulent ibdv strain f52/70 , and measured damage to the bursa of fabricius . in the first comparison ( fig . 1 ) , the majority of the chickens of the low responder parental line 15i ( b15/b15 ) had a high bursal damage score , while nearly all of the chickens of the high responder parental line 61 ( b2/b2 ) had little or no bursal damage . from the backcross 15i f1 ( 61 15i ) family , about one quarter of the birds had a high bursal damage score , and the rest had little or no damage . after typing these chickens , we found that nearly all of the b15/b2 heterozygotes had little or no damage , but that half of the b15/b15 homozygotes had little damage and the other half had a high bursal damage score . since genes outside of the mhc should have segregated randomly , this shows that a single b2 haplotype in a b2/b15 heterozygote chicken confers protection , which was as much as two b2 haplotypes in a b2/b2 homozygote chicken . however , there must be another gene outside of the mhc which confers responsiveness as well since half of the b15/b15 homozygote chickens were protected . thus , there are two genetic loci that confer the responsiveness found in line 61 chickens.fig . 1backcross mapping of protection conferred by fp - ibd1 vaccination to bursal damage by ibdv strain f52/70 challenge in two families derived from line 61 ( mhc haplotype b2 ) , cb-12 ( b12 ) and 15i ( b15 ) . the graphs represent bursal damages score , with 0 as no perceptible damage and 5 as extremely damaged ( x - axis ) against percentage birds with that score ( y - axis ) . upper two panels are birds from the parental lines 15i and 61 , and birds from the backcross family 15i f1(15i 61 ) segregated into the homozygotes and heterozygotes . lower two panels are birds from the parental lines 15i and c - b12 , and birds from the backcross family 15i f1(15i c - b12 ) segregated into the homozygotes and heterozygotes backcross mapping of protection conferred by fp - ibd1 vaccination to bursal damage by ibdv strain f52/70 challenge in two families derived from line 61 ( mhc haplotype b2 ) , cb-12 ( b12 ) and 15i ( b15 ) . the graphs represent bursal damages score , with 0 as no perceptible damage and 5 as extremely damaged ( x - axis ) against percentage birds with that score ( y - axis ) . upper two panels are birds from the parental lines 15i and 61 , and birds from the backcross family 15i f1(15i 61 ) segregated into the homozygotes and heterozygotes . lower two panels are birds from the parental lines 15i and c - b12 , and birds from the backcross family 15i f1(15i c - b12 ) segregated into the homozygotes and heterozygotes in the second comparison ( fig . 1 ) , the majority of the chickens of the low responder parental line 15i ( b15/b15 ) had a high bursal damage score , while the majority of the high responder parental line c - b12 ( b12/b12 ) had little or no bursal damage . from the backcross 15i f1(c - b12 15i ) family , about half of the birds had a high bursal damage score , while half had less damage . after typing these chickens , we found that most of the b15/b15 homozygote chickens had high bursal damage score , but most of the b12/b15 heterozygote chickens had a moderate bursal damage score . since genes outside of the mhc should have segregated randomly , this shows that a single b12 haplotype in a b12/b15 heterozygote chicken confers about half as much protection as two b12 haplotypes in a b12/b12 homozygote chicken . thus , there is one major genetic locus that confers responsiveness found in line c - b12 chickens which is the mhc , but two copies of the c - b12 haplotype are necessary for the highest level of protection under these circumstances . given that the fp - ibd1 vaccine contains ibdv vp2 protein as a fusion protein with -galactosidase and that the mechanism of protection does not involve antibodies , it seemed most likely that the response was directed against peptides presented by class i molecules . having determined peptide motifs for the class i molecules of the b12 and b15 haplotypes ( bf2 * 1201 and the bf2 * 1501 ) , we used these motifs to predict peptides from the vp2 protein sequence that might bind these class i molecules . we found 12 peptides that fit the b12 peptide motif , but only 2 peptides that fit the b15 peptide motif ( fig . 2cell binding and assembly analysis of vp2 peptides predicted to bind b12 and b15 class i molecules . left hand panel , mean fluorescence intensities measuring the increase of f21 - 21 binding to cells after culture overnight with 1 mm peptide compared to culture overnight without added peptide . right hand panel , ratio of the area of peaks corresponding to assembled monomer compared to 2 m from size exclusion chromatography of renaturation assays with indicated peptides cell binding and assembly analysis of vp2 peptides predicted to bind b12 and b15 class i molecules . left hand panel , mean fluorescence intensities measuring the increase of f21 - 21 binding to cells after culture overnight with 1 mm peptide compared to culture overnight without added peptide . right hand panel , ratio of the area of peaks corresponding to assembled monomer compared to 2 m from size exclusion chromatography of renaturation assays with indicated peptides from work on mammals ( and our own work with these same chicken class i molecules in a previous study ) , generally only a fraction of peptides that fit the simple kind of motif used here actually bind the class i molecule with measurable affinity . so , we tested the binding of synthetic peptides to chicken peripheral blood lymphocytes with an assay we have used previously ( wallny et al . 2006 ) , a flow cytometry - based class i stabilisation assay analogous in procedure to the rma - s and t2 stabilization assays using mammalian cells with defective tap genes . as assessed by the fluorescence in comparison with no peptide controls , neither of the two b15 peptides predicted bound to b15 cells , whereas many of the predicted b12 peptides did bind ( fig . 2 ) . in addition , we tested the b12 peptides by an assembly assay , in which b12 heavy chain and 2 m were renatured in the presence of peptide , and the material analysed by size exclusion chromatography , as illustrated in fig . 3 . since the amount of 2 m in the assay was far in excess , ratio of the areas under the peaks of monomer was taken as a measure of the success of the assembly of heavy chain and 2 m dependent on peptide . ten of the 12 predicted b12 peptides supported assembly very well , in agreement with the stabilisation assay ( fig . class i heavy chain was renatured with 2 m and peptide ibdv-8 , the components separated by size exclusion chromatography , and the areas under the peaks corresponding to reassembled monomer and free 2 m ( labelled and indicated by dashed boxes ) determined an example of an assembly assay . class i heavy chain was renatured with 2 m and peptide ibdv-8 , the components separated by size exclusion chromatography , and the areas under the peaks corresponding to reassembled monomer and free 2 m ( labelled and indicated by dashed boxes ) determined having found many peptides from the vaccine fp - ibd1 that bind bf2 * 1201 , the dominantly expressed class i molecule from the b12 haplotype , we wanted to test whether immunisation of c - b12 chickens with one of them would protect against challenge by ibdv . we had previously developed a peptide immunisation protocol that protected against tumour progression after infection of chickens by rous sarcoma virus . we used the same protocol of intramuscular injection of liposomes loaded with quil a and various peptides , in comparison with no immunisation and with fp - ibd1 immunisation . three days after challenge with ibdv strain f52/70 , we measured bursal / body weight ratio , bursal damage score , peak blood viremia and bursal viral load ( figs . 4 and 5).fig . 4means of bursal / body weight ratio ( 1,000 ; upper panel ) , bursal damage score ( second panel ) , peak blood viremia ( log2ro ; third panel ) and bursal viral load ( log2ro ; lower panel ) in line c - b12 birds with no challenge , or after ibdv f52/70 challenge following vaccination with nothing , fp - ibd1 or different peptides . as an indication of variability means are shown with their standard errors ( though the data do not conform to the strict requirements of parametric analysis)fig . 5percentages of line c - b12 birds with different bursal damage scores after no challenge , or after ibdv f52/70 challenge following vaccination with nothing , fp - ibd1 or different peptides means of bursal / body weight ratio ( 1,000 ; upper panel ) , bursal damage score ( second panel ) , peak blood viremia ( log2ro ; third panel ) and bursal viral load ( log2ro ; lower panel ) in line c - b12 birds with no challenge , or after ibdv f52/70 challenge following vaccination with nothing , fp - ibd1 or different peptides . as an indication of variability means are shown with their standard errors ( though the data do not conform to the strict requirements of parametric analysis ) percentages of line c - b12 birds with different bursal damage scores after no challenge , or after ibdv f52/70 challenge following vaccination with nothing , fp - ibd1 or different peptides as expected , without the challenge infection , chickens had a normal bursa to body weight ratio , a low bursal damage score and no virus in the blood or bursa . all four measures rose after infection without vaccination , as well as after vaccination with two peptides , the control peptide from rsv ( rsv6 ) and one of the peptides from fp - ibd1 ( ibdv12 ) . in contrast , protection was evident by all measures after vaccination with fp - ibd1 and even more so after vaccination with another peptide from fp - ibd1 ( ibdv8 ) . thus , immunisation with a peptide from ibdv that binds the dominantly expressed class i molecule bf2 * 1201 will protect from challenge with a virulent ibdv ( figs . 4 and 5 ) . in this paper , we have shown that ( 1 ) response to a molecular defined vaccine containing the vp2 sequence of ibdv can be determined by at least two genetic loci , one of which is the mhc , ( 2 ) absence of vp2 peptides that bind the class i molecule is the basis for the lack of response in a low responder line and ( 3 ) presence of at least one vp2 peptide that binds the dominantly expressed class i molecule is the basis for the protection in a high responder line . the work in this paper grew from earlier observations ( shaw and davison 2000 ) that vaccination with fp - ibd1 could protect some experimental chicken lines and not others . examination of the mechanisms of protection showed that antibody was not involved , which is in accord with the fact that the vp2 protein as a fusion protein with -galactosidase in the vaccine is likely to have a different three - dimensional structure compared to vp2 protein as a part of the ibdv capsid . these examinations also showed that line c - b12 was protected by vaccination with fp - ibd1 but not by the fowlpox virus vector alone , while line 61 was protected somewhat even by the fowlpox virus vector alone . line 15i is a low responder with no protective locus , c - b12 is a high responder with only one major protective locus ( the mhc ) and line 61 is a high responder with two major protective loci ( the mhc which presumably depends on the vp2 fusion protein , and another locus which may determine response to fowlpox virus vector alone , perhaps through an innate antiviral response like inflammation or natural killer cell responses ) . from our peptide predictions and peptide vaccination , line 15i is a low responder because the classical class i molecule bf2 * 1501 does not bind peptides from the vp2 sequence , while line c - b12 is a high responder because the dominantly expressed class i molecule bf2 * 1201 finds at least one vp2 peptide ( ibdv-8 ; gnvlvgegv ) that confers protection to challenge with a virulent ibdv strain . it must be stressed that ibdv-8 may not be the only peptide that is responsible for protection , but that there is at least one such peptide . on the basis of these results , we would predict ( but did not show ) that line 61 is a responder because the dominantly expressed class i molecule bf2 * 0201 presents at least one protective ibdv peptide . first , after vaccination with fp - ibd1 , the protection of b12/b15 heterozygotes in the backcross was half of that of b12/b12 homozygotes in the original line . this most likely indicates that the level of peptide bound to bf2 * 1201 on the surface of cells is a critical factor in priming the immune response ( as has been shown for mammals , see for example bullock et al . 2000 ) , and that the particular conditions of vaccination are just barely sufficient to protect from the particular conditions of challenge . second , vaccination with the vp2 peptide ibdv-12 ( lahaigegv ) did not result in a protective immune response after vaccination , despite the fact that this peptide bound well to bf2 * 1201 . there are no structural differences between ibdv-8 and ibdv-12 which obviously account for this difference . the protective ibdv-8 peptide is located in the linker region between the p and s domains of vp2 , while the non - protective binding cb12-ibdv-12 epitope is located deep in the s domain ( lee et al . 2006 ) and both peptides are well conserved ( indeed identical in a wide variety of ibdv sequences , including strain 52/70 , accession number baa00745 ; strain cu1 , baa00740 ; strain stc , baa00391 ; strain e / del , caa38637 ; strain 23/82 , z21971 ; strain ct , cac35470 ; strain uk661 , cac60256 ; strain 88180 , caj34339 ; isolate 903/78 , afi55462 ; isolate sp8 , afn80509 ) . one explanation might be that there is a hole in the t cell repertoire due to negative selection in the thymus ( as has been reported in mammals , see for example wlfl et al . the previous work ( shaw and davison 2000 ) used our motifs to predict the peptides for the dominantly expressed class i molecule bf2 * 0401 from line c - b4 and concluded in discussion that there were no peptides that would bind , but nevertheless found that fp - ibd1 conferred the same level of protection to line c - b4 as line c - b12 . one potential explanation is that another genetic locus in c - b4 is responsible for the protection , but that seems unlikely since c - b4 and c - b12 chickens are taken ( as individuals or as annual sublines ) from a c line flock , and so all non - mhc genes should segregate freely between the lines . another potential explanation is that the response is due to the minor gene bf1 * 0401 , but this gene has virtually undetectable expression at the level of rna , protein and antigenic peptide . therefore , the most likely explanation for the response of c - b4 is that the previous work used a very stringent peptide motif ( octamers with asp2 , asp5 and glu8 , as we published in kaufman et al . 1995 ) , but in fact the motif can be somewhat wider ( wallny et al . the motif and even individual peptides show that glu is also found at positions 2 and 5 , and certain hydrophobic amino acids found at a low level at position 8 . moreover , some peptides may be longer , and there may be c - terminal overhangs , as we found for b12 and other haplotypes . in fact , this more relaxed motif does indeed predict 4 peptides from the vp2 in fp - ibd1 . these results are all in accord with the concept of a minimal essential mhc ( kaufman et al . 1995 , 1999a , b ; kaufman 2008 ) , in which the structural organisation of the chicken mhc ( compared to most mammals ) has striking effects on function . we have previously shown that properties of the single dominantly expressed class i molecule can explain the responses of experimental chicken lines to rsv and mdv ( wallny et al . 2007 ) , and the results in this paper extend this concept from viral pathogens to viral vaccines . moreover , ibdv continues to be a serious threat to commercial chickens , with both antigenic variation and increase in virulence reported ( reviewed in ingrao et al . while the poultry industry generally controls ibdv through inactivated vaccines that work through class ii molecules ( which also show the phenomenon of mhc - determined differences in response ) , some attenuated viral vaccines are used , particularly in ovo ( reviewed in mller et al . the likelihood that some individual chickens remain poorly protected after either form of vaccination is an unsolved problem . given that the mhc of many , if not most , non - mammalian vertebrates have features first discovered in chickens ( reviewed in kaufman 1999 , 2008 ) , it is likely that this is also a problem in other important contexts , for instance vaccination in some commercial fish ( grimholt et al . 2003 ) or resistance to citrid fungi in amphibians ( savage and zamudio 2011 ) .

in contrast to typical mammals , the chicken mhc ( the bf - bl region of the b locus ) has strong genetic associations with resistance and susceptibility to infectious pathogens as well as responses to vaccines . we have shown that the chicken mhc encodes a single dominantly expressed class i molecule whose peptide - binding motifs can determine resistance to viral pathogens , such as rous sarcoma virus and marek s disease virus . in this report , we examine the response to a molecular defined vaccine , fp - ibd1 , which consists of a fowlpox virus vector carrying the vp2 gene of infectious bursal disease virus ( ibdv ) fused with -galactosidase . we vaccinated parental lines and two backcross families with fp - ibd1 , challenged with the virulent ibdv strain f52/70 , and measured damage to the bursa . we found that the mhc haplotype b15 from line 15i confers no protection , whereas b2 from line 61 and b12 from line c determine protection , although another locus from line 61 was also important . using our peptide motifs , we found that many more peptides from vp2 were predicted to bind to the dominantly expressed class i molecule bf2 * 1201 than bf2 * 1501 . moreover , most of the peptides predicted to bind bf2 * 1201 did in fact bind , while none bound bf2 * 1501 . using peptide vaccination , we identified one b12 peptide that conferred protection to challenge , as assessed by bursal damage and viremia . thus , we show the strong genetic association of the chicken mhc to a t cell vaccine can be explained by peptide presentation by the single dominantly expressed class i molecule .

clinic of obstetrics , clinical center university of sarajevo is a tertiary level of health care with more than 3,000 births per year . during the 1992 - 1995 war the old building of the clinic of obstetrics and gynecology was the target of destruction , so the clinic was relocated into another building within the clinical center university of sarajevo . during 2010 the original building of the clinic of obstetrics and gynecology at the location jezero in sarajevo was rebuild , officially opened and put into operation on november 25 , 2010 . two years later , there was the reorganization of the clinic operation , so in the framework of the existing building was organized a discipline that consists of two clinics or the clinic of obstetrics and gynecology clinic . clinic of obstetrics currently has about 15 doctors , specialist in obstetrics and gynecology , more than 10 doctors on residency and about 20 nurses and other supporting staff . birth room of the clinic of obstetrics is organized to provide services 24 hours a day throughout the year . in the delivery rooms are located the most modern equipment for vaginal delivery , with a total of 10 beds for delivery , with room for delivery in the presence of a spouse ( partner ) . as part of the clinic of obstetrics , there are operating rooms with direct access from the delivery room in case of emergencies . the goal is to show the operation at the birth room of clinic for gynecology and obstetrics for the year 2012 , frequency of completed deliveries by caesarian section , also describe manners of delivery and to provide a description of other techniques and manual interventions that have been applied during childbirth . the study was conducted at the clinic of obstetrics , clinical center university of sarajevo . a database based on the data from the delivery room protocol is made in which are entered all details regarding delivery ( parity , mode of delivery , interventions during birth , information on newborn ) . data were entered in ms excel , imported into the statistical program spps v.20.0 and then statistically analyzed . for the statistical analysis of qualitative data we used chi - square tests , and for quantifying data anova . in the study period of one year at the clinic of obstetrics there were 3216 ( 100% ) deliveries . of the total number of births vaginally completed delivery was in 2101 ( 65% ) cases and cesarean section in 1115 ( 35% ) cases ( figure 3 ) . the analysis of the frequency of the number of births per month reveals that the lowest number of births per month was during february with a total of 234 ( 7.27% ) and the highest percentage in september ( 9.48% ) . the average number of births per day in the 2012 amounted to 8.81 ( figure 4 ) . anova test showed a statistically significant difference in the incidence of cesarean sections on a monthly basis ( p<0.05 ) . in the 2012 the total of 48 pregnant women completed delivery by caesarean section after a failed trial of vaginal delivery ( figure 5 ) . of the total number of pregnant women delivered during the study period in 1603 ( 49.84% ) cases they were nulliparous , second child had 1186 ( 36.87% ) , while third or more delivery was in 427 ( 13.27% ) cases . chi - square test showed a statistically significant difference ( =663.02 , df=2 , p 0.05 ) in the parity of pregnant women who gave birth in the 2012 ( figure 6 ) . the number of births completed by cesarean section in 2012 amounted to 1115 ( 100% ) . in 731 ( 65.56% ) cases it was the first cesarean section , repeated - iterative caesarean section was performed in 353 ( 31.66% ) cases , while reiterative caesarean section was performed in 31 ( 2.78% ) cases with statistically significant difference in the incidence of cesarean section ( =660.02 , df=2 , p<0.05 ) . table 1 shows the number of births in relation to the number of babies . of the total number of births 3156 ( 98.13% ) were singleton pregnancies , twin pregnancy and childbirth was in 59 ( 1.83% ) cases and the triplets were only 1 ( 0.03% ) case . of the total number of births in the 2012 in term deliveries had a total of 2907 ( 89% ) , while premature births had 370 ( 11% ) pregnant women . analysis of infants weight has led to the fact that in 2012 was born 3006 ( 91.73% ) infants with birth weight over 2500 g , with a body weight of 1000 - 2500 g was born 240 ( 7.32% ) infants , while the body weight of 500 - 1000 g had a total of 31 ( 0.95% ) infants . of the total number of deaths , 15 had birth weight from 10002500 g , while 8 had a body weight over 2500 g . of all obstetric surgeries in 2012 , the majority was manual explorations of the uterus ( 197 ) , followed by manual placental lysis ( 49 ) . among other surgeries was performed 21 vacuum extractions , 15 hand assisted births in case of breech births and 3 births with use of forceps . for centuries the pregnancy caused fear and caution of poor outcome for the mother or child . all of us , who have ever attended childbirth , are aware of the fact , that there is nothing more natural and normal than normal birth and that there is nothing abnormally as abnormal childbirth and nothing so astonishingly quickly yarn from one to another ( 1 ) . work of birth room at the clinic of obstetrics is based on a 24-hour working time and involvement of experts in the field of obstetrics . in the 2012 year at the clinic of obstetrics , 3216 woman gave birth . abadzic in his study showed that the percentage of caesarean sections at the clinic of obstetrics , clinical center of sarajevo university is in relation to the total number of births and in the 1996 it was 8.57% while the percentage of 2007 was 27.75% . for a 12 years period the number of caesarean sections is tripled compared to the total number of births ( 2 ) . the upward trend in cesarean sections obstetric practice is witness to the world trend of increasing rates of caesarean sections in the past few years . australia and the united states have the highest rate of caesarean sections in the developed world of 28.5% and 29.1% ( 3 ) . similar trends of increasing rates of caesarean sections occur in latin america , especially in mexico and brazil 25.7% and 27.9% , as well as in other developing countries , such as india ( kerala state ) , 21.4% ( 5 ) . although optimal rate of cesarean sections remains point of many debates . fatusic and colleagues in a retrospective study at clinic of gynecology and obstetrics in tuzla , processed medical data on cesarean sections completed in five years period , from 1984 to 1988 . period which they processed was not affected by the epidemic of cesarean sections . in the study period was performed 6.47% ( 1819 ) caesarean section in relation to the total number of births . of the total percentage of performed caesarean sections 90.22% ( 1641 ) was an unplanned cesarean section while 9.78% ( 178 ) was elective caesarean sections ( 6 ) . the largest number of births in the 2012 year was by primiparous women ( 1603 ) , then the second child ( 1186 ) . perinatal mortality in this period amounted to 7.12 . abadzic in his study showed that the rate of perinatal mortality in 1996 amounted to 16.65 whereas in 2007 it amounted to 9.60 ( 2 ) . the study , the rate of cesarean delivery and perinatal mortality in infants at maichin dom ( 7 ) is performed in bulgaria in the period from 1976 to 2000 and showed that the increase in the cesarean section rate of 4.8% to 24.4% lead to the reduction of perinatal mortality from 27.7 to 11.4 . in the group of women with normal pregnancy , perinatal mortality is 8 - 11 at the frequencies of performed cesarean sections of 15 - 16% , while in high - risk pregnancies perinatal mortality is the same with the rate of performed cesarean sections of 24 - 26% . no correlation between cesarean section rates and perinatal mortality of singleton infants over 2500 g is a study conducted in iceland which provided new data when obstetric trends are in question ( 8) . information is gathered through icelandic birth registry for a period of 20 years from 1987 to 2006 . perinatal mortality for this period amounts to an average of 2 annually and the range is from 0.8 to 4.1 . cesarean section rate varied between 11.9% and 16.7% and did not correlate with perinatal mortality . among primiparas , the rate of cesarean section was increased from 13.1 to 17.8% , without correlation with perinatal mortality , which averaged to 1.7 . the number of births at the clinic of obstetrics , clinical center university of sarajevo has gradually decreased , while the number of caesarean sections is constantly increasing . during the 2012 perinatal mortality has decreased and the percentage of infants with low birth weight ( < 2500 g ) is in limits of the birth of such children in the european union . a small number of completed vaginal deliveries after previous cesarean section warn that we should instead prefer instead of elective cesarean section first a test delivery .

clinic for gynecology and obstetrics , clinical center university of sarajevo represents a tertiary level of health care with more than 3,000 births during one year . the aim of this article is to present the operation of the birth room at the clinic for gynecology and obstetrics in 2012 . data were obtained on the basis of protocol of deliveries from the birth room . material and methods : analyzed are the total number of births , the incidence of obstetric surgeries and other manual interventions . results : over the study period , there were a total of 3216 births , of which by caesarean section was , completed 1115 . the highest number of births was in september ( n=305 ) . the largest number of women who gave birth was nulliparous . also among primiparous is performed the majority of cesarean sections ( n=731 ) . in the study period , multiple births were recorded in a total of 60 ( 59 twins , 1triplets ) . number of premature births was 370 , and the perinatal mortality was 7.12 . number of newborn with birth weight below 2500 g was 271 . among manual interventions in the delivery room mostly was used manual exploration of the uterus followed by the manual lysis of the placenta .

immunoglobulin g4-related disease ( igg4-rd ) is a newly recognized immune - mediated fibro - inflammatory condition that is characterized by enlargement of tissues or organs , abundant igg4 + plasma cell infiltration in damaged organs , and elevated serum igg4 levels . igg4-rd was first characterized in 2001 as sclerosing pancreatitis , and then was referred to as type i igg4-related autoimmune pancreatitis . however , within just a few years a variety of extrapancreatic organ involvement linked by unique histopathological features led to the recognition of igg4-rd as a systemic disease . the comprehensive diagnostic criteria of igg4-rd was first determined in a 2012 study by umehara et al . , which normalized the diagnosis of igg4-rd internationally . due to its relative novelty , the prevalence of igg4-rd remains unclear , as does the exact mechanism of pathogenesis . we previously reported that igg4-rd patients showed disturbed b - cell subsets and dysfunction of regulatory b - cells , whereas wallace et al . described elevated levels of circulating igg4 + plasmablasts , and regarded the levels of such cells as a diagnostic biomarker and reliable indicator of disease activity in igg4-rd patients . these findings indicated that b - cell subsets may play a crucial role in igg4-rd . the clinical features of igg4-rd manifest as single or multiple organ swellings or masses that occur in various sites , including lacrimal glands , salivary glands , pancreas , bile ducts , retroperitoneal tissues , lung , kidney , prostate , pituitary gland , thyroid , and uterus . to date , most studies focused on the clinical and laboratory features of igg4-rd patients , including one of our prospective cohort studies that concerned the clinical characteristics of igg4-rd in 118 chinese patients , which contributed to a more comprehensive understanding of this disease . however , few studies have focused on the correlation between organ involvement and serum igg4 level . the 2015 international consensus guidance statement on the management and treatment of igg4-rd highlighted the necessity of a large cohort study to define the patient 's clinical phenotype and clarify the natural history of igg4-rd . based on previous studies , we conducted a detailed analysis concerning the organ involvement in 200 igg4-rd patients in china that emphasized the types and numbers of organs involved in igg4-rd patients . a multidisciplinary collaborative prospective cohort study of igg4-rd patients was conducted at the peking union medical college hospital ( pumch , beijing , china ) from january 2011 to january 2015 . newly diagnosed igg4-rd patients who fulfilled the 2011 comprehensive diagnostic criteria for definite , probable , or possible igg4-rd were consecutively enrolled . detailed clinical and laboratory data were recorded , including demographic data , initial symptoms , disease duration , history of allergies , physical examination , laboratory assessments , imaging studies , and pathological biopsy . this study was approved by the medical ethics committee of pumch and all patients provided written informed consent . all the patients were tested for complete blood count , liver and renal function tests , erythrocyte sedimentation rate ( esr ) , hyper - sensitivity c - reactive protein ( crp ) , serum igg / iga / igm levels , total ige level , serum igg subclasses , complement , and autoantibodies , including rheumatoid factor ( rf ) , and antinuclear antibodies ( anas ) . serum igg subclasses were measured by immunonephelometry using siemens n igg1 - 2 and igg3 - 4 ( formerly dade behring , marburg , germany ) on a siemens bntmii specific protein analyzer . all the patients underwent imaging examinations , including ultrasonography , computed tomography ( ct ) , or magnetic resonance imaging , and some patients underwent f - fluorodeoxyglucose positron emission tomography / ct . tissue biopsies were performed on 115 patients , and samples were analyzed using previously described pathology methods . organ involvement was assessed by reviewing the patient 's symptoms , past medical history , physical examination findings , laboratory studies , imaging examinations , and/or tissue biopsies . continuous , normally distributed data were shown as mean standard deviation ( sd ) and analyzed by student 's t - test ; all continuous nonnormally distributed data were shown as median ( q1 , q3 ) and evaluated using nonparametric tests . correlations between variables were assessed using pearson 's rank test ( normally distributed ) or spearman 's rank correlation test ( nonnormally distributed ) . the value of p < 0.05 was considered statistically significant . a multidisciplinary collaborative prospective cohort study of igg4-rd patients was conducted at the peking union medical college hospital ( pumch , beijing , china ) from january 2011 to january 2015 . newly diagnosed igg4-rd patients who fulfilled the 2011 comprehensive diagnostic criteria for definite , probable , or possible igg4-rd were consecutively enrolled . detailed clinical and laboratory data were recorded , including demographic data , initial symptoms , disease duration , history of allergies , physical examination , laboratory assessments , imaging studies , and pathological biopsy . this study was approved by the medical ethics committee of pumch and all patients provided written informed consent . all the patients were tested for complete blood count , liver and renal function tests , erythrocyte sedimentation rate ( esr ) , hyper - sensitivity c - reactive protein ( crp ) , serum igg / iga / igm levels , total ige level , serum igg subclasses , complement , and autoantibodies , including rheumatoid factor ( rf ) , and antinuclear antibodies ( anas ) . serum igg subclasses were measured by immunonephelometry using siemens n igg1 - 2 and igg3 - 4 ( formerly dade behring , marburg , germany ) on a siemens bntmii specific protein analyzer . all the patients underwent imaging examinations , including ultrasonography , computed tomography ( ct ) , or magnetic resonance imaging , and some patients underwent f - fluorodeoxyglucose positron emission tomography / ct . tissue biopsies were performed on 115 patients , and samples were analyzed using previously described pathology methods . organ involvement was assessed by reviewing the patient 's symptoms , past medical history , physical examination findings , laboratory studies , imaging examinations , and/or tissue biopsies . continuous , normally distributed data were shown as mean standard deviation ( sd ) and analyzed by student 's t - test ; all continuous nonnormally distributed data were shown as median ( q1 , q3 ) and evaluated using nonparametric tests . correlations between variables were assessed using pearson 's rank test ( normally distributed ) or spearman 's rank correlation test ( nonnormally distributed ) . the value of p < 0.05 was considered statistically significant . the male to female ratio of the patient cohort was 2.08:1 , and the average age at diagnosis was 52.5 14.0 years ( range : 1680 years ) . the majority ( 91% ) of patients were northern chinese . the mean disease duration was 2.18 2.97 years . according to the 2011 comprehensive diagnostic criteria for definite , probable , and possible igg4-rd , the number of corresponding cases was 115 ( 57.5% ) , 7 ( 3.5% ) , and 78 ( 39.0% ) , respectively . the main clinical manifestations were summarized , with the most common being submandibular gland swelling ( 102 , 51.0% ) , lacrimal gland swelling ( 84 , 42.0% ) , superficial lymph node enlargement ( 74 , 37.0% ) , abdominal pain ( 70 , 35.0% ) , parotid gland swelling ( 48 , 24.0% ) , or nasal congestion ( 43 , 21.5% ) . less common manifestations included jaundice ( 28 , 14.0% ) , pruritus ( 27 , 13.5% ) , cough ( 27 , 13.5% ) , low back pain ( 27 , 13.5% ) , dysuresia ( 26 , 13.0% ) , dry mouth and/or dry eye ( 25 , 12.5% ) , or nausea and/or vomiting ( 24 , 12.0% ) . a minority of patients experienced fever ( 18 , 9.0% ) , edema ( 17 , 8.5% ) , exophthalmos ( 11 , 5.5% ) , arthralgia ( 8 , 4.0% ) , or thyroid enlargement ( 5 , 2.5% ) . very few patients experienced chest pain , hoarseness , limb numbness , visual changes , subcutaneous nodules , headache , or hearing loss as initial symptoms , whereas two patients were asymptomatic until a regular medical check - up . notably , 118 ( 59.0% ) patients in this cohort had a history of allergy . the majority of igg4-rd patients had normal white blood cell counts , as well as platelet and hemoglobin tests ( 87.5% , 88.5% , and 95.5% , respectively ) . esr and crp were elevated in 57.7% and 50.6% of patients , and rf was positive in 23.4% of patients . only 13.5% of patients were positive for anas with a titer range from 1:80 to 1:320 among different immunofluorescence types . hypocomplementemia ( low concentration of c3 and/or c4 ) occurred in 31.1% of patients . in immunoglobulin tests , 67.0% of patients had an elevated igg serum level , while most patients had normal serum igm and iga levels ( 92.4% and 94.0% , respectively ) . with respect to serum igg subclasses , igg1 , igg2 , igg3 , and igg4 were above normal levels in 19.7% , 34.8% , 23.2% , and 96.5% of patients , respectively . the relationship between serum igg4 level and igg4/igg ( % ) in this cohort was remarkable ( r = 0.934 , p < 0.001 ) [ figure 1a ] . moreover , the serum igg4 level and igg4/igg ratio were positively correlated with igg values ( r = 0.65 , p < 0.001 and r = 0.413 , p < 0.001 ) , and were negatively correlated with iga and igm values ( iga : r = 0.413 , p < 0.001 and r = 0.501 , p < 0.001 ; igm : r = 0.346 , p < 0.001 and r = 0.375 , p < 0.001 ) [ figure 1b and 1c ] . igg1 levels were positively correlated with serum igg4 value ( r = 0.145 , p = 0.042 ) and igg3 showed remarkable positive correlations with both serum igg4 level and igg4/igg ratio ( r = 0.338 , p < 0.001 and r = 0.202 , p < 0.01 , respectively ) , while only igg2 showed negative correlations with the igg4/igg ratio ( r = 0.239 , p = 0.001 ) [ figure 1d and 1e ] . however , neither serum igg4 level nor igg4/igg ratio showed statistical correlation with age at disease diagnosis , disease duration , esr , or crp ( data not shown ) . correlations between serum igg4 level and igg4/igg value ( a ) ; correlations between serum levels of ig g / a / m / e / g1/g2/g3/eosinophil percentage and serum igg4 level or igg4/igg ( % ) ( b - e ) . values are shown as r , which was determined by spearman 's rank correlation test . ig : immunoglobulin ; igg4-rd : immunoglobulin g4-related disease . notably , eosinophil ( eos ) levels were elevated in 33.7% of cases , and the ige level was increased in 83.6% of igg4-rd patients . both peripheral eos% and serum ige level showed remarkable positive correlations with serum igg4 level and igg4/igg value ( eos% : r = 0.412 , p < 0.001 , r = 0.399 , p < 0.001 ; ige : r = 0.312 , p = 0.001 ; r = 0.271 , p = 0.004 ) [ figure 1 ] . interestingly , relative to patients with or without allergic history , there was no statistical difference in serum ige level and eos% in the patient cohort ( data not shown ) . various sites of the body were involved in igg4-rd patients , including : lymph nodes ( 113 cases , 56.5% ) , submandibular gland ( 102 , 51.0% ) , lacrimal gland ( 84 , 42.0% ) , pancreas ( 77 , 38.5% ) , lung ( including parenchyma , airway and pleura ) ( 64 , 32.0% ) , parotid gland ( 48,24.0% ) , sinus ( 43 , 21.5% ) , bile ducts ( 38 , 19.0% ) , retroperitoneal tissue ( 36 , 18.0% ) , prostate ( 26 , 13.0% ) , kidney ( 20,10.0% ) , artery ( 14 , 7.0% ) , gallbladder ( 11 , 5.5% ) , skin ( 11 , 5.5% ) , inflammatory pseudotumor ( 10 , 5.0% ) , liver ( 6 , 3.0% ) , and others ( including thyroid gland , mediastinum , pituitary gland , spinal cord , endocranium , spleen , ureter , bladder , and gastrointestinal tract ) . the mean number of organs involved was 3.0 1.6 ( range : 110 ) . we divided the igg4-rd patients into four groups according to the number of organs involved as follows ( lymph nodes were not counted except for those patients who had lymphadenopathy as the main clinical manifestation , e.g. , castleman disease ) : only one organ involved ( g1 ) , 2 or 3 organs involved ( g2 ) , 4 or 5 organs involved ( g3 ) , and more than 5 organs involved ( g4 ) . g2 was the most common categorization in this cohort ( 117 cases , 58.5% ) , followed by g3 ( 43 , 21.5% ) and g1 ( 26 , 13.0% ) . g4 was the least common ( 14 , 7.0% ) . comparisons of clinical and laboratory findings among the different groups are shown in figure 2a2j . among each group , there was no significant difference in age at disease diagnosis , disease duration , esr , and crp ( data not shown ) , as well as in igg1 , igg2 , and igg3 . however , serum igg levels were highest for the g4 group . importantly , there were significant differences among each group in serum igg4 level , igg4/igg ratio , and percentage of eos , all of which increased concurrently with an increase in the number of involved organs . serum ige levels also showed the same trend except for the g4 group ; this discrepancy may be due to the small number of patients in this group . patients were divided into four groups according to the number of organs involved as follows : only one organ involved ( g1 ) , 2 or 3 organs involved ( g2 ) , 4 or 5 organs involved ( g3 ) , and more than 5 organs involved ( g4 ) . comparisons of eosinophil percentage , igg4/igg(% ) , igm , iga , igg , igg1 , igg2 , igg3 , igg4 , and ige among different groups were analyzed ( a - j ) . parametric or nonparametric tests were performed to determine the statistical difference among different groups , * p < 0.05 , and p < 0.001 . the types of organs involved were classified according to their different anatomic sites : salivary / lacrimal glands ( mikulicz 's disease [ md ] ) , retroperitoneal / mediastinal fibrosis , pancreatic - hepatobiliary - splenic involvement , urinary system ( kidney / ureter / bladder / prostate ) involvement ( n.b . , ureterostenosis and/or hydronephrosis caused by retroperitoneal fibrosis were excluded ) , respiratory system ( sinus / lung parenchymal / airway / pleura ) involvement , regional and/or systemic lymphadenopathy , central nervous system ( pituitary gland / spinal cord / endocranium / brain ) involvement , and gastrointestinal ( stomach / intestinal tract ) involvement . we also analyzed the discrete constituent ratio of organ involvement types described above [ figure 3 ] . the majority of patients with only one type of organ involved suffered from md , and accounted for up to 13.0% of patients , followed by retroperitoneal / mediastinal fibrosis ( 7.5% ) , and pancreatic - hepatobiliary - splenic system involvement ( 7.0% ) . meanwhile , patients who had only lung or kidney involvement were rare . among patients with two types of involved organs , md combined with another organ type was more frequent ( 26.5% ) , and of these , the respiratory system was most commonly involved ( 16.5% ) , followed by the pancreatic - hepatobiliary - splenic system ( 7.0% ) . combinations of two types of involved organs without md were less common , with each accounting for < 3.0% of cases . in addition , a combination of md with two other types of involved organs represented 14.0% of cases , of which md combined with pancreatic - hepatobiliary - splenic and respiratory system involvement accounted for 6.5% , followed by md combined with the pancreatic - hepatobiliary - splenic system and retroperitoneal / mediastinal fibrosis . however , patients with three types of involved organs without md accounted for only 1.5% of cases . moreover , the constituent ratio of patients with multi - type ( 3 ) organ involvement was 12.0% , and most of these patients also suffered from md . we thus evaluated the level of confidence for igg4-rd indication as strong , moderate , or weak according to the proportion of these types of organ involvement and our experience in diagnosing igg4-rd , which might yield insights into igg4-rd indications [ table 1 ] . constituent ratio of organ involvement in igg4-rd patients . organ involvements were classified according to different anatomic sites : superficial gland swelling , retroperitoneum / mediastinum involvement , hepatobiliary - pancreatic involvement , urinary system involvement , central nervous system involvement , respiratory system involvement , gastrointestinal involvement , and regional and/or systemic lymphadenopathy . the constituent ratio of different types of organ involvement in igg4-rd patients is summarized in a pie chart . confidence level for igg4-rd indication according to type of organ involved md : mikulicz 's disease ; igg4-rd : immunoglobulin g4-related disease . table 2 lists parameters for patients with and without superficial gland involvement in patients with one or two types of involved organs ( n.b . , patients with more than two types of involved organs were not included because of the small number of patients without md ) . the average age at diagnosis showed no difference among the groups . when compared with patients with and without md , patients with md were more likely to be female , have longer disease duration , have higher percentage of eos , and have had a history of allergy ( 64.6% vs. 43.9% , p < 0.001 ) . notably , both serum igg4 levels and igg4/igg ratios were significantly higher in patients with md , whereas iga levels were lower . in addition , patients with salivary / lacrimal gland swelling had lower crp levels than patients without swelling . among serum levels of igg , igm , igg1 , igg2 , igg3 , and ige showed no significant difference between patients with and without md . p value was determined by fisher 's exact test ( categorical ) , student 's t - test ( parametric ) and nonparametric tests ( nonparametric ) ; parametric test values were shown as mean sd , and nonparametric test values were shown as median ( percentiles 25 , percentiles 75 ) ; md : mikulicz 's disease ; esr : erythrocyte sedimentation rate ; crp : c - reactive protein ; ig : immunoglobulin . the male to female ratio of the patient cohort was 2.08:1 , and the average age at diagnosis was 52.5 14.0 years ( range : 1680 years ) . the majority ( 91% ) of patients were northern chinese . the mean disease duration was 2.18 2.97 years . according to the 2011 comprehensive diagnostic criteria for definite , probable , and possible igg4-rd , the number of corresponding cases was 115 ( 57.5% ) , 7 ( 3.5% ) , and 78 ( 39.0% ) , respectively . the main clinical manifestations were summarized , with the most common being submandibular gland swelling ( 102 , 51.0% ) , lacrimal gland swelling ( 84 , 42.0% ) , superficial lymph node enlargement ( 74 , 37.0% ) , abdominal pain ( 70 , 35.0% ) , parotid gland swelling ( 48 , 24.0% ) , or nasal congestion ( 43 , 21.5% ) . less common manifestations included jaundice ( 28 , 14.0% ) , pruritus ( 27 , 13.5% ) , cough ( 27 , 13.5% ) , low back pain ( 27 , 13.5% ) , dysuresia ( 26 , 13.0% ) , dry mouth and/or dry eye ( 25 , 12.5% ) , or nausea and/or vomiting ( 24 , 12.0% ) . a minority of patients experienced fever ( 18 , 9.0% ) , edema ( 17 , 8.5% ) , exophthalmos ( 11 , 5.5% ) , arthralgia ( 8 , 4.0% ) , or thyroid enlargement ( 5 , 2.5% ) . very few patients experienced chest pain , hoarseness , limb numbness , visual changes , subcutaneous nodules , headache , or hearing loss as initial symptoms , whereas two patients were asymptomatic until a regular medical check - up . notably , 118 ( 59.0% ) patients in this cohort had a history of allergy . the majority of igg4-rd patients had normal white blood cell counts , as well as platelet and hemoglobin tests ( 87.5% , 88.5% , and 95.5% , respectively ) . esr and crp were elevated in 57.7% and 50.6% of patients , and rf was positive in 23.4% of patients . only 13.5% of patients were positive for anas with a titer range from 1:80 to 1:320 among different immunofluorescence types . hypocomplementemia ( low concentration of c3 and/or c4 ) occurred in 31.1% of patients . in immunoglobulin tests , 67.0% of patients had an elevated igg serum level , while most patients had normal serum igm and iga levels ( 92.4% and 94.0% , respectively ) . with respect to serum igg subclasses , igg1 , igg2 , igg3 , and igg4 were above normal levels in 19.7% , 34.8% , 23.2% , and 96.5% of patients , respectively . the relationship between serum igg4 level and igg4/igg ( % ) in this cohort was remarkable ( r = 0.934 , p < 0.001 ) [ figure 1a ] . moreover , the serum igg4 level and igg4/igg ratio were positively correlated with igg values ( r = 0.65 , p < 0.001 and r = 0.413 , p < 0.001 ) , and were negatively correlated with iga and igm values ( iga : r = 0.413 , p < 0.001 and r = 0.501 , p < 0.001 ; igm : r = 0.346 , p < 0.001 and r = 0.375 , p < 0.001 ) [ figure 1b and 1c ] . igg1 levels were positively correlated with serum igg4 value ( r = 0.145 , p = 0.042 ) and igg3 showed remarkable positive correlations with both serum igg4 level and igg4/igg ratio ( r = 0.338 , p < 0.001 and r = 0.202 , p < 0.01 , respectively ) , while only igg2 showed negative correlations with the igg4/igg ratio ( r = 0.239 , p = 0.001 ) [ figure 1d and 1e ] . however , neither serum igg4 level nor igg4/igg ratio showed statistical correlation with age at disease diagnosis , disease duration , esr , or crp ( data not shown ) . correlations between serum igg4 level and igg4/igg value ( a ) ; correlations between serum levels of ig g / a / m / e / g1/g2/g3/eosinophil percentage and serum igg4 level or igg4/igg ( % ) ( b - e ) . values are shown as r , which was determined by spearman 's rank correlation test . notably , eosinophil ( eos ) levels were elevated in 33.7% of cases , and the ige level was increased in 83.6% of igg4-rd patients . both peripheral eos% and serum ige level showed remarkable positive correlations with serum igg4 level and igg4/igg value ( eos% : r = 0.412 , p < 0.001 , r = 0.399 , p < 0.001 ; ige : r = 0.312 , p = 0.001 ; r = 0.271 , p = 0.004 ) [ figure 1 ] . interestingly , relative to patients with or without allergic history , there was no statistical difference in serum ige level and eos% in the patient cohort ( data not shown ) . various sites of the body were involved in igg4-rd patients , including : lymph nodes ( 113 cases , 56.5% ) , submandibular gland ( 102 , 51.0% ) , lacrimal gland ( 84 , 42.0% ) , pancreas ( 77 , 38.5% ) , lung ( including parenchyma , airway and pleura ) ( 64 , 32.0% ) , parotid gland ( 48,24.0% ) , sinus ( 43 , 21.5% ) , bile ducts ( 38 , 19.0% ) , retroperitoneal tissue ( 36 , 18.0% ) , prostate ( 26 , 13.0% ) , kidney ( 20,10.0% ) , artery ( 14 , 7.0% ) , gallbladder ( 11 , 5.5% ) , skin ( 11 , 5.5% ) , inflammatory pseudotumor ( 10 , 5.0% ) , liver ( 6 , 3.0% ) , and others ( including thyroid gland , mediastinum , pituitary gland , spinal cord , endocranium , spleen , ureter , bladder , and gastrointestinal tract ) . the mean number of organs involved was 3.0 1.6 ( range : 110 ) . we divided the igg4-rd patients into four groups according to the number of organs involved as follows ( lymph nodes were not counted except for those patients who had lymphadenopathy as the main clinical manifestation , e.g. , castleman disease ) : only one organ involved ( g1 ) , 2 or 3 organs involved ( g2 ) , 4 or 5 organs involved ( g3 ) , and more than 5 organs involved ( g4 ) . g2 was the most common categorization in this cohort ( 117 cases , 58.5% ) , followed by g3 ( 43 , 21.5% ) and g1 ( 26 , 13.0% ) . g4 was the least common ( 14 , 7.0% ) . comparisons of clinical and laboratory findings among the different groups are shown in figure 2a2j . among each group , there was no significant difference in age at disease diagnosis , disease duration , esr , and crp ( data not shown ) , as well as in igg1 , igg2 , and igg3 . however , serum igg levels were highest for the g4 group . importantly , there were significant differences among each group in serum igg4 level , igg4/igg ratio , and percentage of eos , all of which increased concurrently with an increase in the number of involved organs . serum ige levels also showed the same trend except for the g4 group ; this discrepancy may be due to the small number of patients in this group . patients were divided into four groups according to the number of organs involved as follows : only one organ involved ( g1 ) , 2 or 3 organs involved ( g2 ) , 4 or 5 organs involved ( g3 ) , and more than 5 organs involved ( g4 ) . comparisons of eosinophil percentage , igg4/igg(% ) , igm , iga , igg , igg1 , igg2 , igg3 , igg4 , and ige among different groups were analyzed ( a - j ) . parametric or nonparametric tests were performed to determine the statistical difference among different groups , * p < 0.05 , and p < 0.001 . the types of organs involved were classified according to their different anatomic sites : salivary / lacrimal glands ( mikulicz 's disease [ md ] ) , retroperitoneal / mediastinal fibrosis , pancreatic - hepatobiliary - splenic involvement , urinary system ( kidney / ureter / bladder / prostate ) involvement ( n.b . , ureterostenosis and/or hydronephrosis caused by retroperitoneal fibrosis were excluded ) , respiratory system ( sinus / lung parenchymal / airway / pleura ) involvement , regional and/or systemic lymphadenopathy , central nervous system ( pituitary gland / spinal cord / endocranium / brain ) involvement , and gastrointestinal ( stomach / intestinal tract ) involvement . we also analyzed the discrete constituent ratio of organ involvement types described above [ figure 3 ] . the majority of patients with only one type of organ involved suffered from md , and accounted for up to 13.0% of patients , followed by retroperitoneal / mediastinal fibrosis ( 7.5% ) , and pancreatic - hepatobiliary - splenic system involvement ( 7.0% ) . meanwhile , patients who had only lung or kidney involvement were rare . among patients with two types of involved organs , md combined with another organ type was more frequent ( 26.5% ) , and of these , the respiratory system was most commonly involved ( 16.5% ) , followed by the pancreatic - hepatobiliary - splenic system ( 7.0% ) . combinations of two types of involved organs without md were less common , with each accounting for < 3.0% of cases . in addition , a combination of md with two other types of involved organs represented 14.0% of cases , of which md combined with pancreatic - hepatobiliary - splenic and respiratory system involvement accounted for 6.5% , followed by md combined with the pancreatic - hepatobiliary - splenic system and retroperitoneal / mediastinal fibrosis . however , patients with three types of involved organs without md accounted for only 1.5% of cases . moreover , the constituent ratio of patients with multi - type ( 3 ) organ involvement was 12.0% , and most of these patients also suffered from md . we thus evaluated the level of confidence for igg4-rd indication as strong , moderate , or weak according to the proportion of these types of organ involvement and our experience in diagnosing igg4-rd , which might yield insights into igg4-rd indications [ table 1 ] . organ involvements were classified according to different anatomic sites : superficial gland swelling , retroperitoneum / mediastinum involvement , hepatobiliary - pancreatic involvement , urinary system involvement , central nervous system involvement , respiratory system involvement , gastrointestinal involvement , and regional and/or systemic lymphadenopathy . the constituent ratio of different types of organ involvement in igg4-rd patients is summarized in a pie chart . confidence level for igg4-rd indication according to type of organ involved md : mikulicz 's disease ; igg4-rd : immunoglobulin g4-related disease . table 2 lists parameters for patients with and without superficial gland involvement in patients with one or two types of involved organs ( n.b . , patients with more than two types of involved organs were not included because of the small number of patients without md ) . the average age at diagnosis showed no difference among the groups . when compared with patients with and without md , patients with md were more likely to be female , have longer disease duration , have higher percentage of eos , and have had a history of allergy ( 64.6% vs. 43.9% , p < 0.001 ) . notably , both serum igg4 levels and igg4/igg ratios were significantly higher in patients with md , whereas iga levels were lower . in addition , patients with salivary / lacrimal gland swelling had lower crp levels than patients without swelling . among serum levels of igg , igm , igg1 , igg2 , igg3 , and ige showed no significant difference between patients with and without md . p value was determined by fisher 's exact test ( categorical ) , student 's t - test ( parametric ) and nonparametric tests ( nonparametric ) ; parametric test values were shown as mean sd , and nonparametric test values were shown as median ( percentiles 25 , percentiles 75 ) ; md : mikulicz 's disease ; esr : erythrocyte sedimentation rate ; crp : c - reactive protein ; ig : immunoglobulin . the clinical , laboratory , imaging , and histopathological features of igg4-rd patients have been discussed by many studies and case reports . based on previous cohort studies , we further expanded the number of igg4-rd patients in the cohort examined here so as to supplement the clinical features and laboratory findings , such as rarely involved organs . to further our understanding of igg4-rd , we focused on an analysis of clinical and laboratory features among patients with different numbers and types of involved organs . on the whole , the disease predominantly occurred in middle - aged to elderly men and patients with involvement of two or three organs were the most common case types . salivary / lacrimal gland swelling , which is manifested as md , was the most common among igg4-rd patients , followed by involvement of the pancreatic gland , respiratory system , retroperitoneal / mediastinum , and urinary system . meanwhile , the thyroid gland , pituitary gland , central nervous system , and spleen were rarely affected in igg4-rd patients . thus , except for typical characteristics that include salivary and/or lacrimal gland swelling , the presence of lymph node enlargement , abdominal pain , jaundice , nasal congestion , cough , or low back pain , which might indicate the involvement of salivary / lacrimal glands , abdominal organs , the respiratory system , and retroperitoneal fibrosis , respectively , as well as some rare clinical symptoms , such as those associated with the central nervous system , should also be taken into account when diagnosing igg4-rd . lymphadenopathy was very common in igg4-rd patients , whereas lymphadenopathy manifested as castleman disease was present in only a few patients . although this study involved multidisciplinary cooperation , selection bias might have occurred in part because the majority of our patients were enrolled from rheumatology unit . elevated serum igg and igg4 levels were the most important laboratory features of igg4-rd , and the serum igg4 level and igg4/igg ratio increased with an increasing number of involved organs . patients with hypocomplementemia were not uncommon in our cohort , which is consistent with previous reports , and this situation was more remarkable in patients with kidney disease . given that the igg4 molecule lacks complement - binding activity , wallace et al . speculated that an alternate igg subtype might be responsible for the low complement levels in igg4-rd patients . however , we found no relationship between esr and either serum igg4 or crp levels , which indicated that inflammatory factors may not play crucial roles in igg4-rd progression . indeed , the majority of our patients had normal serum iga and igm levels . although serum igg3 levels were normal in most patients of our cohort , its value increased with igg4 level , although the mechanistic explanation of this observation is unclear . elevated eos numbers and total serum ige levels were also observed in 33.7% and 83.6% of igg4-rd patients , respectively , which is consistent with a previous study . in addition , both the eos percentage and serum ige level were significantly correlated to serum igg4 level and igg4/igg value and increased as the number of involved organs increased . these findings support the view that igg4-rd itself rather than atopy contributes to the elevation in eos percentages and ige level . although nearly 60% of our igg4-rd patients had allergic history , there was no significant difference in eos numbers and serum ige levels relative to patients without allergic disease . the presentation of various manifestations and organ involvement in igg4-rd patients highlights the potential interest of classifying clinical phenotypes . we believe that the characteristics of the different organ types that are involved in this disease are not only useful for indicating igg4-rd rather than malignancies or other benign diseases , but also for understanding the nature of the disease . in addition , the first international consensus guidance statement on the management and treatment of igg4-rd , which proposed research priorities to achieve advances in the management and treatment of igg4-rd , emphasized the necessity of defining the patient 's clinical phenotype . in reviewing the literature , we found only one recent study that analyzed organ involvement correlations in 132 igg4-rd patients , although this correlation was restricted to serum igg4 level and organ involvement . in this study , patients were classified into different types according to the numbers and anatomic sites of involved organs , with two main types of organ involvement being the most common while patients with involvement of multiple ( 4 ) organs were the rarest . notably , in patients with one type of involved organ , salivary / lacrimal gland swelling was the most common clinical feature , followed by retroperitoneal / mediastinal fibrosis and pancreatic - hepatobiliary - splenic system involvement . in patients with two types of organ involvement , salivary / lacrimal gland swelling along with respiratory system involvement was the most common pattern . in addition , salivary / lacrimal gland swelling combined with hepatobiliary - pancreatic and respiratory system involvement was the most common in patients who had three types of organs involved . patients who had only one type of internal organ involved accounted for only a small portion of this cohort . the clinical types of organ involvement could thus provide valuable clues for the diagnosis and differential diagnosis of igg4-rd . in analyzing differences between patients with salivary / lacrimal gland and internal organ involvement , we found no difference in gender distribution , while elderly male patients were more inclined to have internal organ involvement . koizumi et al . , which reported that patients with lacrimal and/or salivary gland lesions had higher serum igg4 levels than patients who did not , our data revealed that patients with salivary / lacrimal gland swelling had higher serum igg4 concentrations and igg4/igg values than those with internal organ involvement . higher percentages of eos and allergic history was also more frequent in patients with salivary / lacrimal gland involvement . together these findings suggested that lacrimal and salivary gland involvement in igg4-rd translated into different disease manifestations relative to cases that did not involve these glands . this outcome might thus provide clues about igg4-rd pathogenesis although additional study will be needed . first , this is a single - center prospective study , and most of our patients were enrolled from the rheumatology unit . as such , a multicenter prospective study is needed to evaluate more broadly the clinical and laboratory features of igg4-rd . the number of patients who underwent tissue biopsy was relatively small , and represented only 60% of cases . furthermore , we encountered some difficulties in classifying involvement of various organs , such as lymph nodes , as well as those patients with rare disease manifestations . in conclusion , patients with different types of organ involvement revealed distinct clinical and laboratory features in some important ways , which might be helpful for confirming the presence of igg4-rd rather than malignancies or other benign diseases . our findings would also be useful for understanding the nature of igg4-rd while the prognosis and outcome of igg4-rd patients with different types of organ involvement deserves further long - term follow - up study . this work was supported by grants from the national natural science foundation of china ( nos . this work was supported by grants from the national natural science foundation of china ( nos .

background : immunoglobulin g4-related disease ( igg4-rd ) is a newly recognized systemic disease that can involve multiple organs and various clinical phenotypes . the purpose of this study was to analyze different types of organ involvement in igg4-rd patients in china.methods:we conducted a prospective cohort study on igg4-rd patients to analyze the clinical manifestations and rare features of igg4-rd . patients were grouped into different types according to organ involvement regarding organ number and organ site . the constituent ratio in different types was also analyzed.results:a total of 200 igg4-rd patients , with a male : female ratio of 2.08:1 , were grouped into different types . cases having involvement of two or three organs were the most common whereas the fewest number of patients had multi - organ ( 4 ) involvement . serum igg4 and ige levels , igg4/igg ratio , and percentage of eosinophils increased as the number of involved organs increased . in addition , constituent ratio analysis revealed that patients with salivary gland / lacrimal gland swelling , who also constituted the largest number of igg4-rd patients , had higher serum igg4 concentrations and igg4/igg values , had higher percentage of eos , and were more likely to have had a history of allergies relative to patients with internal organ involvement.conclusions:the characteristic feature of igg4-rd is multiple organ involvement with various clinical manifestations and different types . although serum igg4 levels increased with the number of involved organs , serum igg4 levels were higher for those patients with salivary gland / lacrimal gland swelling compared with those with internal organ involvement . thus , valuable clues to the differential diagnosis of igg4-rd could be obtained by examining the clinical patterns of organ involvement .

ethical approval was acquired and informed consent was obtained from the patient or the family of the patient . in addition to patient samples , control pancreatic blocks and isolated islet sections were prepared from five deceased donors without diabetes ( three women ; age 2461 years ; bmi 2534 kg / m ) . sections were stained with hematoxylin and eosin in addition to sirius red collagen staining using standard procedures . indirect immunofluorescence staining was performed on 4-m sections after deparaffinization , rehydration , and heat - mediated antigen retrieval using citrate buffer . after blocking with 10% fcs , sections were incubated with guinea pig anti - insulin ( 1:500 ; abcam , cambridge , u.k . ) , rabbit antivimentin ( 1:250 ; abcam ) , or mouse antiglucagon ( 1:1,000 ; sigma - aldrich , gillingham , u.k . ) overnight . sections were incubated with anti - guinea pig fluorescein isothiocyanate , anti - mouse af543 , or anti - rabbit af488/af543 secondary antibodies ( invitrogen , paisley , u.k . ) . for negative control subjects , patient 1 was a 65-year - old woman whose pancreas was procured during deceased organ donation after brain death after intracranial hemorrhage . bmi was 32 kg / m , with random plasma glucose of 8.1 mmol / l . patient 2 was an 81-year - old woman who underwent distal pancreatectomy for an intraductal papillary mucinous neoplasm . she had experienced two episodes of pancreatitis 12 months and 7 years before pancreatic resection but had no chronic symptoms or evidence of pancreatic exocrine deficiency . patient 3 was a 52-year - old woman whose pancreas was procured for clinical islet isolation during deceased organ donation after brain death after intracranial hemorrhage . there was no history of known diabetes , but a diagnostic hba1c test performed on admission indicated hba1c of 63 mmol / mol ( hba1c 7.9% ) with random glucose of 8.7 morphological analysis after hematoxylin and eosin staining of pancreatic sections showed islet size , distribution , and integrity comparable with those of nondiabetic control subjects . there was no evidence of fibrosis in islets or exocrine pancreatic tissue with patterns of collagen deposition comparable with those of control samples on sirius red staining . immunofluorescence staining clearly demonstrated cells within intact islets expressing both insulin and vimentin in the cytoplasm . d . as shown , cells expressing insulin and vimentin were present in 40% of islets , constituting 5% of insulin - positive cells in affected islets . in cells expressing both phenotypic markers , confocal imaging confirmed coexpression within individual cells and maintained characteristic cytoplasmic insulin and filamentous vimentin staining patterns . the boxed area in panel a indicates the region that is magnified in panels b d . arrows indicate cells expressing both insulin ( b , c ) and vimentin ( b , d ) . e : cytofluorogram derived from this image confirming colocalization of insulin and vimentin in islet -cells . comparative cytofluorogram from stained , normal , nondiabetic pancreas ( f ) confirming no colocalization of insulin ( green ) and vimentin ( red ) . i : representative images show islet -cells from patient 1 coexpressing the -cell marker glucagon . the boxed area in panel g indicates the magnified region in panels h and i , with arrows indicating cells expressing both insulin ( h ) and glucagon ( i ) . the boxed area in panel j indicates the region that is magnified in panels k and l. arrows indicate cells expressing both glucagon ( k ) and vimentin ( l ) . in contrast , no coexpression of vimentin in insulin - positive cells within or outside islets was detected in nondiabetic control sections . islet cells coexpressing insulin and glucagon within the cytoplasm were identified in pancreatic sections from patient 1 ( fig . l ) . both of these phenotypes were rare , constituting 1% of all islet cells . in contrast , neither of these mixed phenotypes could be detected on pancreatic sections from nondiabetic control subjects . macroscopic examination of resected pancreatic tail demonstrated a cystic area with a maximum diameter of 8 mm . staining of pancreatic sections confirmed intraductal papillary mucinous neoplasm without high - grade dysplasia or evidence of malignancy . there was evidence of lobular atrophy and granulomatous inflammation in the surrounding pancreas , but islet endocrine morphology was reported as being within normal limits . islets stained positive for insulin , with categorical evidence of cells coexpressing insulin and vimentin , insulin and glucagon , and vimentin and glucagon ( supplementary fig . 5% of insulin - positive cells in affected islets coexpressed insulin and vimentin . in patient 2 , although cells expressing only insulin , glucagon , or vimentin could be clearly identified , many cells coexpressed insulin and glucagon and vimentin and glucagon in virtually all islets . immunofluorescence staining of isolated islets enabled clear differentiation of individual cells , particularly at the periphery . cells coexpressing insulin and vimentin , insulin and glucagon , and vimentin and glucagon within the cytoplasm were identified ( supplementary fig . 2 ) . absence of any of these phenotypes in control islets isolated from nondiabetic donors was confirmed . patient 1 was a 65-year - old woman whose pancreas was procured during deceased organ donation after brain death after intracranial hemorrhage . bmi was 32 kg / m , with random plasma glucose of 8.1 mmol / l . patient 2 was an 81-year - old woman who underwent distal pancreatectomy for an intraductal papillary mucinous neoplasm . she had experienced two episodes of pancreatitis 12 months and 7 years before pancreatic resection but had no chronic symptoms or evidence of pancreatic exocrine deficiency . patient 3 was a 52-year - old woman whose pancreas was procured for clinical islet isolation during deceased organ donation after brain death after intracranial hemorrhage . there was no history of known diabetes , but a diagnostic hba1c test performed on admission indicated hba1c of 63 mmol / mol ( hba1c 7.9% ) with random glucose of 8.7 morphological analysis after hematoxylin and eosin staining of pancreatic sections showed islet size , distribution , and integrity comparable with those of nondiabetic control subjects . there was no evidence of fibrosis in islets or exocrine pancreatic tissue with patterns of collagen deposition comparable with those of control samples on sirius red staining . immunofluorescence staining clearly demonstrated cells within intact islets expressing both insulin and vimentin in the cytoplasm . d . as shown , cells expressing insulin and vimentin were present in 40% of islets , constituting 5% of insulin - positive cells in affected islets . in cells expressing both phenotypic markers , confocal imaging confirmed coexpression within individual cells and maintained characteristic cytoplasmic insulin and filamentous vimentin staining patterns . the boxed area in panel a indicates the region that is magnified in panels b d . arrows indicate cells expressing both insulin ( b , c ) and vimentin ( b , d ) . e : cytofluorogram derived from this image confirming colocalization of insulin and vimentin in islet -cells . comparative cytofluorogram from stained , normal , nondiabetic pancreas ( f ) confirming no colocalization of insulin ( green ) and vimentin ( red ) . i : representative images show islet -cells from patient 1 coexpressing the -cell marker glucagon . the boxed area in panel g indicates the magnified region in panels h and i , with arrows indicating cells expressing both insulin ( h ) and glucagon ( i ) . the boxed area in panel j indicates the region that is magnified in panels k and l. arrows indicate cells expressing both glucagon ( k ) and vimentin ( l ) . in contrast , no coexpression of vimentin in insulin - positive cells within or outside islets was detected in nondiabetic control sections . islet cells coexpressing insulin and glucagon within the cytoplasm were identified in pancreatic sections from patient 1 ( fig . l ) . both of these phenotypes were rare , constituting 1% of all islet cells . in contrast , neither of these mixed phenotypes could be detected on pancreatic sections from nondiabetic control subjects . macroscopic examination of resected pancreatic tail demonstrated a cystic area with a maximum diameter of 8 mm . staining of pancreatic sections confirmed intraductal papillary mucinous neoplasm without high - grade dysplasia or evidence of malignancy . there was evidence of lobular atrophy and granulomatous inflammation in the surrounding pancreas , but islet endocrine morphology was reported as being within normal limits . islets stained positive for insulin , with categorical evidence of cells coexpressing insulin and vimentin , insulin and glucagon , and vimentin and glucagon ( supplementary fig . 5% of insulin - positive cells in affected islets coexpressed insulin and vimentin . in patient 2 , although cells expressing only insulin , glucagon , or vimentin could be clearly identified , many cells coexpressed insulin and glucagon and vimentin and glucagon in virtually all islets . immunofluorescence staining of isolated islets enabled clear differentiation of individual cells , particularly at the periphery . cells coexpressing insulin and vimentin , insulin and glucagon , and vimentin and glucagon within the cytoplasm were identified ( supplementary fig . 2 ) . absence of any of these phenotypes in control islets isolated from nondiabetic donors was confirmed . morphological analysis after hematoxylin and eosin staining of pancreatic sections showed islet size , distribution , and integrity comparable with those of nondiabetic control subjects . there was no evidence of fibrosis in islets or exocrine pancreatic tissue with patterns of collagen deposition comparable with those of control samples on sirius red staining . immunofluorescence staining clearly demonstrated cells within intact islets expressing both insulin and vimentin in the cytoplasm . d . as shown , cells expressing insulin and vimentin were present in 40% of islets , constituting 5% of insulin - positive cells in affected islets . in cells expressing both phenotypic markers , confocal imaging confirmed coexpression within individual cells and maintained characteristic cytoplasmic insulin and filamentous vimentin staining patterns . the boxed area in panel a indicates the region that is magnified in panels b d . arrows indicate cells expressing both insulin ( b , c ) and vimentin ( b , d ) . e : cytofluorogram derived from this image confirming colocalization of insulin and vimentin in islet -cells . comparative cytofluorogram from stained , normal , nondiabetic pancreas ( f ) confirming no colocalization of insulin ( green ) and vimentin ( red ) . i : representative images show islet -cells from patient 1 coexpressing the -cell marker glucagon . the boxed area in panel g indicates the magnified region in panels h and i , with arrows indicating cells expressing both insulin ( h ) and glucagon ( i ) . the boxed area in panel j indicates the region that is magnified in panels k and l. arrows indicate cells expressing both glucagon ( k ) and vimentin ( l ) . in contrast , no coexpression of vimentin in insulin - positive cells within or outside islets was detected in nondiabetic control sections . islet cells coexpressing insulin and glucagon within the cytoplasm were identified in pancreatic sections from patient 1 ( fig . l ) . both of these phenotypes were rare , constituting 1% of all islet cells . in contrast , neither of these mixed phenotypes could be detected on pancreatic sections from nondiabetic control subjects . macroscopic examination of resected pancreatic tail demonstrated a cystic area with a maximum diameter of 8 mm . staining of pancreatic sections confirmed intraductal papillary mucinous neoplasm without high - grade dysplasia or evidence of malignancy . there was evidence of lobular atrophy and granulomatous inflammation in the surrounding pancreas , but islet endocrine morphology was reported as being within normal limits . islets stained positive for insulin , with categorical evidence of cells coexpressing insulin and vimentin , insulin and glucagon , and vimentin and glucagon ( supplementary fig . 5% of insulin - positive cells in affected islets coexpressed insulin and vimentin . in patient 2 , although cells expressing only insulin , glucagon , or vimentin could be clearly identified , many cells coexpressed insulin and glucagon and vimentin and glucagon in virtually all islets . immunofluorescence staining of isolated islets enabled clear differentiation of individual cells , particularly at the periphery . cells coexpressing insulin and vimentin , insulin and glucagon , and vimentin and glucagon within the cytoplasm were identified ( supplementary fig . 2 ) . absence of any of these phenotypes in control islets isolated from nondiabetic donors was confirmed . consistent with a role for dedifferentiation in the pathogenesis of -cell dysfunction in diabetes , we describe previously unreported coexpression of mesenchymal and -cell phenotypic markers in insulin - positive cells in two patients with recently diagnosed noninsulin - requiring diabetes and in one patient with previously undiagnosed diabetes . it recently has been postulated from a series of -cell fate - marking studies of transgenic mice that dedifferentiation is the primary mechanism underlying -cell failure in nonautoimmune diabetes ( 5 ) . specifically , the investigators proposed that metabolic stress leads to activation of mesenchymal markers in endocrine cells , a phenomenon well described in human -cells after establishment in adherent proliferative culture but not previously described in vivo in preclinical studies or in situ in humans ( 7,8 ) . ( 5 ) demonstrated that a number of dedifferentiated -cells undergo conversion to other endocrine phenotypes , leading them to suggest that -cell reprogramming to -cells may explain the apparent reciprocal association of insulinopenia with hyperglucagonemia in early type 2 diabetes ( 9 ) . the presence of cells coexpressing glucagon and insulin in the patients reported here is in keeping with this hypothesis . we also detected cells expressing both glucagon and vimentin , a phenotype reported in the preclinical studies . dedifferentiation of nonendocrine pancreas with coexpression of epithelial and mesenchymal markers has been recognized in human sections , with occasional cells coexpressing vimentin and glucagon within the ducts of patients with type 2 diabetes ( 10 ) . our data provide circumstantial evidence for the recently reported phenomenon of -cell dedifferentiation and possible reprogramming to -cells in humans . whether this process is reversible in vivo , contributing to the rapid recovery of -cell function after calorie restriction or bariatric surgery , requires further study . if so , then this may provide a new target for the development of disease - modifying drugs that restore -cell mass and function in type 2 and secondary diabetes through redifferentiation .

objectiverelative contributions of reversible -cell dysfunction and true decrease in -cell mass in type 2 diabetes remain unclear . definitive rodent lineage - tracing studies have identified -cell dedifferentiation and subsequent reprogramming to -cell fate as a novel mechanism underlying -cell failure . the aim was to determine whether phenotypes of -cell dedifferentiation and plasticity are present in human diabetes.research design and methodsimmunofluorescence colocalization studies using classical endocrine and mesenchymal phenotypic markers were undertaken using pancreatic sections and isolated islets from three individuals with diabetes and five nondiabetic control subjects.resultsintraislet cytoplasmic coexpression of insulin and vimentin , insulin and glucagon , and vimentin and glucagon were demonstrated in all cases . these phenotypes were not present in nondiabetic control subjects.conclusionscoexpression of mesenchymal and -cell phenotypic markers in human diabetic islet -cells has been confirmed , providing circumstantial evidence for -cell dedifferentiation and possible reprogramming to -cells in clinical diabetes .

enhanced inflammation is well - established in patients with chronic kidney disease ( ckd ) . several factors have been implicated in such inflammation in patients with ckd including uremic state , elevated serum inflammatory markers , oxidative stress , etc . ( 1 - 3 ) . systemic inflammation has important implications in renal failure and can lead to potentially severe conditions such as premature atherosclerosis . premature atherosclerosis is one of the main causes of death in hemodialysis patients ( 4 , 5 ) . some studies suggested that hemodialysis itself increases the production of free radicals ( 6 - 8 ) . in addition to inflammation , malnutrition has been considered as an important determinant factor in clinical outcome of patients with ckd ( 9 , 10 ) . among various nutritional elements in fact , there is extensive evidence that selenium level is depleted in patients with ckd , especially those under hemodialysis ( 4 , 5 , 12 ) . this nutrient is an essential part of enzymes of the body 's cells against free radicals by participating in the structure of glutathione peroxidase . free radicals are formed during natural oxygen metabolism process of body and have widespread destructive effects on body . one of the essential functions of selenium is preventing heart disease and elevated blood pressure ( 13 , 14 ) . in fact , the highest concentration of selenium inside the human body ( per gram tissue ) is found in the thyroid ( 15 ) . for long time this is mainly due to special selenoproteins inside the thyroid gland and activity of thyroid enzyme deiodinase , which requires selenium for its proper function ( 16 ) . abnormal changes reported include reduction in serum level of total and free t3 and t4 , increase in rt3 and tsh reduction ( 17 ) . in fact , hypothyroidism has been documented to occur in a higher rate in patients with ckd than general population ( 18 ) . considering the abnormal changes in tfts in patients with ckd , some authors advised mathematical calculations to correct tfts measurements so that a subnormal free t4 in a non - thyroidal disease would not cause confusion in diagnosing hypothyroidism ( 19 ) . since micronutrients are important components in metabolism pathways of proteins and enzymes and have many metabolic functions , any changes in their serum level can have profound impact on other important and critical parts of body . in view of the fact that selenium may influence inflammation and thyroid function , we decided to evaluate the role of selenium supplementation regarding any possible changes in inflammatory markers and tfts in patients with ckd undergoing hemodialysis . in this double - blinded clinical trial , 64 consecutive patients presented to out tertiary care center to undergo hemodialysis and six months had passed from hemodialysis initiation were included . exclusion criteria were acute viral infections in past three months , previous or current diseases of thyroid , taking steroidal and non - steroidal anti - inflammatory drugs , taking vitamins e and c and zinc during two past months prior to the study . before hemodialysis selenium measurement was performed via atomic absorption analysis using vapor generation accessory ( vga ) . patients with low levels of selenium were randomly divided ( using random number table ) into experimental ( 32 cases ) and control ( 32 cases ) groups . experimental group received selenium capsules ( 200 g / day ) for three months , while control group received placebo capsules for the same period . for laboratory markers , 3 ml of venous blood this was performed twice first at the start of study and again after three months . the following markers were measured ; esr ( erythrocyte sedimentation rate ) , ferritin , quantitative c - reactive protein ( crp ) and thyroid function tests ( thyroid - stimulating hormone ( tsh ) , t3 resin uptake ( t3ru ) and free t4 ) . after completion of hemodialysis , weight was measured with light clothing using a weight scale with precision of 1 kg . height was also measured without shoes by a meter installed on the wall with precision of 1 cm . mean and standard deviation ( sd ) were used . to compare the data between experimental and control groups , independent t - test , paired t - test , wilcoxon after normal distribution of dependent variables and ascertaining proportional odds , ordinal regression was used , otherwise logistic regression was applied . the study protocol was confirmed by the ethics committee of research of kermanshah university of medical sciences , kermanshah , iran . written informed consent was obtained from patients . both selenium and placebo were delivered to patients free of charge . in case of detecting any abnormality in laboratory tests , mean ( sd ) age of experimental and control groups were 57.34 ( 13.23 ) and 59.53 ( 14.68 ) years , respectively ( p = 0.53 ) . there were 15 males ( 46.9% ) in experimental group and 17 females ( 53.1% ) in the control group . mean ( sd ) body mass index ( bmi ) values in experimental and control groups were 23.78 ( 4.83 ) and 24.46 ( 3.68 ) kg / m , respectively ( p = 0.54 ) . mean ( sd ) serum selenium levels before the intervention in experimental and control groups were 32.81 ( 10.5 ) and 39.09 ( 13.18 ) g / dl , respectively ( p = 0.049 ) . ancova showed that by controlling selenium effect before the intervention between the two groups , selenium change after the intervention was significant ( p < 0.001 ) . mean ( sd ) selenium levels after the intervention in experimental and control groups were 182.16 ( 42.4 ) and 51.16 ( 36.37 ) g / dl , respectively . table 1 presents the comparison between acute phase reactants measured at baseline and after three months . as seen , selenium supplementation did not have any significant effect on acute phase reactants . in table 2 , mean sd values of thyroid function tests are presented at baseline and after intervention . abbreviations : crp : c - reactive protein , esr : erythrocyte sedimentation rate . we studied the effects of selenium supplementation in hemodialysis patients who had low serum levels of selenium . according to the obtained results , although serum selenium level increased significantly after supplementation and was higher after three months in experimental group compared to control group , selenium supplementation did not have any significant effect on acute phase reactants or tfts . at baseline , after three months , we observed increase in crp level in the both groups , but change in crp level in experimental group was less prominent than that of control group , though the difference was insignificant . ( 11 ) studied 80 hemodialysis patients in a randomized double - blind trial and followed them for three months to determine the effects of selenium supplementation on inflammatory markers . they reported no significant differences between selenium and placebo groups regarding changes in serum levels of lipoproteins , high - sensitivity crp , homocysteine , ferritin and transferrin . in their study , mean ferritin level in selenium group was 819.4 ng / ml with an interquartile range of 439.95 to 1180 , which changed by a mean of 23 ng / ml ( 95% ci = -26.67 to 168.58 ) and was not statistically different from the placebo group . mean hs - crp in selenium group was 4.5 with an interquartile range of 1.72 to 19.97 . after supplementation , hs - crp showed a mean decrease by 0.85 g / ml , which did not show significant difference to the control group . the only inflammatory marker decreased significantly in that study in selenium group was il-6 . in support of the concept of beneficial effects of selenium on oxidative stress and inflammation , previous reports recommended selenium supplementation in hemodialysis patients ( 20 , 21 ) . in another trial ( 22 ) , 45 mild , moderate and severe ckd patients of at least six - month duration were studied to find out the effect of selenium supplementation on red blood cell glutathione peroxidase ( gsh - pxs ) enzyme activity . the authors reported that this intervention was effective in increasing mean gsh - pxs level as well as plasma selenium level significantly regardless of ckd severity . we did not find any study about the effect of selenium supplementation on thyroid function in patients with ckd . there is only one study performed on patients with acute renal failure ( 23 ) . in the mentioned study , the role of kidney disease and its effect on tfts is supported further by reports which observed improvement in thyroid hormones and morphology after kidney transplantation ( 17 ) . we recommend to perform future studies with larger sample sizes to address controversy in the literature . in addition , the study period was relatively short . therefore , we recommend to perform future studies in longer periods on more patients . besides , various factors could affect acute phase reactants like infections , which were not able to monitor in this study . it is recommended to control confounding variables , which can adversely affect acute phase reactants such as crp and esr to yield better assessment of selenium effect on these inflammatory markers . the effect of selenium on inflammatory markers and tfts in hemodialysis patients is controversial . according to our findings , selenium supplementation via oral route for

background : selenium deficiency is a common problem in patients with chronic kidney disease ( ckd ) . this micronutrient has anti - inflammatory and anti - oxidant effects . selenium is also found in high concentrations in the thyroid gland.objectives:to determine the effect of selenium supplementation on thyroid function tests and acute phase reactants in hemodialysis patients.patients and methods : in this double - blinded randomized clinical in 3 months , 64 hemodialysis patients with selenium deficiency were divided into experimental ( received selenium supplementation ; 32 cases ) or control group ( received placebo ; 32 cases ) . erythrocyte sedimentation rate ( esr ) , ferritin , quantitative c - reactive protein ( crp ) and thyroid function tests ( tfts ) including thyroid stimulating hormone ( tsh ) , t3 resin uptake ( t3ru ) , and free t4 were measured before and after the intervention and compared between experimental and control groups.results:at baseline , no significant difference was found between experimental and control groups regarding crp , esr and ferritin serum levels . likewise , after intervention , no significant difference was found between experimental and control groups for crp ( 14.77 17.93 vs. 18.29 21.56 mg / l ) , esr ( 32.90 32.62 vs. 33.91 31.15 mm / h ) and ferritin ( 528.6 423.07 vs. 519.52 345.59 ng / ml ) . at baseline , no significant difference was found between experimental and control groups regarding tfts . likewise , after intervention , no significant difference was found between experimental and control groups for tsh ( 3.7 2.22 vs. 2.84 1.88 u / ml ) , free t4 ( 7.19 1.98 vs. 7.02 1.87 g / dl ) and t3ru ( 30.04 2.28% vs. 29.2 1.98%).conclusions : oral selenium supplementation for three months did not have any significant effect on thyroid function tests or acute phase reactants .

female genital tuberculosis ( fgtb ) is common in developing countries and associated with significant morbidity in the form of menstrual dysfunction , infertility , tubal block , peritubal adhesions , intrauterine adhesions , and perihepatic adhesions . fallopian tubes are involved in about 90% of the cases with findings such as congested tubes , tubercles , or caseous nodules on them , peritubal adhesions , and tubal blockage at various levels such as cornual block , mid - tubal block , multiple tubal blocks , or fimbrial block , hydrosalpinx , pyosalpinx , and tubo - ovarian masses . a 25-year - old woman with family history of pulmonary tb was presented to the gynecology outpatient department with primary infertility and oligomenorrhea of 3 years . on examination , vaginal examination demonstrated anteverted uterus with fullness and induration in both fornices . on investigations , hysterosalpingogram which was already performed by the referring doctor for infertility before diagnosis of fgtb was made showed bilateral tubal block . endometrial aspirate performed in premenstrual phase showed positive polymerase chain reaction based on the amplification of the 240 bp region of the mpt 64 gene using primers mpt 1 ( 59-tccgctgccagtcgtcttcc-39 ; nt 460479 ) and mpt 2 ( 59-gtcctcgcgag tctaggcca-39 ; nt 700681 ) with equipment using amplitron thermocycler ( barnstead / thermolyne ) , it also tested positive for culture for mycobacterium tuberculosis using mycobacterium growth indicator tube-960 . , there were findings of tb in the pelvis in the form of multiple tubercles on tubes and uterus , bilateral hydrosalpinx with beading of tubes , and few caseous nodules . when methylene blue dye was injected in the uterus , the fallopian tubes became distended and blue colored with beading looking with alternate constrictions and dilatation looking - like blue pythons diagnosis of genital tb was made , and she was started on anti - tb therapy . her postoperative period and follow - up were uneventful . left fallopian tube with distension , multiple ballooning , and blue coloration with fimbrial block showing blue python sign although gold standard in diagnosis of fgtb is demonstration of mycobacterium tuberculosis either on microscopy or culture of endometrial biopsy or presence of epithelioid granuloma on histopathology , they are positive in only few cases leading on to missing of diagnosis in many cases . polymerase chain reaction on endometrial biopsy is a sensitive and rapid method for detecting mycobacterium dna ( mpt 64 gene ) but can be false positive and may not be able to differentiate between infection and disease . gene xpert has recently been introduced in both pulmonary and extra - pulmonary tb as a sensitive and specific method , but its data in fgtb are very sparse . imaging modalities such as ultrasound , computed tomography scan , magnetic resonance imaging , and positron emission tomography scan have main role in tubo - ovarian masses . hysterosalpingography is generally avoided in a suspected case of fgtb due to risk of flare up of the disease but may demonstrate tubal block and other tubal patterns , especially in advanced case . hysteroscopy is useful in endometrial disease and may show pale looking endometrium with endometrial cavity being partially or completely obliterated by adhesions of varying grade ( asherman 's syndrome ) which may involve ostia . however , laparoscopy is the most reliable tool to diagnose fgtb , especially for tubal , ovarian , and peritoneal disease . there can be tubercles on peritoneum or tubes , tubo - ovarian masses , caseous nodules , encysted ascites , various grades of pelvic adhesions , hydrosalpinx , pyosalpinx , beaded tubes , tobacco pouch appearance , and inability to see tubes due to adhesions . other authors have also found laparoscopy very useful in diagnosis and management of genital and peritoneal tb with the advantage of avoidance of laparoscopy which is more traumatic and hazardous especially in a case of abdominopelvic tb . in a case of fgtb , if there is no cornual block , there is a partial or complete fimbrial block with multiple blocks at various parts of tube as is typical of fgtb , dye enters the tube but remains in various parts of tubes with alternate dilatation and constriction making the tube look like a blue python as happened in the present case . the new sign case is easily diagnosed by gynecologists during routine laparotomy performed for infertility patients with suspected fgtb and can aid in its diagnosis

female genital tuberculosis ( fgtb ) is an important cause of infertility in developing countries . various type of tb salpingitis can be endosalpingitis , exosalpingitis , interstitial tb salpingitis , and salpingitis isthmica nodosa . the fallopian tubes are thickened enlarged and tortuous . unilateral or bilateral hydrosalpinx or pyosalpinx may be formed . a new sign python sign is presented in which fallopian tube looks like a blue python on dye testing in fgtb .

recent genome - wide transcriptome studies have revealed that the majority of the mammalian genome is transcribed , thereby giving rise to a range of coding and noncoding rna ( ncrna ) transcripts . the latest human gencode release ( version 24 ) , presented by the encyclopedia of dna elements consortium ( encode ) , showed that the human genome is composed of 60554 genes ( figure 1 ) . among those , 19815 are protein - coding genes , whereas the remaining two - thirds of the genome represents noncoding rna genes ( 15941 long noncoding rna genes , 9882 small noncoding rna genes , and 14505 pseudogenes ) . noncoding rnas are predominant in the genome and are classified as either housekeeping or regulatory noncoding rnas . housekeeping noncoding rnas are constitutively expressed and they function as key regulatory molecules of many cellular processes . this group includes ribosomal ( rrna ) , transfer ( trna ) , small nuclear ( snrna ) , and small nucleolar rnas ( snornas ) . the second group , regulatory noncoding rnas , is divided into short ncrnas ( < 200 nucleotides ) or long noncoding rnas ( lncrnas , > 200 nucleotides ) . short ncrnas are represented by micrornas ( mirnas ) , small interfering rnas ( sirnas ) , and piwi - associated rnas ( pirnas ) , and , in few cases , by antisense rnas and enhancer rnas ( ernas ) . long noncoding rnas group includes antisense rnas and enhancer rnas ( ernas ) [ 13 ] ( figure 2 ) . in this review , this class of ncrnas was first discovered in the mouse during large - scale sequencing of full - length cdna libraries . generally , lncrnas were characterized as transcripts longer than 200 nucleotides with some features typical for protein - coding mrna . they are transcribed by rna - polymerase ( pol ) ii , capped on the 5 end , polyadenylated , and commonly expressed as alternatively spliced variants . moreover , lncrnas were found to have the same trimethylation marks of h3k4 and h3k36 at their promoters and transcribed regions . in contrast to mrna , lncrnas usually contain intron / exon structure , they are expressed at lower levels , often in a tissue - specific manner , and their sequence is poorly evolutionary conserved . additionally , they do not possess open reading frames ( orfs ) , 3 utr , and termination regions ; thus the majority have limited coding potential . nevertheless , recent studies have demonstrated that some lncrnas can be associated with polysomes and ribosomes , suggesting that they may act as coding transcripts and give rise to small peptides . an emerging number of studies related to the discovery of new lncrnas and their position to the nearest protein - coding genes have resulted in the introduction of a new nomenclature for these molecules . long noncoding rnas may be classified into five categories : ( 1 ) sense lncrnas that overlap the nearest protein - coding genes at the same strand ; ( 2 ) antisense lncrnas located across the exons of protein - coding genes from the opposite strand ; ( 3 ) bidirectional lncrnas transcribed on the opposite strand within 1 kb from the nearest protein - coding gene ; ( 4 ) intronic lncrnas that overlap intronic regions of coding genes in either the sense or antisense orientation ; ( 5 ) intergenic lncrnas that represent the largest group of lncrna ; they are located between protein - coding genes but are at least 1 kb away from the nearest protein - coding gene ( figure 3 ) . in recent years , numerous studies have aimed to identify functions of newly discovered long noncoding rnas . although the primary role of lncrnas was their epigenetic regulation of protein - coding gene expression , only a limited number of such long noncoding transcripts have been identified . it was soon discovered that lncrnas play a role in variety of biological process , acting in the nucleus , or in the cytoplasm , or in both . functions of lncrnas are manifested by three types of interaction : rna - rna , rna - dna , and rna - protein , all of which depend on the site of action . in the nucleus , for example , the main role of lncrnas is assumed to be epigenetic imprinting . one of the best - described mechanisms of lncrnas action during the epigenetic regulation is the x chromosome inactivation , in which x chromosome inactive - specific transcript ( xist ) plays a key role . recent studies demonstrated that lncrnas can interact with chromatin remodeling complexes including polycomb repressive complex ( prc2 ) 2 , trithorax / mll , and h3k9 methyltransferase g9a , acting as docking platforms to specific genomic loci . these protein complexes modify dna methylation patterns via trimethylation leading to transcriptional repression ( prc2 ) or activation ( trithorax / mll ) of the lncrna target genes . numerous lncrnas have been recognized as scaffolds for inhibitory prc2 including xist , kcnq1ot1 , and hotair [ 8 , 25 ] . other lncrnas , like hottip , directly bind to protein complexes of trithorax / mll . in these situations , , it has been postulated that these lncrnas are guides for chromatin - modifying complexes , where they first recognize the target localizations at the chromatin , bind to them , and then form docking platforms for protein partners . in addition to regulation of genomic imprinting , lncrnas are also involved in the control of gene expression at the transcriptional level . lncrnas can directly interact with transcription factors , acting as decoys or inhibitors for their binding to the target dna sequence . for example , two lncrna molecules , lethe and p50-associated cox-2 extragenic rna ( pacer ) , have been shown to interact with different subunits of nf-b , thus preventing it from binding to the promoter region of the target gene [ 10 , 27 ] . moreover , lncrnas exert their regulatory function during mrna processing and stability , interacting with the heterogeneous nuclear ribonucleoproteins ( hnrnps ) . an example of this type of rna - protein linkage is demonstrated for several lncrnas including lincrna - p21 , lincrna - cox2 , or thril ; thus they affect either activation or repression of target genes . other actions of lncrna in the nucleus are manifested by alternative splicing regulation and subnuclear compartment formation ( figure 4 ) . in the cytosol , lncrnas exert their function by interacting with target mrnas ( or mirnas ) through base - pairing . in this manner , lncrnas may either stabilize ( e.g. , bace1-as prevents mirna - induced repression of bace1 transcript ) or decay ( e.g. , 1/2-sbsrnas bind to the alu element of smd target mrnas within the 3 utr region ) target transcripts . moreover , lncrnas promote translation of transcripts ( e.g. , antisense uchl1 interacts with uchl1 mrna , resulting in recruitment of ribosomes ) or repress this process ( e.g. , lincrna - p21 binds to target mrna causing recruitment of translation repressors ) ( figure 4 ) . inflammation is a complex process , aiming to defend the body against harmful agents by removing or neutralizing them in order to restore tissue homeostasis . the complexity of this process results from the broad spectrum of inflammatory pathways , various inflammatory inducers , sensors , and mediators . in the classic view , the inflammatory response is induced immediately after stimulation by the inflammatory stimuli , such as a bacterial pathogen . cells , such as neutrophils , dendritic cells ( dcs ) , and macrophages , express evolutionarily conserved toll - like receptors ( tlrs ) on their surface . the tlr family is composed of several classes of transmembrane proteins and recognizes specific structures present on the microbes , known as pathogen - associated molecular patterns ( pamps ) . induction of tlrs triggers a multistep signaling cascade , thereby resulting in the modulation of gene expression involved in the immune response . key processes regulated by tlrs are related to the secretion of various mediators by immune cells , phagocytosis , cell migration , metabolic reprogramming , and tissue repair . in general , based on the length , the inflammatory response can be classified into acute or chronic . acute inflammation usually develops locally , and its role is to eliminate toxic agents , repair damaged tissue , and restore homeostasis . in some circumstances , local inflammation might be greater , therefore called a generalized inflammatory response , which is associated with the response of the whole organism . although the acute phase leads to the beneficial recovery of the injured tissue and healing , this nonspecific immune process could have also negative effects . without the proper resolution phase , it can easily turn into a chronic state . chronic inflammation may appear after the switch from the acute phase , and it is characterized by the specific , long - term cellular , and humoral immune response present at the site of tissue injury . in consequence , it may significantly contribute to dangerous pathophysiological changes and initiate the development of chronic diseases . each inflammatory stage one such factor is nf-b ( nuclear factor kappa - light - chain - enhancer of activated b cells ) , representing a family of transcription factors . nf-b plays an important role in gene expression regulation of multiple factors involved in immune , acute phase , and inflammatory responses . nf-b , in addition to controlling the development and function of the immune system , is required in other physiological processes such as suppression of apoptosis and cell proliferation and differentiation . in mammals , the active form of nf-b is composed of homo- and heterodimers of the nf-b family members , which can be divided into five different subunits : p50 , p52 , p65 ( rela ) , c - rel , and relb . all of these share homology of the specific n - terminal domain responsible for the dimerization and binding to target dna sequences . depending on the local cytokines produced by the immune cells , nf-b also controls macrophage relocalization , activation , and differentiation into two different phenotypes : proinflammatory ( m1 ) or anti - inflammatory ( m2 ) . in response to proinflammatory cytokines , nf-b is also involved in the activation of t lymphocytes , which proliferate and secrete cytokines such as interferon gamma ( ifn ) , interleukin 6 ( il-6 ) , or tnf-. the result of nf-b activation is stimulation of proinflammatory cytokine production by macrophages and t lymphocytes , thereby enhancing the inflammatory response . it is characterized by an excessive fat accumulation and enhanced production of proinflammatory cytokines / chemokines by adipose tissues . it is commonly caused by a combination of excessive food intake , lack of physical activity , and genetic susceptibility . a great majority of obese individuals present features of the metabolic syndrome : ( a ) increased waist circumference , ( b ) insulin resistance , ( c ) hyperglycemia , ( d ) hypertension , and ( e ) hypertriglyceridemia . appearance of these symptoms is associated with a high risk of serious disease development , including type 2 diabetes , cardiovascular diseases , , nonalcoholic fatty liver disease ( nafld ) , and even some cancers . the mechanism of metabolic disorder development is still not fully understood ; nevertheless numerous studies have suggested an adipocyte dysfunction as a main cause of metabolic system failure . in this pathological state , adipose tissue secretes various mediators , including cytokines , inducing a chronic , low - grade inflammatory response that results in recruitment of immune cells into the tissue . the excessive accumulation of tissue fat leads to disturbance both in the secretory and in the storage functions of adipocytes . it is postulated that the excessive fat mass induces the production of reactive oxygen species and the development of chronic oxidative stress in patients with obesity and metabolic dysfunction [ 44 , 45 ] . in adipose tissue of obese patients is observed severe inflammatory response , characterized by high production of cytokines and increased infiltration of immune cells , including monocytes , stimulated to differentiate into macrophages and lymphocytes t , activated to proliferate and secrete cytokines [ 46 , 47 ] . cytokines secreted by activated macrophages act on tlrs present on the surface of adipocytes , stimulating them to produce adipokines and cytokines . based on these observations , it has been found that the reactions of both the immune and metabolic systems are closely connected , and they play an important role in the development of metabolic disorders . adipocytes play a central role in energy homeostasis by fine - tuning the equilibrium between nutrient deposition ( white adipose tissue , wat ) and energy expenditure ( brown adipose tissue , bat ) . additionally , adipose tissue acts as an endocrine organ by secreting factors ( adipokines ) that regulate whole body energy and glucose homeostasis . adipogenesis is a complex process governed by a network of transcription factors , cofactors , and signaling intermediates from numerous pathways . the transcriptional cascade that regulates adipocyte differentiation is predominantly driven by peroxisome proliferator - activated receptor gamma ( ppar ) , shown to be both necessary and sufficient for adipogenesis . importantly , adipogenesis is highly regulated not only by ppar but also by the coordinated effects of other transcription factors including ccaat / enhancer - binding proteins ( c / ebps ) , kruppel - like factors ( klfs ) or wingless proteins ( wnt ) , and transcriptional cofactors [ 5254 ] . in recent years , there has been growing attention paid to noncoding rnas as a novel , cell intrinsic regulatory mechanism . specifically , lncrnas are now emerging as important regulators of gene expression both on the transcriptional and on the posttranscriptional levels . long noncoding rnas are being discovered using modern techniques of microarray or next - generation sequencing . however , only small portions have been fully characterized . in the case of adipogenesis , there is wide expression of lncrnas , controlling a variety of genes involved in the formation , differentiation , and activation of adipocytes ( figure 5 ) . steroid receptor rna activator ( sra ) lncrna was described for the first time as a molecule regulating adipogenesis . it was shown that lncrna sra , initially identified as a coactivator of steroid receptors , also acts as a transcriptional coactivator of ppar [ 55 , 56 ] . lncrna sra promotes adipocyte differentiation by binding to the n - terminal portion of ppar and enhances its transcriptional activity . additionally , its overexpression significantly increased mrna and protein levels of adipocyte master regulators ppar and c / ebp as well as ppar target genes . moreover , gene expression profiling showed that lncrna sra regulates expression of genes related to various cellular processes including cell cycle and insulin transduction pathways . further experiments proved that lncrna sra overexpression inhibits phosphorylation of p38 mitogen activated protein kinase ( mapk ) and c - jun nh2-terminal kinase ( jnk ) in the early differentiation of st2 mesenchymal precursor cells . in contrast , knockdown of lncrna sra increased p38 and jnk phosphorylation while reducing insulin receptor mrna and protein levels , which led to decreased downstream signaling . supporting that data , sra knock - out mice ( sra ) are resistant to high fat diet ( hfd ) induced obesity . after 14 weeks of hfd , sra mice are characterized by reduced wat mass and decreased expression of adipocyte genes such as adiponectin or fatty acid - binding protein 4 . the lean phenotype is also associated with smaller adipocytes in wat compared to wt mice , reduced liver mass , fewer lipid droplets in the liver , and decreased expression of lipogenesis - associated genes . finally , sra animals have improved insulin sensitivity . in a recent paper , xiao and coworkers identified lncrna adinr ( adipogenic differentiation induced noncoding rna ) that , in cis , transcriptionally activates c / ebp. it was upregulated 2030-fold during the course of adipogenesis and was transcribed from a position ~450 bp upstream of the c / ebp gene . knockdown of lncrna adinr with sirna resulted in a dramatic adipogenic defect as shown by a decreased number of oil red o positive cells and reduced adipogenic transcripts c / ebp , ppar , fatty acid - binding protein 4 , and lipoprotein lipase . importantly , inhibition of adipogenesis caused by depletion of lncrna adinr was rescued by overexpression of c / ebp. the lncrna adinr mechanism of action relies on its binding to pa1 followed by recruitment of mll3/4 histone methyltransferase complexes . in turn , that causes an increase in h3k4me3 and a decrease in h3k27me3 histone modification in the c / ebp locus during adipogenesis . expression of the pu.1 gene gives rise to both the mrna encoding pu.1 protein and antisense ( as ) lncrna originating from a promoter in the antisense strand of intron 3 . the protein pu.1 was originally demonstrated to be a key transcription factor regulating hematopoiesis , but recent studies revealed that it is also expressed in 3t3-l1 preadipocytes and murine adipocytes isolated from wat [ 60 , 61 ] . gain and loss of function studies established the pu.1 protein as an inhibitor of 3t3-l1 preadipocyte differentiation , acting via downregulation of ppar . on the other hand , pu.1 as lncrna promotes adipogenesis through preventing pu.1 mrna translation by binding to pu.1 mrna to form an mrna / as lncrna duplex . formation of this sense - antisense rna duplex was also confirmed in porcine adipocytes by an endogenous ribonuclease protection assay combined with rt - pcr . in line with the previous data , expression of ppar and fatty acid synthase ( fasn ) was significantly upregulated in the lncrna pu.1 shrna treated group . in a number of studies , global transcriptome profiling was used to select specific lncrnas either involved in adipogenesis or characteristic for adipocytes specifically isolated from wat or bat . in a seminal study done by sun and coworkers , the transcriptome of primary brown and white adipocytes , preadipocytes , and cultured adipocytes was profiled . scientists identified 175 lncrnas that were specifically regulated during adipogenesis . to further validate lncrnas that are functionally important for adipogenesis , researchers selected the top 20 lncrna genes based on the following criteria : ( i ) significant upregulation in both brown and white fat cultures , ( ii ) binding to ppar or cepb promoters , and ( iii ) independent validation of adipose - specific expression . by using rnai - mediated loss of function , the top 10 most important lncrnas were selected and called regulated in adipogenesis ( lncraps ) in order to highlight their key role in proper differentiation of adipocyte precursors . in comparison to white adipocytes , brown adipocytes have a higher mitochondrial content and express uncoupling protein 1 ( ucp1 ) , which disperses chemical energy through heat production . brown adipose tissue helps to regulate energy expenditure in rodents and newborn babies , but it has been considered to have no physiologic relevance in adults [ 65 , 66 ] . however , recent studies demonstrated that metabolically functional bat is also present in adult humans . as a result , an understanding of bat physiology might provide an effective treatment of obesity or other metabolic disorders . identification of the lncrnas crucial for bat differentiation and function was done using whole transcriptome rna sequencing . next , gain and loss of function studies established lncrna blnc1 as a potent activator of thermogenic adipocyte differentiation . it functions upon formation of rna - protein complex with the early b cell factor 2 ( ebf2 ) transcription factor to stimulate bat formation . additionally , lncrna blnc1 itself is a target of ebf2 , thereby forming a feed - forward regulatory loop . on the other hand , despite its stimulatory effects on brown preadipocyte differentiation , lncrna blnc1 failed to promote differentiation of 3t3-l1 cells into ucp1 positive adipocytes . another global profiling of gene expression during mouse brown fat cell differentiation , followed by candidate lncrnas genomic context analysis , pathway analysis , and gene ontology enrichment of their associated protein - coding genes was recently described . in this study , scientists identified three lncrnas ( gm15051 , tmem189 , and cebpd ) associated with their flanking coding genes ( hoxa1 , c / ebp , and c / ebp ) that participated in adipose commitment . more recently , rna - seq analysis of murine brown , inguinal white , and epididymal white fat allowed for identification of lncrna cluster enriched in bat . among them , authors showed that bate1 binds to the heterogeneous nuclear ribonucleoprotein u , a factor also required for bat formation . finally , interactions of lncrnas with micrornas in the context of adipogenesis were also described . as demonstrated by gernapudi and coworkers , the mir-140/lncrna neat1 signaling network is necessary for adipogenesis . adipocyte derived stem cells isolated from mir-140 knock - out mice had dramatically decreased adipogenic capabilities found to be associated with the downregulation of lncrna neat1 expression . they also found that the physical interaction with mir-140 in nucleus led to increased expression of neat1 . excessive fat accumulation not only causes pathological expansion of adipose tissue but also leads to triglyceride deposition in the liver . presence of lipid droplets in excess of 5% of total hepatocytes is a diagnostic hallmark of nonalcoholic fatty liver disease ( nafld ) . although most patients suffer from a mild course of illness , approximately 25% of cases progress and can subsequently lead to the development of steatohepatitis , hepatic fibrosis , liver cirrhosis , and hepatoma . scientists and physicians estimate that between 20% and 30% of west 's general population suffers from nafld , which makes this affects more than a billion people worldwide . to address questions about the global expression pattern and functional contribution of lncrnas during nafld , sun and coworkers analyzed microarray expression using rna extracted from liver biopsies of healthy patients and those suffering from nafld . in this analysis , 535 lncrnas were found upregulated in nafld samples compared with controls and 1,200 were downregulated in nafld ( figure 5 ) . out of these , seven nafld samples that had highly up- or downregulated gene expression were validated by quantitative real - time pcr ( qpcr ) . three lncrna results obtained by qpcr were different in comparison to the microarray data , showing no change between analyzed groups . however , changes in other selected hits were confirmed , highlighting the importance of microarray data validation . pancreatic islets , through the secretion of endocrine hormones such as insulin and glucagon , play a key role in metabolic homeostasis . however , one of the most common malfunctions of -cells is caused by lack of insulin , leading to the development of diabetes . additionally , diabetes may develop as a result of insulin resistance , a condition in which the body can not effectively use the insulin produced by -cells . as a result , blood sugar levels are deregulated and various tissues are exposed to prolonged hyperglycemia that , over time , causes serious damage to many of the body 's systems . it should be emphasized that hyperglycemia , a hallmark of diabetes , can cause both acute and long - term complications like cardiovascular disease , stroke , foot ulcers , ketoacidosis , or hyperosmolar coma . based on etiology , who classifies diabetes into four categories , but the vast majority of cases fall into two broad etiopathogenetic groups : type 1 diabetes mellitus ( t1 dm ) or type 2 diabetes mellitus ( t2 dm ) . accounting for 510% of all cases , t1 dm is characterized by the loss of insulin producing -cells due to an autoimmune reaction . type 2 diabetes mellitus represents 9095% of all cases and , unlike t1 dm , is strongly associated with patients ' lifestyle . a number of lifestyle factors are known to be important for development of t2 dm including obesity ( bmi above 30 ) , lack of physical activity , poor diet , or stress . it is a chronic disease that begins with insulin resistance , a condition in which cells ( mostly adipocytes , hepatocytes , and muscle cells ) fail to properly absorb and metabolize glucose . as a result , -cells increase insulin production to overcome hyperglycemia , but this compensatory mechanism is transient and generally fails over time . intensified metabolism of -cells leads to a gradual loss of their endocrine function and finally causes -cell apoptosis . who , in 2014 , the global prevalence of diabetes was estimated to be 9% among adults aged over 18 years and it is expected to double before 2030 [ 75 , 80 ] . additionally , experts project that , in 2030 , diabetes will be the 7th leading cause of death . initial studies aimed at identification of specific lncrnas expressed in murine and human pancreatic islets were done in 2012 [ 81 , 82 ] . by using next - generation sequencing , scientists revealed a large collection of 1359 intergenic lncrnas expressed in murine -cells ( figure 5 ) . many of them were highly tissue - specific , but their function was not evaluated , and future experiments are still needed . similar numbers of 1128 intergenic and antisense lncrnas were identified in human -cells and many of them ( e.g. , hi - lnc12 and hi - lnc77 ) were highly tissue - specific . additionally , lncrna - encoding genes were preferentially located near genes encoding important regulators of -cell function , development , and transcription . one of them , lncrna hi - lnc25 , conserved between mouse and human , was shown to positively regulate glis3 mrna . glis3 encodes a pancreatic islet transcription factor that is mutated in monogenic diabetes and contains t2 dm risk variants [ 83 , 84 ] . in the same set of experiments , scientists also showed that selected lncrnas ( e.g. , hi - lnc12 and hi - lnc25 ) are linked to -cell differentiation program as their expression was significantly higher in human islets when compared to the embryonic pancreas . moreover , the addition of glucose to in vitro -cell culture induced expression of some lncrnas , suggesting their importance for mature islet cell physiology . finally , authors showed deregulation of islets ' lncrnas in t2 dm by comparing profiles of lncrnas in islets from 19 nondiabetic and 16 t2 dm donors . two lncrnas , namely , kcnq1ot1 and hi - lnc45 , were significantly increased or decreased in t2 dm islets , respectively . seven lncrnas specific for -cells , as reported by morn and coworkers , were also discussed in another study that analyzed pancreatic islets from 89 individuals with or without diabetes . microarray analysis , rna sequencing , and exome sequencing methods enabled researchers to identify 493 lncrnas that were differentially expressed in the pancreatic islets . out of those , 17 long intergenic noncoding rnas ( lincrnas ) were significantly associated with donor 's hba1c levels , and two ( loc283177 and snhg5 ) were also involved in gene expression regulation . to obtain insight into functional target genes of lncrnas loc283177 and snhg5 , the authors performed a coexpression analysis , linking their expression with all other genes in pancreatic islets . lncrna loc283177 levels correlated with expression of genes that play a key role in islet function , namely , synaptotagmin 11 , map - kinase activating death domain ( madd ) , and paired box 6 ( pax6 ) . synaptotagmin 11 is known to regulate the exocytosis of insulin and madd proinsulin synthesis , and pax6 is involved in development of pancreatic islets [ 85 , 86 ] . supporting these findings , lncrna loc283177 expression was found to be directly associated with insulin exocytosis in the islets . insulin is secreted from -cells in response to glucose , while other nutrients such as free fatty acids and amino acids can augment glucose - induced insulin secretion . in addition , various hormones ( e.g. , leptin , growth hormone , glucagon like peptide-1 , and estrogen ) also regulate insulin secretion . as a result , -cells are called a metabolic hub in the body that coordinate nutrient metabolism with endocrine system . insufficient insulin production , or insulin resistance , is a prime cause of diabetic complications . among the many tissues affected by hyperglycemia , endothelial cells are very important because they are implicated in pathogenesis of diabetes - related microvascular and macrovascular complications . one of these complications , diabetic retinopathy , was recently shown to be influenced by lncrna malat1 ( metastasis - associated lung adenocarcinoma transcript 1 ) ( figure 5 ) . in a mouse model of streptozotocin - induced diabetes microarray analysis , followed by qpcr validation of selected genes , allowed for identification of 303 differentially expressed lncrnas including 214 downregulated and 89 upregulated genes in diabetic versus nondiabetic samples . one gene , lncrna chr19 : 57956895802671 , a murine ortholog of human lncrna malat1 , had a greater than 10-fold upregulation in diabetic retinas . lncrna malat1 was previously described in humans to be deregulated in several solid tumors and was associated with cancer metastasis and recurrence . it was shown to be significantly upregulated in a rf/6a cell model of hyperglycemia , in the aqueous tumor samples and in fibrovascular membranes of diabetic patients . to further investigate the molecular mechanism of lncrna malat1 action , scientists decided to analyze its function in retinal vasculature and endothelial cell dysfunction in diabetes mellitus . lncrna malat1 knockdown in retinas of db / db mice resulted in amelioration of diabetic retinopathy as manifested by reduced apoptosis of retinal cells and pericytes . importantly , after administration of lncrna malat shrna , scientists observed decreased retinal vascular permeability as measured by evans blue leakage . moreover , in vitro tests showed that lncrna malat1 knockdown decreased retinal endothelial cell proliferation , migration , and tube formation . in rf/6a endothelial cells , lncrna malat1 acts via induction of p38 mapk signaling , and its silencing reduced phosphorylated p38 level but had no effect on phosphorylated erk1/2 or jnk1/2 . pretreatment of rf/6a cells with sb203580 , a p38 mapk pathway inhibitor , strongly blocked the effect of lncrna malat1-induced cell proliferation . more recently , the same research group reported that lncrna miat ( myocardial infarction - associated transcript ) also regulates diabetes mellitus microvascular dysfunction , acting as a competing endogenous rna ( figure 5 ) . similar to a previous report , lncrna miat expression was increased in diabetic retinas and endothelial cells cultured in high glucose medium . as shown by in vivo tests , lncrna miat knockdown ameliorated diabetes mellitus induced retinal microvascular dysfunction . in line with in vivo data , silencing of lncrna miat in endothelial cells cultured in vitro led to an inhibition of proliferation , migration , and tube formation . lncrnas may act as an mrna molecular sponge and regulate mirnas available for binding their target mrnas . bioinformatics suggested that the lncrna miat sequence contains 4 putative mirna binding sites including mir-29a-3p , mir-29b-3p , mir-29c-3p , and mir-150 - 5p . one of these , mir-150 - 5p , was proven to directly target lncrna miat in endothelial cell both in vitro and in vivo . this particular mirna is of great importance for angiogenesis because it regulates the expression of vegf , a key angiogenic factor involved in physiological and pathological angiogenesis . yan and coworkers proved that miat regulates the expression of mir-150 - 5p target gene vegf creating a critical regulatory loop for endothelial cell function . during angiogenesis , lncrna miat is significantly upregulated , which in turn alleviates the mir-150 - 5p repression effect , thereby upregulating the level of mir-150 - 5p target gene , vegf . in contrast to lncrna miat , lncrna meg3 ( maternally expressed gene 3 ) expression was significantly downregulated in the retinas and endothelial cells of stz - induced diabetic mice upon high glucose and oxidative stress conditions . its knockdown regulated retinal endothelial cell proliferation , migration , and tube formation in vitro . as demonstrated in rf/6a endothelial cells , inhibition of lncrna meg3 by sirna significantly increased levels of phosphorylated pi3k and phosphorylated akt at thr and ser . addition of pi3k inhibitors to cell cultures abrogated the observed phenotype , proving that lncrna meg3 regulates hyperproliferation of retinal endothelial cells through pi3k / akt signaling . additionally , its activation influences endothelial cell biology by modulating angiogenesis , proliferation , and microvascular permeability .

ribonucleic acids ( rnas ) are very complex and their all functions have yet to be fully clarified . noncoding genes ( noncoding rna , sequences , and pseudogenes ) comprise 67% of all genes and they are represented by housekeeping noncoding rnas ( transfer rna ( trna ) , ribosomal rna ( rrna ) , small nuclear rna ( snrna ) , and small nucleolar rna ( snorna ) ) that are engaged in basic cellular processes and by regulatory noncoding rna ( short and long noncoding rna ( ncrna ) ) that are important for gene expression / transcript stability . in this review , we summarize data concerning the significance of long noncoding rnas ( lncrnas ) in metabolic syndrome related disorders , focusing on adipose tissue and pancreatic islands .

fascin is an actin - binding protein that is known to regulate the parallel bundling of actin filaments ( vignjevic et al . , 2006 ) , stabilize filopodia and invadopodia ( jayo et al . , 2012 , regulate adhesion dynamics in migrating cells ( elkhatib et al . , 2014 ) . fascin has received considerable attention in recent years as its expression is very low or absent in normal adult epithelia , but it is dramatically upregulated at both transcript and protein levels in all forms of human carcinomas studied to date ( hashimoto et al . , 2005 ) . thus , fascin is emerging as an excellent prognostic marker and a potential therapeutic target for metastatic disease ( tan et al . , 2013 , adams , 2015 ) . despite this recognized clinical importance , there is still very little molecular detail available defining the mechanisms underpinning fascin - dependent cell invasion , thus significantly limiting strategic approaches for therapeutic design . it is also unclear whether these defined roles for fascin in tumorigenesis rely upon the classical f - actin - bundling function or whether other roles may exist that coordinate cell invasion . fascin comprises four tandem -trefoil domains that form a bilobed structure , with -hairpin triplets located symmetrically on opposite sides of each lobe that are proposed to act as the actin - binding domains ( sedeh et al . , 2010 ) . these actin bundles , whether in the form of filopodia extending beyond the cell edge or microspikes within lamellae of migrating cells or neuronal growth cones , are involved in controlling cell migration in vitro ( adams , 2004 ) and embryonic development in vivo ( wood and martin , 2002 , mattila and lappalainen , 2008 , hashimoto et al . , invasion of carcinoma cells is a highly coordinated process that depends largely on alterations to cell - cell and cell - extracellular matrix ( ecm ) adhesion and organization of the actin cytoskeleton ( guo and giancotti , 2004 ) . carcinoma cells migrating in 3d ecm and in living tissues assemble membrane protrusions and specialized ecm - degrading adhesions termed invadopodia to enable tunneling through the matrix ( friedl and wolf , 2003 , condeelis et al . we and other groups have shown that loss of fascin function in a range of cell types results in reduced assembly of actin protrusions , more stable adhesions , and reduced migration and invasion in vivo ( hashimoto et al . however , it remains unclear whether these reported functions for fascin depend upon actin bundling within filopodia alone , or whether other roles for fascin exist within normal and metastatic cells that promote motility . physicochemical properties of the ecm play an important role in the regulation of cell migration ( charras and sahai , 2014 , friedl and alexander , 2011 ) and cancer cells have been shown to have great plasticity , enabling them to adapt their migratory strategies to external cues ( sanz - moreno et al . , 2008 , wolf et al . , 2003 , several studies have demonstrated that nuclear size and deformation act as limiting factors of cell migration in physically confined environments ( wolf et al . , 2013 , rowat et al . , 2013 , davidson et al . , 2014 ) . contractile force generation , cytoskeleton - driven force transmission to the nucleus , and nuclear stiffness ( harada et al . , 2014 , lammermann et al . , 2008 , lombardi et al . , 2011 , alam et al . , 2015 ) can together create a migratory threshold ( isermann and lammerding , 2013 , swift and discher , 2014 ) . the linker of the nucleus and cytoskeleton ( linc ) complex connects the cytoskeleton to the nuclear inner lamina ( ( chang et al . , 2015 , meinke and schirmer , 2015 ) ) and is formed by kash proteins ( klarsicht , anc-1 , and syne homology proteins , nesprins ) at the outer nuclear envelope ( ne ) and sun proteins ( sad1 and unc-84 ) at the inner ne . this complex is known to play an essential role in force transmission ( lombardi et al . ne response to physical strain ( guilluy et al . , 2014 ) , and nuclear localization in migrating cells ( luxton et al . , 2010 , meinke et al . , 2014 ) . mutations in the linc complex have been associated mainly with muscular dystrophies and cardiomyopathies ( isermann and lammerding , 2013 , zhang et al . , 2007 ) , but despite its role in cell motility , the potential contribution to cancer cell invasion and metastatic disease is currently unknown . here , we report that nesprin-2 binds directly to the fascin -trefoil3 domain through spectrin repeats ( srs ) 5153 located at its c - terminal domain . nesprin-2 recruits fascin to the ne both in vitro and in vivo , where it regulates f - actin connection to the linc complex , modulating nuclear localization in fibroblasts and nuclear deformation in cancer cells . these effects are independent of the fascin f - actin - bundling activity , since the disruption of fascin - nesprin-2 interaction by the overexpression of -trefoil3 domain does not alter f - actin dynamics . together , these results provide a role for fascin in coupling the ne to the cytoskeleton to assist in coordinated force transmission during migration . to define potential unexplored regulators of fascin function , we performed mass spectrometry analysis of specific isolated proteins in complex with gfp - fascin immunoprecipitated from mda mb 231 human breast carcinoma cells . within the reproducible hits that were significantly represented in the resulting peptide analysis , we identified nesprin-1 , a member of the linc complex ( figure s1a ) . we have previously shown that fascin localizes to the nucleus and the nuclear periphery both during drosophila development and in mammalian cells ( groen et al . , the function associated with this specific localization remains unknown ; therefore , we chose to further characterize the potential interaction with the nesprin family of proteins . initial experiments did not reveal a detectable interaction with endogenous full - length nesprin-1 , but as this analysis was performed at low detergent concentration , we can not exclude that this was due to retained co - association between other cytoskeletal - associated ne proteins in complex with fascin . based on our previously published data on fascin subcellular localization , we then focused on the c - terminal domains of the proteins nesprin-1 and -2 , as these regions are present in all nesprin-1 and -2 isoforms anchored to the ne and encompass the most conserved regions across the whole sequence ( autore et al . , 2013 ) ( figure s1b ) . co - immunoprecipitation ( coip ) experiments with either gfp - nes1(6674 ) or gfp - nes2(4956 ) demonstrated that fascin was able to specifically form a complex with the nesprin-2 c - terminal region ( figure 1a ) . we confirmed this interaction in intact cells by quantifying fluorescence resonance energy transfer ( fret ) using fluorescence lifetime imaging microscopy ( flim ) between gfp - tagged nesprins and monomeric rfp ( mrfp)-tagged fascin . this analysis demonstrated a specific and direct interaction in whole cells between gfp - nes2(4956 ) , but not gfp - nes1(6674 ) or the inner ne protein sun2 ( figure 1b ) . to define the region of nesprin-2 that associates with fascin , we used recombinant glutathione s - transferase ( gst)-tagged regions of tandem srs from nesprin-1 and nesprin-2 as bait to pull down endogenous fascin from lysates of nih 3t3 fibroblasts . western blotting revealed that fascin was only found in a complex with sr5153 from nesprin-2 ( figure 1c ) . we verified that fascin directly interacted with sr5153 of nesprin-2 through in vitro binding assays ( figure 1d ) and far - western blot analysis of gst - nesprin sr proteins probed with his - tagged fascin ( figure s1c ) . moreover , gfp - mini - n2g-sr3 - 50 containing the sr5153 region ( schematic representation in figure s1b ) showed specific coip with endogenous fascin in nih 3t3cells ( figure 1e ) . collectively these data demonstrate that fascin forms a complex specifically with nesprin-2 in vitro and in cells , and this binding occurs directly and through sr5153 in nesprin-2 . we next wanted to determine whether fascin localization at the ne was common across different cell types and conditions . confocal images of nih 3t3 cells demonstrated recruitment of fascin at the ne where it colocalized with nesprin-2 ( figure 1f ) . interestingly , fascin localization also showed significant enrichment within the perinuclear area together with nesprin-2 in human cancer cells grown as xenograft tumors in nude mice ( figure s1d ) and with the ne marker protein sun-2 in spontaneous colon carcinomas ( figure s1e ) . we have previously shown that fascin localizes to the nucleus of nurse cells of the developing drosophila ovarian follicle ( groen et al . , 2015 ) ( figure s1 g ) , suggesting that the role of fascin at the ne is evolutionarily conserved . to determine whether nesprin-2 was responsible for fascin localization at the ne or vice versa , we knocked down each protein in fibroblasts and analyzed their respective localization by confocal microscopy . nesprin-2 knockdown ( kd ) , including giant and smaller isoforms ( figure s1f ) , resulted in loss of fascin localization at the ne , but nesprin-2 localization was unaffected by fascin kd ( figure 1h ) . similarly in drosophila embryos , complete disruption of the linc complex through homozygous klaroid mutation ( technau and roth , 2008 ) or loss of only the nesprin homolog msp-300 ( yu et al . , g ) , while loss of fascin did not affect linc complex localization ( figure s1h ) . together , these data demonstrate that fascin localizes to the ne in a nesprin-2-dependent manner . fascin - dependent f - actin bundling is known to be partly controlled by phosphorylation of ser39 by protein kinase c ( pkc ) such that mutation of ser39 to a phospho - dead alanine mimic results in constitutive f - actin bundling by fascin ( ono et al . 2003 ) . to determine whether fascin - nesprin-2 binding was dependent upon fascin - f - actin bundling , we performed coip and fret / flim analysis of wild - type ( wt ) or s39a fascin and nes2(4956 ) . both assays demonstrated that significantly lower levels of s39a fascin bound to nes2(4956 ) compared with wt fascin ( figures 2a and 2b ) . analysis of total fret efficiency and that associated to the ne showed that most of the interaction was restricted to the perinuclear area ( figure 2b ) . s39a fascin localization was associated with a more prominent cytoplasmic recruitment and less enrichment at the ne in hela cells ( figure 2b ) , and a similar mutation in drosophila fascin ( s52a ) also reduced fascin localization to the ne ( figure s2a ) . we thus hypothesized that non - s39 phosphorylated fascin may conformationally restrict the interaction with nesprin-2 . to test this in a cell - free in vitro system , we used far - western blot assays with recombinant fascin mutants and nesprin-2 and nesprin-1 most c - terminal gst - tagged sr triplets . s39a mutant fascin did not show significantly impaired binding to sr5153 ( figure 2c ) , suggesting that any conformational change in this region does not significantly alter binding . this further implies that the stable f - actin bundling and recruitment to filopodia by this mutant may be incompatible with fascin localization to the ne and interactions with nesprin-2 . to further define the region(s ) on fascin required for the interaction with nesprin-2 , we generated four constructs that mapped to the four distinct -trefoil domains in fascin ( figure 2d ) . in vitro pull - down assays with purified gst - nes2(5153 ) and recombinant full - length fascin and -trefoil subdomains revealed that this interaction occurred specifically through the -trefoil3 subdomain ( figure 2e ) . gfp - nes2(4956 ) immunoprecipitations also showed a complex formation with overexpressed -trefoil3 subdomain but not -trefoil1 ( figure 2f ) , and fascin - nes2(5153 ) interaction could be competed in vitro with increasing amounts of recombinant -trefoil3 subdomain ( figure s2b ) . we then co - expressed flag - tagged -trefoil subdomains with gfp - nes2(4956 ) and mrfp - fascin and measured fascin - nesprin-2 binding by fret / flim in hela cells . quantification of fret values demonstrated that overexpression of the -trefoil3 significantly inhibited nesprin-2-fascin binding , whereas the other three domains had no effect on formation of the complex ( figures 2 g and 2h ) . moreover , expression of mcherry - tagged versions of -trefoil3 , but not 1 , 2 , or 4 , led to displacement of endogenous fascin from the ne in nih 3t3cells ( figure 2i ) . conversely , expressing mcherry - tagged sr(5153 ) domain in nih 3t3cells displaced fascin localization from the ne ( figure s2d ) . the formin fhod1 has been reported to interact with nesprin-2 at its n - terminal domain ( amino acids 1,1301,724 ) , and plays a role creating an additional binding site for actin cables to the ne during nuclear movement . to determine potential interdependence of these two actin - binding proteins in connecting the actin cytoskeleton and linc complex , we first explored the role of fhod1 in the interaction of fascin with nesprin-2 . immunoprecipitation assays showed that silencing fhod1 did not affect fascin binding to the gfp - mini - n2g-sr3 - 50 construct ( figure s2e ) . moreover , fret between gfp - nes2(4956 ) with mrfp - fascin after silencing fhod1 showed no change in the level of interaction ( figures s2f s2h ) . taken together , these results demonstrate that fascin binds directly to the region of nesprin-2 encompassing srs 5153 through its third -trefoil domain . this interaction is required for fascin localization at the ne and is independent from the nesprin-2 interaction with fhod1 . we next sought to determine the functional consequences of fascin localization to the ne and its association with nesprin-2 . nesprin-2 has been shown to be important for active positioning of the nucleus during cell migration . starved fibroblasts require stimulation with serum factors such as lpa to establish their nucleo - centrosomal axis during cell polarization after scratch wounding ( gomes et al . , 2005 ) . this process relies in the rearward displacement of actin cables that associate with nesprin-2 at the transmembrane actin - associated ( tan ) lines . these structures are responsible for coupling the nuclei to the moving actin cables , inducing the nuclear positioning at the cell rear while the centrosome remains stationary at the cell centroid ( luxton et al . , 2010 , kutscheidt et al . , 2014 , lombardi et al . , 2011 ) . we therefore hypothesized that the fascin - nesprin complex may play a role in nuclear positioning during migration . to test this confocal microscopy and fluorescence intensity line scan analysis demonstrated that overexpressed myc - tagged fascin showed significant overlap with both f - actin and gfp - mini - n2g-sr3 - 54 in cables positioned over the nucleus ( figure 3a ) , endogenous fascin colocalized with gfp - mini - n2gsr3 - 50 ( figure s3a ) , and gfp - fascin and f - actin cable displacement both correlated with nuclear movement during cell polarization ( figure s3b ) . moreover , stable kd of fascin in nih 3t3cells ( figure s3c ) resulted in a significant reduction in nuclear rearward movement during polarization , and this defect was restored by re - expression of human wt gfp - fascin but not s39a mutant ( figures 3b and 3c ) correlating with fascin binding to nesprin-2 . the reduction in rearward nuclear displacement was also coupled with a reduction in the speed of actin fiber moving over the nucleus ( figures 3d and 3e ; movie s1 ) . this demonstrates that fascin localization at the ne is required for not only efficient f - actin - nuclear coupling but also normal actin rearward movement during cell polarization . to further confirm that this fascin - dependent defect in nuclear movement was dependent upon fascin - nesprin binding , we expressed mcherry - tagged -trefoil domains 1 or 3 of fascin in nih 3t3 and analyzed nuclear movement . our data demonstrated that -trefoil3 , which specifically competes for fascin - nesprin-2 binding , but not -trefoil1 , resulted in a significant reduction in nuclear rearward movement during fibroblast polarization ( figures 3f and 3 g ) . this was specific to fascin - nesprin disruption , as no further defect in nuclear movement was seen in fascin kd cells expressing -trefoil3 compared with mcherry alone ( figure s3d ) . the lack of rescue of phenotype in fascin - depleted cells expressing -trefoil3 also demonstrates that this domain is not sufficient to regulate nesprin-2-f - actin linkage . expression of -trefoil3 did not alter the speed of retrograde actin fiber displacement , suggesting that these defects seen in fascin kd or s39a - fascin - expressing cells are due to global fascin - f - actin coupling ( figures 3h and 3i ; movie s2 ) . importantly , -trefoil domains of fascin expressed in fibroblasts did not localize to f - actin or change stress fibers or filopodia formation ( figures s3e and s3f ) . this demonstrated that the expression of single -trefoils does not affect f - actin cytoskeleton and that the role of fascin at the ne is uncoupled and mechanistically distinct from that at the cell periphery . these data combined demonstrate that fascin - nesprin binding at the ne is specifically required for nuclear polarization and movement during polarization . work from our laboratory and others has previously shown that fascin contributes to invasion of human cancer cells ( li et al . , 2010 , hashimoto et al . , 2007 , schoumacher et al . , 2010 ) . we confirmed that kd of fascin leads to significantly reduced invasion of mda mb 231 cells through 3d collagen gels , an effect that was restored upon re - expression of small hairpin rna ( shrna)-resistant gfp - fascin ( figures 4a and s4a ) . previous reports have suggested that this pro - invasive effect is due to fascin - dependent stabilization of filopodia and possibly invadopodia . however , a number of recent studies have shown that nuclear deformation and movement in tumor cells are required to enable efficient cell navigation through changing ecm environments ( davidson et al . , 2014 , zwerger et al . , 2011 , wolf et al . , 2013 ) . we therefore reasoned that our discovery of fascin - dependent nuclear movement could represent a new way for fascin to control cell invasion independently of f - actin bundling . to explore this further , we examined the morphology and volume of nuclei of control or fascin kd cells in stiff 2d surfaces and embedded in 3d collagen . quantification revealed that in 3d matrices , but not on 2d surfaces , nuclear volume was significantly reduced in fascin - depleted cells , and that this was restored in cells rescued with shrna - resistant gfp - fascin ( figures 4b4d ) suggesting that fascin is required to maintain nuclear architecture in 3d environments . acto - myosin force generation and transmission are required to induce nuclear shape change in a range of migrating cells ( lammermann et al . , 2008 , wolf et al . , 2013 ) , but intrinsic nuclear mechanical properties can modify cell responses to force , acting as a limiting factor of nuclear deformability and , thus , migration ( harada et al . , 2014 , davidson et al . , 2014 ) . to determine whether fascin loss correlated with altered nuclear stiffness , we used atomic force microscopy ( afm ) . to avoid overall contribution from cytoplasmic dorsal actin fibers to nuclear stiffness , we averaged several local measurements performed with a quadratic pyramidal - tipped cantilever per nuclei instead of a broader spherical tip ( figures s4b s4d , see experimental procedures ) . in mda mb 231 cells plated on a rigid surface , kd of fascin did not change nuclear stiffness ( figure 4e ) . spatial confinement on 2d matrices is known to modulate nuclear size and volume , but the contribution of the linc complex and microfilaments to this process remain unclear at present ( khatau et al . , 2009 , li et al . , 2015 ) . our results show that fascin kd only affects nuclear volume in cells embedded in 3d matrices and indicates that fascin contributes to nuclear deformation under conditions of 3d spatial confinement . this suggests that fascin plays a role in the transduction of mechanical forces to nesprin-2 and ne without affecting bulk mechanical properties , and that this plays a more profound role in 3d environments . to study this further in an environment with controlled spatial constrictions , we used a microfluidic device with arrays of channels of different sizes ( figure 4f ) ( malboubi et al . , 2015 ) . similar devices have been previously used to study the ability of mda mb 231 cells to invade in 3d , showing that nesprin-2 plays an important role in nuclear translocation through confined spaces ( thomas et al . , 2015 ) . in our system , control mda mb 231 cells ( scr ) showed equally efficient nuclear translocation through channel widths of 15 m , 10 m , and 7 m , but decreased significantly in 5-m wide gaps , where only 30% of nuclei entered the channels ( figures 4 g and 4h ) . stable fascin kd cells , however , showed a significant reduction in nuclear translocation into channels with widths smaller than 10 m , suggesting that loss of fascin limits nuclear deformation , a phenotype that was rescued by the stable re - expression of shrna - resistant gfp - fascin ( figures 4 g and 4h ) . nuclear translocation through microchannels of different widths fits to a sigmoidal distribution in which the inflection point of the curve , where 50% of the cells translocate , can be used as a relative cut - off size for cell translocation . in accordance with our previously published data ( malboubi et al . , 2015 ) , control mda mb 231 showed a cut - off size of 6.99 m , while in fascin kd cells this increased to 9.77 m . conversely , stable expression of shrna - resistant gfp - fascin induced a decrease in the cut - off size to 6.45 m , close to the control levels ( figures s4e and s4f ) . our experimental setup did not allow us to discriminate rescued cells overexpressing fascin levels above the wt , and thus we can not discard that this partially enhanced effect in nuclear translocation might be due to the levels of fascin achieved in the fascin kd cell line re - expressing gfp - fascin ( figure s4a ) . these data suggest that fascin regulates nuclear translocation into spatially confined environments ( figures s4e and s4f ) . to further determine whether this defect in fascin - dependent cell movement and adaptation was due to fascin - nesprin binding , we analyzed wt cells expressing mcherry alone versus mcherry - tagged -trefoil3 domains of fascin in the microfluidic chambers . our analysis demonstrated that specifically competing the fascin - nesprin-2 complex with -trefoil3 resulted in a significant impairment of nuclear translocation through gaps smaller than 10 m wide compared with control or mcherry - expressing cells ( figures 4i , s4e , and s4f ) . -trefoil3 expression in mda mb 231 cells or fibroblasts did not induce any change in actin cytoskeleton organization , morphology , or focal adhesion formation cells , all features closely related to acto - myosin contractility ( figure s4j ) . fascin kd is known to increase non - muscle myosin ii activity and cell contractility ( elkhatib et al . , 2014 ) , which , together with nesprin-2 tethering to actin cytoskeleton , favors cancer cell invasion and nuclear translocation into spatially confined 3d matrices ( thomas et al . , 2015 ) . this phenomenon could partially counteract the negative effects of fascin depletion in cell invasion and mask some of these observed effects . in our experimental setup , we specifically disengaged fascin binding to nesprin-2 from other fascin - dependent effects . the larger channel cut - off size for -trefoil3-expressing cells further suggests that acute disruption of the nesprin - fascin complex severely impairs the transmission of cytoplasmic forces and , thus , nuclear deformability . in line with this notion , our data demonstrated that silencing nesprin-2 in mda mb 231 cells resulted in impaired invasion through 3d gels ( figures s4h and s4i ) . we therefore sought to determine whether disruption of the fascin - nesprin complex , and subsequent defects in nuclear deformation might play a role in cancer cell invasion through 3d ecm . analysis of mda mb 231 cells showed that -trefoil3 expression resulted in a significant reduction in invasion into collagen gels ( figures 4j and 4k ) . moreover , expression of -trefoil3 in fascin kd cells did not alter invasion levels , demonstrating that this effect was specific , but also that this domain of fascin is not sufficient to enable coupling of nesprin-2 and f - actin to permit efficient nuclear displacement and cell invasion ( figures 4j and 4k ) . importantly , focal adhesion formation and migration speed of cells expressing of -trefoil3 on 2d surfaces was not significantly altered ( figures s4j and s4k ) , further suggesting that the inhibition of invasion in 3d is due to nuclear deformation and force transmission defects and not an overall loss of actin - based motility . together , these results show that fascin plays a key role in coordinating the link between nesprin-2 and actin that in turn facilitates nuclear deformation and the ability of tumor cells to navigate through complex 3d matrices . here we have uncovered a filopodia - independent role for fascin in controlling cell invasion through coupling nesprin-2 to f - actin at the ne . our data also identify a non - f - actin - bundling functional role for fascin in cells . these findings support a model in which fascin can occupy at least two distinct roles in cells to regulate migration and invasion : ( 1 ) f - actin bundling , through two distinct f - actin - binding sites within filopodia at the cell periphery ; and ( 2 ) f - actin and nesprin-2 binding at the ne to couple f - actin retrograde flow and nuclear translocation in 3d environments . the notion of these distinct and separable functions for fascin is supported by our ability to uncouple filopodia formation from nuclear movement through overexpression of the -trefoil3 domain of fascin . fascin has a number of known binding partners that play a role in regulating f - actin bundling , stability , or localization ( anilkumar et al . , 2003 , zhang et al . , 2009 , jayo et al . , 2012 ) . our data demonstrate that nesprin-2 is a fascin - interacting protein that recruits it to a yet uncharacterized subcellular localization . interestingly this interaction is direct , and occurs through the -trefoil3 domain , but constitutive actin bundling and filopodia formation competes with this interaction in whole cells . the -trefoil3 domain contains a number of residues shown to be important for actin bundling ( zanet et al . , 2012 , yang et al . , 2013 , jansen et al . , 2011 ) , but it is unclear whether it sits on an actin - binding site or plays a structural regulatory role ( jansen et al . our -trefoil3 overexpression approach did not substantially alter actin microfilament cytoskeleton while disrupting the interaction with nesprin-2 , showing that the nature of this interaction is structurally distinct from the binding to the actin filament and that a larger structural domain is needed for f - actin filament binding . in this context , constitutive f - actin bundling by s39a - fascin impeded an efficient interaction with nesprin-2 , showing that pkc - mediated phosphorylation at s39 is required for both normal actin dynamics and f - actin cytoskeleton connections with the linc complex . future studies of this molecular switch will shed further light on how fascin acts as a linker between f - actin bundles and nesprin-2 . actin - dependent nuclear movement in fibroblasts relies on the interaction of actin filaments with the n - terminal site of the nesprin-2 giant isoform ( luxton et al . , 2010 ) . more recently , an interaction with fhod1 formin , another actin - binding protein , was reported to play a role in tan - line formation and nuclear movement ( kutscheidt et al . , 2014 ) . these findings and data presented here support the emerging concept that the actin - ne connection is supported by more than one f - actin - nesprin-2 interaction site through its actin - binding domain . fhod1 interacts with nesprin-2 through a domain close to its n - terminal region , and we have shown that fhod1 and fascin - nesprin-2 interactions are not interdependent . these additional binding sites may play a role in strengthening the link and bearing a higher strain exerted by the cytoskeleton under certain conditions ( antoku et al . , 2015 ) . our data would suggest that this is even more evident in the context of migration into 3d environments where the nuclear deformability acts as a limiting factor ( wolf et al . several factors modulate the nuclear deformability in a wide range of cells , such us contractility ( lammermann et al . , 2008 ) , nuclear stiffness ( harada et al . , 2014 ) , and nucleo - cytoskeletal force transduction ( lombardi et al . , 2011 , alam et al . , 2015 ) . in our model , while loss of fascin did not induce any change in the mechanical properties of the nuclei , disruption of fascin - nesprin interactions led to a severe defect in nuclear translocation into physically confined environments . this function of fascin appears to be essential in this process , since despite the increased myosin activity induced by fascin depletion ( elkhatib et al . , together , these findings suggest a role for fascin as a mechano - transducer of cytoplasmic forces and provide evidence of its role in nuclear deformation and movement during cell migration . as fascin is a prognostic marker in many different cancer types and a potential therapeutic target , our data may provide new avenues to target fascin for anti - metastatic therapy . see supplemental experimental procedures for details . for the proteomic analysis of fascin - interacting proteins . gfp and gfp - fascin were transfected into fascin kd mda mb 231 cells , cells were lysed in a buffer ( 0.5% triton x-100 , 150 mm nacl , 15 mm tris [ ph 7.4 ] , and propidium iodide ) and gfp - fascin - containing complexes were immunoprecipitated overnight with a polyclonal anti - gfp antibody - coated protein ag agarose beads . single bands of proteins interacting specifically with gfp - fascin were excised and sent for mass spectrometry analysis ( university of aberdeen ) . his - fascin was prepared as previously described ( zanet et al . , 2012 ) . for gst - tagged nesprin constructs , protein production was induced in bl21 escherichia coli strains overnight at 16c with 200 mm isopropyl -d-1-thiogalactopyranoside . pellets were resuspended in 10 mm phosphate buffer , 300 mm nacl , 1 mm dtt , and 25 mm imidazole in the presence of protein inhibitors , sonicated , and cleared by centrifugation . gst - tagged proteins were bound to glutathione sepharose beads for 2 hr , washed , and proteins eluted in 10 mm phosphate buffer , 150 mm nacl , 1 mm dtt , 25 mm imidazole , and 3 mg / ml reduced glutathione ( ph 7.5 ) for 30 min at 4c . details of immunostaining procedures for cells , tissues , and fly samples are provided in supplemental experimental procedures . fret efficiency was quantified from hela cells transiently expressing donor and acceptor fluorophores by measuring time - domain fluorescence lifetime with a multi - photon microscope system ( te2000 , nikon ) . in brief , cells were fixed in 3.6% formaldehyde for 15 min , permeabilized with 0.1% triton x-100 , and quenched with 1 mg / ml sodium borohydride for 10 min at room temperature . fluorescence lifetime was measured as described previously ( zanet et al . , 2012 ) , and histogram data show mean fret efficiency from at least 12 cells per condition in three independent experiments . when indicated , fret efficiency was calculated from the area masked around the cell nucleus , using tri2 analysis software ( paul barber , university of oxford ) . quantification of protein localization at the ne was performed from single confocal slides of several cells by using fiji software ( schindelin et al . , 2012 ) . in brief , a 10-m line was drawn from the nucleus to the cytoplasm , while the middle point of the line was made to coincide with the nucleus - cytoplasm boundary using the dapi channel as nuclear marker . fluorescence values over the line were plotted using the plot profile function , and the fluorescence values over different cells were later normalized . fluorescence intensity plots of drosophila nurse cell nuclei were generated from a single slice of 63 confocal images using imagej software ( abramoff et al . , the image background was subtracted using imagej ( 50-pixel rolling - ball radius ) , then a line segment was drawn across one of the nurse cell nuclei and the plot profile function was used to generate a fluorescence intensity plot for each desired channel . the raw data files generated by these plot profiles were analyzed in microsoft excel , where each plot line was normalized to the peak value within that plot , creating intensity plots where the maximum observed fluorescence of a given line is represented by a value of 1.0 ; the relative fluorescence intensity . the ne was marked as the set of data points in which the wheat - germ agglutinin value was at least 0.5 . fibroblast polarization was quantified as previously described ( chang et al . , 2013 , 2014 ) . in brief , a monolayer of nih 3t3 fibroblasts was starved for 48 hr ; later a scratch wound was performed with a sterilized pipette tip , and cells were treated with 10 m lysophosphatidic acid ( lpa ) . two hours later cells were fixed and stained for tubulin , f - actin , and dna , confocal snapshots were taken on a nikon eclipse ti - e inverted microscope and images were uploaded and analyzed by custom - made software to define cell boundaries and relative position of the nucleus and centrosome with respect to the cell centroid ( chang et al . , 2013 ) . for the study of retrograde movement of actin fibers , fibroblasts were plated on 35-mm imaging dishes ( ibidi ) and cells were imaged for 2 hr directly after lpa addition under a 60 oil objective in a nikon eclipse ti - e inverted microscope equipped with an su-1 spinning - disk confocal and an andor ixon3 em - ccd camera . fiji software was used to quantify the speed of displacement of the fibers ( schindelin et al . , 2012 ) . in brief , time - lapse acquisition files were rotated so that the actin flow was reoriented in an up - down direction . kymographs of a 10-m - wide region of interest were then generated , and the coordinates of the fibers over time were annotated to calculate the displacement rate over time . at least two fibers per cell and ten cells per condition in three independent experiments were quantified . filopodia number and length quantification was performed as described previously ( zanet et al . , 2012 , fascin - containing filopodia were quantified in live fibroblasts growing in complete medium expressing both gfp - fascin and mcherry--trefoil domains . snapshots from a single confocal plane by a cell were taken from several cells and quantification was performed using fiji software ( schindelin et al . , 2012 ) . finger - like , thin , and straight projections protruding from the cell edge with increased gfp - fascin content were considered as filopodia and measured by drawing a straight line with fiji from the cell edge to the tip . command was later used for the quantification of the length of each filopodia . the area covered by f - actin fibers in fibroblasts was quantified from snapshots of time - lapse movies acquired at the same time range from fibroblasts expressing gfp - lifeact after 1 hr of 10 m lpa stimulation . using fiji software , the area covered by f - actin fibers was drawn and measured , and the results were plotted as the percentage of total area covered by fibers . for the invasion assays in matrigel , a previously described method was used ( scott et al . , 2011 ) . for collagen invasion assays , the described method was partially adapted : 1.6 mg / ml collagen gel supplemented with 10 g / ml fibronectin and 2% fetal bovine serum ( fbs ) . more details are provided in supplemental experimental procedures . mda mb 231 cells were plated on 2d coverslips or embedded in 1.6 mg / ml collagen gels supplemented with 10 g / ml fibronectin and 2% fbs . when the gels polymerized , ham s f-12 medium supplemented with 10% fbs was added on top and the cells were allowed to migrate for 24 hr . gels were then fixed in 3.6% formaldehyde , permeabilized with 0.1% triton x-100 , and incubated with phalloidin-647 and syto-16 overnight at 4c . imaging was performed with a nikon a1r confocal system using a 40 water immersion objective . reconstruction of 3d stacks was performed using nis elements software ( nikon instruments ) , and nuclear volume was quantified using the quantification of nuclear stiffness was performed as previously described ( harris and charras , 2011 ) . more details are provided in supplemental experimental procedures . the microfluidic device used in this study first , the outliers were removed following the rout method for outlier removal from non - linear regressions . when the populations followed a parametric distribution , anova analysis followed by dunnett 's multiple comparison test or student 's t test for two - groups mean comparison was used . otherwise , data not following normal distributions were analyzed with a non - parametric kruskal - wallis test followed by dunn 's multiple comparison test . significance was measured as p < 0.05 , p < 0.01 , p < 0.001 , and p < 0.0001 . all experiments were conceived by m.p . and a.j . , with input from g.g.g . , g.c . , t.t . , and s.a . experiments were performed and analyzed primarily by a.j . , with input on fret / flim from m.p . , on adhesion from k.p . , on microchannel device experiments from m.m . , and on afm experiments from g.c . nesprin-2 western blots , tan lines localization , and nesprin-2 kd invasion assays were performed by s.a .

summaryfascin is an f - actin - bundling protein shown to stabilize filopodia and regulate adhesion dynamics in migrating cells , and its expression is correlated with poor prognosis and increased metastatic potential in a number of cancers . here , we identified the nuclear envelope protein nesprin-2 as a binding partner for fascin in a range of cell types in vitro and in vivo . nesprin-2 interacts with fascin through a direct , f - actin - independent interaction , and this binding is distinct and separable from a role for fascin within filopodia at the cell periphery . moreover , disrupting the interaction between fascin and nesprin-2 c - terminal domain leads to specific defects in f - actin coupling to the nuclear envelope , nuclear movement , and the ability of cells to deform their nucleus to invade through confined spaces . together , our results uncover a role for fascin that operates independently of filopodia assembly to promote efficient cell migration and invasion .

, blood flukes , are parasitic helminths found mainly in developing countries with a tropical or subtropical climate and affect 200 million people worldwide . schistosoma mansoni , japonicum , and mekongi harbor in veins of the portal system and lay eggs in the blood vessels . the deposition of numerous eggs in the intestines and liver results in intestinal and hepatic granulomatous lesions , fibrosis , portal hypertension , and hepatosplenomegaly . in contrast , schistosoma haematobium mainly harbors in the venous plexus of the bladder and/or rectal venous plexus . this worm usually causes bloody urine but is also considered to have an etiological relationship with bladder cancer . because of the extensive distribution of schistosomes and morbidity due to egg deposition , researchers have been interested in the influences of schistosome infections on concomitant diseases . as mentioned , s. haematobium is an important carcinogenic factor of bladder cancer although the worm itself does not have mutagenic activity . this parasite is also suggested to be a risk factor for the transmission of hiv . these effects are attributable to pathological lesions caused by the parasites . on the other hand , most helminthic parasites including schistosomes are known to induce robust th2-polarization . especially in schistosome infections , egg deposition in the host tissues was reported to be the major stimulus of th2 responses , and egg proteins ( e.g. , omega-1 , ipse , and peroxiredoxin ) are involved in the th2-biasing activity [ 711 ] . in addition , schistosome eggs have immunomodulatory potential inducing the alternative activation of macrophages and regulatory t - cell expansion . because of its robust systemic th2-inducing and immunomodulatory ability , this worm has been studied most extensively for its bystander effects on various immunological phenomena in vivo . in this paper , we summarize the effects of a concurrent infection of schistosomes on immunological disorders and parasitic / microbial infections . effects of helminths ( including schistosomes ) on allergic diseases have been studied extensively in both experimental and epidemiological settings . in experimental asthma or airway hypersensitivity models , schistosome infections and antigen administrations have been consistently shown to protect the animals from the diseases ( table 1 ) . in most studies on the antiasthmatic effects of schistosomes , cellular infiltration ( including eosinophils ) into bronchoalveolar lavage fluid ( balf ) was diminished and simultaneously il-4 , il-5 , and ige levels were reduced . in contrast , an increase in treg cells and augmentation of il-10 production were observed . fallon and mangan designated this kind of th2 response ( treg dominant and il-10 dominant ) a they demonstrated that in infected mice , il-10-producing cd1d b cells induce treg cells and consequently ameliorate allergic airway inflammation . the authors also showed the importance of b cells , and il-10 in the suppression of systemic anaphylaxis by schistosomes . smits et al . found that in adoptive transfer experiments , spleen cells ( especially b cells and cd4 t cells ) from chronically infected mice could confer protection against pulmonary infiltration by white blood cells , especially eosinophils . in addition , administration of anti - il-10r antibody cancelled out the effects of the cell transfer . these studies seem to support the critical importance of b cells , treg cells and il-10 in schistosome - induced protection against airway allergic inflammation . in studies by another group , however , il-10 signaling was not essential for the antiallergic immunomodulation by schistosomes [ 18 , 19 ] . the reason for this discrepancy is unclear , but as the authors point out , other immunomodulatory factors may be able to compensate the absence of il-10 . antiallergic effects of schistosomes have also been demonstrated in humans . in a study in brazil , both asthmatic symptoms and skin reactivity to indoor allergens were reduced in s. mansoni - infected asthmatics compared to non - infected individuals . in another study , higher expression levels of hla - dr , il-10r ( in monocytes ) , ctla-4 and cd40l ( in cd4 t cells ) were observed in s.mansoni-infected asthmatics . according to that paper , the main sources of il-10 were monocytes and treg cells . a meta - analysis of current parasite infections and atopy revealed that schsitosomiasis was protective against allergic skin sensitization as well as some other helminths ( ascaris lumbricoides , trichuris trichiura , hookworm ) . the same research group also reported a meta - analysis of interrelationships between helminths and asthma . hookworm infections were shown to be protective , but no beneficial effect was found for schistosome infections , probably due to the insufficient number of studies ( only two ) . collectively , anti - asthmatic effects of schistosomes have been confirmed in animal models and suppressive effects on allergic skin reactivity have been confirmed in humans . however , more epidemiological ( especially intervention ) studies are necessary to conclude whether schistosome infections have beneficial or detrimental effects on asthmatic patients . autoimmune disorders had been considered th1-mediated diseases for a long time . as the th2-biasing ability of parasitic helminths and consequent downregulation of th1 responses have been well known , antiautoimmune effects of such parasites had been attributed to the downregulation of th1 responses in infected animals . however , some major autoimmune diseases are now considered to be dependent on th17 , a newly found pathogenic t - cell subset that mainly produces il-17 . with this finding , downmodulation of th17 responses by parasitic helminths has been reported [ 24 , 36 , 47 , 48 ] . if the suppressive activity on both th1 and th17 is common to parasitic helminths , helminths may become ideal sources of drug screening for treatment of autoimmune disorders . that is because both t - cell subsets are involved in the pathogenesis of some autoimmune diseases . an upregulation of il-4 and downregulation of ifn- responses are almost commonly observed . in addition , responses of il-17 and/or tnf- , both of which play pathological roles in autoimmune arthritis [ 24 , 26 ] and hapten - induced colitis [ 36 , 40 ] , were also downregulated by schistosome infections . moreover , our study in mice with collagen - induced arthritis ( cia ) revealed that the disease - associated local augmentation of bone - destructive cytokines ( i.e. , il-6 and rankl ) was abrogated in infected animals . these results indicate that schistosomes have suppressive effects on both th1/th17 and inflammatory cytokines . schistosomes decreased levels of anti - insulin igg , anti - tshr igg2a , and anticollagen igg [ 24 , 25 ] . recently reported that s. haematobium infection intensity was inversely related to autoreactive antinuclear antibody ( ana ) levels . reported the presence of antinuclear antibodies in s. mansoni - infected mice and that sera from patients with systemic lupus erythematosus ( sle ) reacted with cercarial antigens , suggesting that schistosomes trigger some kinds of autoimmunity . in conclusion , large - scale cross - sectional studies may be necessary to reveal the interrelationships between schistosomiasis and autoimmunity . it is reasonable to hypothesize that the downmodulation of proinflammatory cytokines and pathogenic antibodies is involved in the antiautoimmune activity of schistosomes , at least partially . as regulatory cytokines are known to downregulate proinflammatory cytokines and pathogenic antibodies , it is important to determine the essential regulatory cytokines in each disease models for elucidation of suppressive mechanisms . for this purpose , it is necessary to perform experiments of cytokine neutralization with specific antibodies or experiments using gene - targeted animals . in th1/th17-dependent autoimmunity , il-4 , the key cytokine of th2 responses , may be responsible for the alleviation of the disease symptoms . for instance , by using gene - targeted mice , stat6 ( a key signaling molecule in the response to il-4 and il-13 ) was shown to be indispensable to the s. mansoni egg - induced suppression of experimental autoimmune encephalomyelitis ( eae ) and of tnbs - induced colitis . in contrast , il-4 and il-13 were dispensable to the suppression of dss - induced colitis ( th2 cytokine dominant and macrophage - mediated colitis ) by male worms of s. mansoni . in the same study , authors demonstrated that il-10 and tgf- were also dispensable to the anticolitic effects of schistosome , by using specific antibodies against il-10r and tgf-. il-10 was not a crucial regulatory cytokine also in other helminthic infections , that is , piroxicam - induced colitis was suppressed by heligmosomoides polygyrus , and eae was suppressed by fasciola hepatica , both in il-10-deficient mice . in the latter study , however , tgf- was shown to be responsible for anti - eae effects of the worms . taken together , the involvements of il-4 , il-10 , and tgf- in antiautoimmune effects of helminths depend on the disease models and helminth species . further investigations using various autoimmunity models and gene - targeted animals would be necessary for comprehensive elucidation of the suppressive mechanisms by regulatory cytokines in schistosome infections . regarding the participation of regulatory cell populations in schistosome - induced antiautoimmune effects , treg cells , macrophages , and other types of cells ( e.g. , nkt cells ) have been suggested . although treg cell population is known to expand by schistosome infection or sea administration [ 13 , 32 ] , their involvement seems to depend on the disease models . for instance , cooke et al . have been studying antidiabetic effects of s. mansoni using a spontaneous t1d model ( nod mouse ) , and they demonstrated that treg cells were essential in the suppression of t1d by cell transfer experiments . the authors ( zaccone et al . ) showed that splenocytes from nontreated nod mice successfully transmitted diabetes into nod / scid recipients , whereas splenocytes from sea - treated nod mice had a reduced capacity to transmit diabetes . they also showed that sea had various immunomodulatory effects on dendritic cells ( dcs ) , macrophages , and t cells of nod mice [ 12 , 32 ] : for example , increased expressions of tgf- , galectins , pd - l1 , and so forth . in contrast to this t1d model , smith et al . showed that depletion of treg cells did not influence the suppressive effect of schistosomes on dss - induced colitis . they demonstrated that macrophages ( but not alternatively activated macrophages ) played an essential role in the amelioration of the colitis . in some of the studies in table 1 , the injection of eggs or sea was not effective [ 27 , 39 , 40 ] . likewise , in our study of cia , sea injection was not effective . taken together with findings that mice infected with male worms become resistant to dss - induced colitis , egg - derived substances are not sufficient to explain all of the immunomodulatory activities of schistosomes . indeed , injection of soluble worm antigens ( swas ) could prevent t1d in nod mice and tnbs - induced colitis . therefore , differential effects of worms and eggs should be further elucidated for a precise understanding of the immunomodulatory mechanisms of schistosomes . in addition , further studies on differences between single - sex infections and mixed - sex infections may be necessary . in tropical developing countries , infections with multiple microbes consequently , the immune responses and/or pathological lesions caused by one pathogen may affect the outcome of other infections . therefore , influences of parasitic infections on concomitant diseases have been studied . in this section , consequently , there are more than a few experimental studies on the interrelationships between schistosomiasis and malaria ( table 2 ) . the influence of schistosome infections depends on the species of the malarial parasites and mouse strains used in the experiments . for instance , in cba / ca mice , s. mansoni protected against p. chabaudi infection , worsened p. yoelii infection , and had no effect on p. berghei infection . even when the same parasite , p. chabaudi , was used , schistosomes exacerbated the parasitemia and mortality in c57bl/6 mice but ameliorated the outcome in a / j mice [ 54 , 55 ] . although these complicated outcomes should be taken into consideration , in general , schistosome infections seem to exacerbate rodent malaria , that is , increase of parasitemia , mortality , and hepatosplenomegaly . detrimental effects of schistosome infections on malarial outcome are also reported in humans [ 57 , 58 ] . in kenyan school children , even a light s. mansoni infection was shown to exacerbate hepatosplenomegaly of malarial patients . in a study in senegal however , the modification of antibody responses by concomitant schistosomiasis is controversial [ 59 , 60 ] . the mechanism responsible for the exacerbation ( or amelioration ) of malaria is clear in neither mice nor humans . suggested diminished production of tnf- in schistosome - infected mice to have contributed to the increase in parasitemia of p. chabaudi . extensively analyzed possible mechanisms of protective effects of schistosomes against p. chabaudi in a / j mice . they observed an enhanced th1 response to p. chabaudi in schistosome - infected mice and that an anti - ifn- antibody abrogated the schistosome - induced protective effect against p. chabaudi . as monoinfection with s. mansoni induced a th2-dominant response , upregulation of ifn- seemed to derive from the mixed infections of both parasites . they also observed the upregulation of inos gene expression in spleen from mice with mixed infections , suggesting an increase of splenic nitric oxide production to have contributed to the protection from p. chabaudi . although the mechanism of the exacerbation of rodent malaria by schistosome is yet to be sufficiently analyzed , treg cells have been shown to play an important role in the exacerbation of p. yoelii infection by preceding h. polygyrus infection . likewise , the induction / expansion of treg cells by schistosome might be involved in the increased susceptibility to rodent malaria . the th1 immune response protects against malarial parasites , but it also plays pathological roles in malaria , especially cerebral malaria . thus , schistosome as a representative th2-biasing helminth is expected to suppress brain pathology . indeed , in a model of cerebral malaria using mice infected with p. berghei anka , moreover , the administration of ipse / alpha-1 , a th2-inducing schistosomal egg protein [ 9 , 10 ] , also delayed death . bucher et al . reported that brain pathology was reduced in schistosome - infected mice although they did not observe any beneficial effect on mortality . influence of schistosomes on other protozoan infections has also been investigated ( table 2 ) . regarding leishmania major , reports vary , with yoshida et al . finding no effects of schistosome on the outcome of l. major infection , and la flamme et al . reporting an exacerbation of experimental leishmaniasis the reason for the discrepancy is not clear but might be intensity of the s. mansoni infection ( 20 cercariae in the former versus 70 cercariae in the latter ) . in the case of l. donovani , schistosome - preinfected mice failed to control growth of the protozoa in the liver . in the coinfected mice , hepatic egg granulomas were shown to provide a favorable microenvironment for the growth of the amastigotes . in general , schistosome infections seem to be detrimental to animals infected with protozoan parasites . in marked contrast to that for protozoan parasites , protective immunity against intestinal helminths therefore , the th2-dominant environment produced by schistosomes can be expected to protect against intestinal helminths . indeed , as summarized in table 2 , schistosome infections protected mice from intestinal helminths . likewise , in a human study in brazil , s. mansoni egg counts were inversely correlated with a. lumbricoides and t. trichiura . although protection against intestinal helminths is commonly observed in experimental settings , the mechanisms differ in each case . for instance , protection against a lung - migratory parasite , strongyloides venezuelensis , mainly involved eosinophil - mediated killing of larvae in the lungs . in addition , intestinal mucosal mastocytosis induced by s. japonicum infection rendered mice resistant to harboring of adult worms . antigen cross - reactivity between schistosomes and s. venezuelensis [ 67 , 76 ] may have also contributed to the protection . in contrast , in the case of hymenolepis diminuta and trichuris muris ( nonmigratory intestinal helminths ) , the protection seems to be dependent on accelerated expulsion from the intestines [ 73 , 74 ] . overall , schistosome infections appear to be beneficial to animals infected with intestinal helminths . prolonged bacteremia in schistosomiasis patients was first reported more than 50 year ago , and the relationship between enterobacteria infections and schistosomiasis has been long studied . the schistosome - induced exacerbation of enterobacterial infections has also been observed in experimental settings [ 77 , 78 ] . as most studies were conducted before the molecular immunology age , the mechanisms of exacerbation of bacterial infections have not been sufficiently elucidated . however , the mechanisms are likely similar to those responsible for the schistosome - induced increase in susceptibility to protozoan parasites , that is , a reduction in th1-dependent protective immunity as a consequence of augmented th2 responses . indeed , a th1-inducing protozoan parasite , l. donovani , did not affect the growth of salmonella paratyphi a in infected hamsters . moreover , impairment of the bactericidal function of macrophages from schistosome - infected mice was reported . in addition to the immunomodulation by schistosomes , there are direct schistosome - bacteria interactions providing worm bodies as foci for bacterial multiplication [ 3 , 80 , 81 ] . the mycobacterium bovis bcg - induced protective response against mycobacterium tuberculosis in mice was reduced by s. mansoni infection . the authors also reported increased susceptibility to intravenous bcg inoculations and lung pathology in s. mansoni - infected mice . in these studies , decrease in ifn- and nitric oxide in response to ppd were observed . as hepatotropic viruses , that is , hepatitis b virus ( hbv ) and hepatitis c virus ( hcv ) , cause liver cirrhosis during chronic infections , the synergistic exacerbation of hepatic pathology is expectable outcome of concurrent infections of hbv / hcv and schistosomes . because of a lack of suitable animal models for infections of these hepatotropic viruses , major findings in schistosome - hbv / hcv coinfections have been obtained from epidemiological studies . regarding hbv , schistosomiasis ( especially the severe hepatosplenic form ) was correlated with a higher frequency of hbv infection [ 3 , 84 , 85 ] . this observation could be explained by an increased susceptibility to hbv caused by schistosome infections . on the other hand , there are reports of no relationship between schistosomiasis and hbv [ 86 , 87 ] . in addition , experiments with animals do not support increased susceptibility to hbv in schistosomiasis . some evidence comes from an experiment using woodchucks infected with both schistosomes and woodchuck hepatitis virus ( whv ) . as hbv and whv belong to the same family ( family hepadnaviridae ) , a concurrent infection by schistosomes and whv in woodchucks is a good model of concurrent infections of schistosomes and hbv in humans . another paper on hbv transgenic mice reported an inhibition of hbv replication during schistosome infection . in that study , overall , it seems premature to conclude the presence of certain positive or negative effects of schistosomes on hbv infection . regarding hbv vaccines ( both serum derived and recombinant ) , they were immunogenic in schistosomiasis patients although reduced responses to vaccination were observed in hepatosplenic schistosomiasis [ 9092 ] . in contrast to the controversial effects on hbv infections , detrimental effects of schistosomes on hcv infections have been clearly demonstrated , that is , schistosomes weaken anti - hcv immune responses and worsen liver disease . according to studies in egypt , patients with coinfections were characterized by a more advanced liver pathology , greater viral burden , higher levels of anti - hcv antibodies , and progression to chronic hepatitis [ 9395 ] . moreover , schistosomiasis was shown to be inversely correlated with hcv - specific cd4 t cells , cd8 t cells , and/or th1 cytokine responses [ 95100 ] . in addition to the modulatory effects of schistosomes on hcv - specific immune responses , sea of s. mansoni and s. haematobium were shown to enhance in vitro viral replication in a hepatoblastoma cell line ( hepg2 ) and peripheral blood mononuclear cells ( pbmcs ) , respectively . likewise , in other viral infections , schistosomes were shown to suppress specific ctl and cytokine responses and to prevent viral clearance [ 103107 ] . it is interesting that the granulomas in s. mansoni - infected mice provide a microenvironment suitable for viral expansion , as in the case of a hepatic infection with the protozoan parasite l. donovani . based on the experimental and epidemiological findings reviewed here , it can be concluded that schistosome infections are generally beneficial to patients with intestinal helminth infections and detrimental to patients with bacterial , viral , or protozoan infections . in most tropical or subtropical countries where schistosomiasis is endemic , more serious infectious diseases ( e.g. , hiv / aids , tuberculosis , and malaria ) are also endemic . therefore , control of schistosomiasis ( especially infections of s. mansoni and s. haematobium ) has been given low priority compared to control of such infectious diseases . however , if the detrimental bystander effects of schistosomes on concomitant bacterial , viral , or protozoan infections are properly considered , the importance of controlling schistosomiasis should be more emphasized . in 2002 , j. f. bach summarized epidemiological trends of allergic and autoimmune diseases during recent several decades in developed countries . according to the paper , the prevalence of the immunological disorders such as asthma , t1d , multiple sclerosis ( ms ) , and crohn 's disease ( cd ) was increasing , whereas the prevalence of infectious diseases such as rheumatic fever , hepatitis a , tuberculosis , mumps , and measles was decreasing . these phenomena may be explained by the hygiene hypothesis in which microbial and helminthic infections prevent immunological disorders . along with this hypothesis , schistosome infections are expected to prevent or alleviate symptoms of immunological disorders . according to the experimental studies until now , antiallergic and antiautoimmune effects of schistosomes are also plausible in humans . however , schistosomes could not be directly used for therapeutic treatment because of their pathogenicity . instead indeed , considerable numbers of helminths ' products have been shown to protect against experimental immunological disorders . the immunomodulators exert their effects via toll - like receptors ( tlrs ) and/or c - type lectin receptors ( clrs ) . taken together with the finding that systemic administration of tlr agonists could prevent experimental autoimmunity and allergy , appropriately synthesized tlr agonists

schistosomiasis is an important tropical disease affecting approximately 200 million people worldwide . because of its chronicity and robust immunomodulatory activity , the effects of schistosomes on other diseases , such as allergies , autoimmunity , and infectious diseases , have been studied extensively in both epidemiological and experimental settings . in this paper , we summarize the beneficial and harmful effects of schistosomes . the importance of controlling schistosomiasis is also discussed .

celiac disease ( cd ) is an immune - mediated enteropathy caused by permanent gluten intolerance . it affects genetically susceptibile subjects , with a higher prevalence in females ( f / m : 2.5/1 ) . epidemiological studies have shown that the prevalence of cd ranges between 1/100 and 1/150 in both europe and the usa . the histological hallmarks of cd are villous subtotal or total atrophy , crypt hyperplasia and lymphoplasmacellular infiltrate in the intestinal lamina propria , with an increased production of igm , igg and iga . the mechanisms on the basis of the gluten - related damage of small intestinal mucosa and malabsorption are not clarified yet . the immunological hypothesis is the most credited ; it is supported by the characteristic histopathologic and serologic alterations , the association with specific genetic markers ( hla - dq2 and hla - dq8 ) , the improvement of intestinal damage after steroid therapy , and the association of cd with other immune - mediated diseases . the pathogenetic mechanism is an immunological reaction to peptides of the gliadin molecule : their high glutamine and proline content renders them resistant to digestion by intestinal enzymes . because of a dysregulation of the zonulin system that causes the opening of tight junctions , gliadin peptides cross the intestinal barrier to the lamina propria and are deamidated by tissue transglutaminase . the deamidated gliadin subsequently binds to either dq2 or dq8 molecules on antigen - presenting cells , causing myd88-dependent release of both zonulin and cytokines . gliadin peptides are also presented to t lymphocytes , initiating an aberrant humoral and cell - mediated immune response , ultimately responsible for the autoimmune process resulting in villous injury . regarding the clinical presentation we can have different forms of cd : classical and oligosymptomatic cd , dominated by symptoms and sequelae of gastrointestinal malabsorption , a positive serologic test and villous atrophy on biopsy ; silent cd , referring to patients who are asymptomatic but have a positive serologic test and villous atrophy on biopsy ; latent cd , defined by positive serological tests but not histological changes on biopsy ; and finally , potential cd , characterized by hla predisposition without clinical , serological or histopathological signs of disease . the most frequent symptoms are : gastrointestinal symptoms such as diarrhea , abdominal pain , flatulence , weight loss , steatorrhea , anorexia and , less frequently , nausea and vomiting and , in children , impaired growth , short stature and pubertal delay ; in females : menarche delay , menstrual irregularities , recurrent abortion , intrauterine fetal growth retardation , infertility and early menopause ; in males : infertility with altered spermatic motility and erectile dysfunction . other extraintestinal manifestations , due to micro- and macronutrient malabsorption , immune - mediated or of unknown origin , are the following : ( 1 ) hematological : iron and folate deficiency anemia , splenic atrophy and thrombocytosis , thromboembolism risk ; ( 2 ) skin : duhring 's dermatitis herpetiformis , psoriasis , clubbing and onychodystrophy , skin hyperpigmentation , alopecia , follicular keratosis , hypertrichosis lanuginosa , peripheral edema , cutaneous elasticity reduction ; ( 3 ) psychiatric / psychological : irritability , poor school performance , anxiety , depression ; ( 4 ) musculoskeletal : osteopenia , osteoporosis , fractures , dental enamel hypoplasia , arthritis , myopathy , cramps , tetany ; ( 5 ) neurological : peripheral neuropathy with paresthesia , tremors and fatigue , cerebellar ataxia , epilepsy with or without cerebral calcifications , migraine , night blindness and hemeralopia , dementia , multifocal leukoencephalopathy , chorea , sensorineural hearing loss ; ( 6 ) laboratory and instrumental abnormalities : hypocalcemia , prolonged pt , aspecific ecg alterations , hypertransaminasemia . several cd - associated autoimmune disorders have been described and reported , including hashimoto 's thyroiditis , type 1 diabetes mellitus , autoimmune hepatitis and cholangitis , primary biliary cirrhosis , sjogren syndrome , addison 's disease , peripheral neuropathy , psoriasis , idiopathic dilated cardiomyopathy and autoimmune myocarditis . untreated cd is associated with several complications : splenic hypotrophy or atrophy , ulcerative jejunitis , enteropathy - associated t cell lymphoma , increased risk for malt lymphoma , esophageal and oropharyngeal carcinomas , small bowel adenocarcinoma , collagenous colitis and lymphocytic colitis . endoscopic evaluation and small bowel biopsy are the gold standard for cd diagnosis and should always be performed if the clinical suspicion for cd is strong . multiple biopsies either from the second portion of the duodenum or the proximal jejunum should be obtained because the histological alterations may be focal . recently , a new endoscopic technology with additional magnification has raised high expectations for a substantial improvement in endoscopic recognition of villous atrophy . iga anti - tissue transglutaminase or endomysial antibody have a high sensitivity and specificity ( approx . 90% ) . in patients with selective iga deficiency , who are known to be at high risk for cd , igg class antibodies ( for tissue transglutaminase or endomysial antibody ) should be determined instead of iga antibodies . other hematologic , biochemical and imaging abnormalities may lead to the suspicion and eventually to the diagnosis of cd , such as the following ones : ( 1 ) steatorrhea in stool examination ; ( 2 ) hematologic abnormalities such as the presence of howell - jolly bodies and target cells in the peripheral smear , reflecting hypersplenism associated with cd ; ( 3 ) anemia , thrombocytopenia , leukopenia , and hypoprothrombinemia ; ( 4 ) decreased levels of serum iron , calcium , folate , vitamin d and albuminemia , and abnormal liver enzymes as the aminotransferases ; ( 5 ) radiographic changes associated with cd including dilatation of the small intestine and loss of the mucosal folds ; ( 6 ) computerized bone mineralometry can show diffuse demineralization and decreased bone mineral density . the treatment of cd patients consists in a lifelong strict adherence to a gluten - free diet , sometimes associated with supplemental therapy ( iron , calcium , zinc , magnesium , various vitamins , especially of the b complex ) . the prognosis of cd is usually favorable since the gluten - free diet determines clinical and histological recovery . however , 30% of cd patients do not present clinical and histological improvement after a gluten - free diet . this group of patients is considered affected by refractory sprue. this condition is rare and its most common cause is the failure to adhere to a strict gluten - free diet . refractory sprue is associated with uncommon complications of cd , such as intestinal ulceration , mesenteric lymph node cavitation , t cell lymphomas , and collagenous sprue , with an unfavorable prognosis and splenic atrophy , with a consequent increased risk for infections and/or thromboembolic complications . a 34-year - old woman came under our observation due to the appearance of intermittent claudication , initially displayed by a sense of fatigue when walking . lately no signs worth noting emerged from the anamnesis : both parents were in good health , as was the patient 's 9-year - old daughter . her menstrual cycle was regular and the patient had never suffered from particular ailments , except for rare cases of diarrhea , meteorism and reported difficulty in gaining weight. on objective examination the patient was found to be in a good general condition , alert and well oriented in time and space with normal facies , negligible decubitus and no signs of bilateral peripheral edemas , apart from an imposing thinness , despite the fact that the patient claimed she ate a balanced diet and did not pay any special attention to her weight . the skin had a normal blood supply and was normally hydrated , but the subcutaneous panniculus adiposus appeared underrepresented yet normally distributed . the results of the hematochemical examinations were normal , as were the virological tests , the dysplastic markers and the autoimmune pattern . the culture and parasitological test of the feces was negative , as was the vidal - wright serodiagnosis . however the test for antiendomysial and anti - transglutaminase antibodies was positive at 227 u / ml , in addition to minimum serum traces of cryoglobulin the thrombophilia evaluation results ( factor v leiden , prothrombin 20210 gene mutation , lupus anticoagulant , anticardiolipin antibodies , protein c , protein s , and homocysteine ) were negative . following these results , the patient underwent an esophagogastroduodenoscopy and an ileoscopy that showed : normal esophagus , hypotonic cardia ; normal gastric mucosa with the exception of moderate antral hyperemia , patent pylorus ; a normal duodenal bulb with sub - atrophic villi and , especially in the second duodenal portion , marked villous atrophy and flattened folds with a histological examination of the duodenal biopsies showed : in the gastric antrum chronic phlogistic process with no evidence of helicobacter pylori , with modest activity levels , and in the second duodenal portion chronic phlogistic process with marked villous atrophy and discreet activity levels , compatible with cd . for the circulatory study the patient underwent a global angio - ct of the chest - abdominal - pelvic vessels and the lower limbs that showed normal thoracic aorta without parietal thrombotic appositions , such as the epiaortic arterial trunks and the intracranial circulation ; the calibre of the abdominal aorta , in the suprarenal tract , was at the lowest limit with evidence of mainly right - lateral parietal thrombotic apposition and the celiac tripod displayed a significant reduction in calibre at the origin with poststenotic dilation ; the superior mesenteric artery had a slightly reduced calibre at the origin ; the renal arteries were normal ; aortic parietal thrombotic apposition with a marked reduction in the vessel calibre of the inferior mesenteric artery and severe stenosis at the level of the carrefour with a residual lumen of 0.3 cm : the stenosis extended to the origin of the common iliac arteries . parietal atheromasia , in part calcified , with a moderate degree of stenosis in the mid - istal tract of the iliac arteries . the internal and external iliacs and the common , superficial and deep femorals were of reduced calibre . , the patient was put on a gluten - free diet , after positioning multiple stents at the level of the inferior mesenteric artery with excellent hemodynamic compensation , return of the distal flow and elimination of the claudication . at present the patient 's condition is good and we evaluated the regression of myointimal proliferation after the beginning of the gluten - free diet with a new angio - ct of the chest - abdominal - pelvic vessels after one year . six months and one year later the patient underwent a new esophagogastroduodenoscopy with ileoscopy that showed , especially in the second duodenal portion , reduction of villous atrophy . as mentioned in the introduction , cd is caused by a reaction to gliadin , a gluten protein found in wheat . upon exposure to gliadin , the enzyme tissue transglutaminase modifies the protein , and the immune system cross - reacts with the bowel tissue , causing an inflammatory reaction and leading to flattening of the lining of the small intestine , which interferes with the absorption of nutrients . about 20 - 30% of people without cd have inherited an abnormal hla - dq2 allele , which suggests that additional factors are nedeed for cd to develop . furthermore , about 5% of those people who do develop cd do not have the dq2 gene . these are storage proteins rich in proline and glutamine that dissolve in alcohols and are resistant to pepsin and chymotrypsin , the two main digestive proteases in the gut . gliadin in wheat is the best - known member of this family , but other prolamins exist . classic symptoms of cd include diarrhea , weight loss , and fatigue ; bowel symptoms may be limited or even absent . abdominal pain and mouth ulcers may be present . the symptoms are frequently ascribed to irritable bowel syndrome and are only later recognized as cd ; a small proportion of patients with symptoms of irritable bowel syndrome have underlying cd , and screening may be justified . cd leads to an increased risk of both adenocarcinoma and lymphoma of the small bowel , which returns to baseline with diet . the changes in the bowel make it less capable of absorbing nutrients , minerals and fat - soluble vitamins . cd has been linked with a number of conditions such as iga deficiency [ 8 , 9 ] , dermatitis herpetiformis , epilepsy , ataxia , myelopathy , peripheral neuropathy and schizophrenia [ 11 , 12 ] , infertility , hyposplenism , diabetes mellitus type 1 , autoimmune thyroiditis , primary biliary cirrhosis and microscopic colitis . serology test is useful both in diagnosing cd with a sensitivity of about 98% and a specificity of over 95% . several serological blood tests exist for cd , but the most commonly used ones detect an antibody of the iga type against particular antigens in the small bowel as gliadin , and against endomysium or tissue transglutaminase . it is important that a total serum iga level is checked in parallel because an iga deficiency is possible [ 18 , 19 , 20 ] . however the gold standard in the diagnosis of cd is endoscopy with multiple biopsies of the duodenum or jejunum . today the only effective treatment is a life - long gluten - free diet and only a tiny minority of patients suffer from refractory disease . multiple cases of intra - abdominal venous thrombosis have also been reported in patients with cd in the literature such as a case of nonischemic central retinal vein occlusion associated with cd that suggests that although patients with an inherited or acquired predisposition to thrombosis have a lifelong risk , they only experience clinical thrombosis occasionally , and that additional exogenous insults are necessary to precipitate a clinical event ; in fact thrombotic episodes are likely multifactorial events in patients who have an inherent predisposition to thrombosis [ 24 , 25 , 26 ] . francis et al . noted that in their patients dehydration due to diarrhea was associated with central retinal vein occlusion ; prothrombotic predisposition is caused by hyperviscosity from the circulating antigliadin antibodies or some other yet unknown cause in patients with cd . the literature also describes an association between the budd - chiari syndrome and cd , as in northern africa and in turkey . here the unexplained hypercoagulable state in cd may affect the hepatic veins , the mesenteric veins or the splenoportal axis , causing different syndromes ; it is merely a different expression of the same abnormality related to different sites . the authors believe that the reason for the hypercoagulable state may be hyposplenism , and cd with abdominal venous thrombosis may be more frequently found if carefully researched . in fact other authors said that the budd - chiari syndrome , in its secondary form , refers to the syndrome caused by such diseases as myeloproliferative disorders [ 32 , 33 , 34 ] , including polycythemia vera , antiphospholipid antibodies [ 36 , 37 ] , paroxysmal nocturnal hemoglobinuria [ 38 , 39 ] , systemic lupus erythematosus , behet disease [ 41 , 42 ] , cd , hepatocellular carcinoma , renal cell carcinoma , and those after use of oral contraceptives [ 46 , 47 ] and pregnancy [ 48 , 49 ] . the present case report suggests an unusual association of myointimal proliferation ( thrombosis / atheromasia ) and cd . despite the fact that no definitive relationship between these diseases could be explained , we think this association must be remembered especially in cases of young and tenuous women with these vascular abnormalities .

celiac disease ( cd ) is an autoimmune disorder of the small bowel that occurs in genetically predisposed people of all ages , from middle infancy , and is caused by a reaction to gliadin , a gluten protein . some patients are diagnosed with symptoms related to the decreased absorption of nutrients or with various symptoms which , although statistically linked , have no clear relationship with the malfunctioning bowel . classic symptoms of cd include diarrhea , weight loss , and fatigue ; bowel symptoms may be limited or even absent . in this article we describe the case of a young woman with cd who presents with myointimal proliferation . however multiple cases of vessel thrombosis have been reported in patients with cd . despite the fact that no definitive relationship between these diseases could be explained , we think this association must be remembered especially in cases of young and tenuous women with these vascular abnormalities .

nephropathy arising from animal origin includes toxins of snakes , scorpions , hymenoptera , jelly fish , spiders , centipedes , caterpillars and grass carp ( raw bile ) . acute renal failure is the commonest nephropathy , but subnephrotic proteinuria , vasculitis , haemolytic we recently admitted a 64-year - old lady with oedema and reduced urine output of 3 days duration . she had been stung by a red scorpion on her left thumb 10 days previously and she had then applied lime to the site of the sting . at admission , she had a linear healing ulcer on her thumb , anasarca , pallor and hypertension of 160/90 mmhg . g / l , total counts 5.7 10/l , platelets 150 10/l , erythrocyte sedimentation rate 35 mm / h , urea nitrogen 12.48 mmol / l , creatinine 97.24 mol / l , total cholesterol 2.98 mmol / l , triglycerides 1.23 mmol / l , low - density lipoprotein 1.4 mmol / l , high - density lipoprotein 0.93 mmol / l , thyroid - stimulating hormone 2.2 miu / l , c3 1.06 g / l ( 0.150.45 ) , negative anti - nuclear antibodies , ds - dna , antineutrophil cytoplasmic autoantibodies , cytoplasmic ( c - anca ) and hepatitis panel and normal liver function tests . dipstick urine testing revealed albumin 3 + and 24 h urinary protein was 735 mg ; urine microscopy showed 12 red blood cells ( rbcs ) , 23 pus cells and 68 epithelial cells without rbc casts . renal biopsy was performed on the seventh day of admission and showed focal irregularity of the basement membrane , mild interstitial infiltrate of lymphocytes , plasma cells and neutrophils with fraying of some tubules and without granulomas or inclusion bodies . we had treated her with diuretics , calcium channel blockers and her renal functions and urinalysis were normal at a follow - up visit 6 weeks later . therefore , we had reviewed the topic of renal failure following scorpion sting envenomation . in the following review , we will discuss the epidemiology of scorpionism , its venom and toxins , systemic effects of venom especially in relation to the kidney and describe the known reports of scorpion sting nephropathy . venom and kidney in the following databases pubmed , science direct , ebsco , wiley online , springerlink , ovid and google scholar . scorpions are ancient creatures that have not seen much evolutionary changes over millions of years . they are nocturnal beings and are exclusively carnivorous , sensing their prey through their pedipalps and also through hearing . the order scorpionidae ( class arachnida ) constitutes of 18 families and 1500 species . the buthidae family is the largest and has the most number of species that are toxic to humans . except antartica the genera of the old world include androctonus ( north africa , saudi arabia and turkey ) , hottenta ( morocco and india ) , buthus ( east mediterranean ) , leiurus ( africa and middle east ) , parabuthus ( south africa ) , hemiscorpius ( iran and iraq ) and mesobuthus ( turkey and iran ) . the new world genera comprises of centruroides ( southern united states and mexico ) and tityus ( brazil , argentina and venezuela ) . health hazards are the greatest in the indian subcontinent , mexico and the north african countries . there are 134 species in mexico and only the centruroides species ( 8 in numbers ) are poisonous . about 250 000 scorpion events are registered in mexico every year , with the highest mortality rate in the world [ 2 , 8 ] . centruroides exilicauda ( formerly centruroides sculpturatus ) found in arizona is the only poisonous species in the usa [ 7 , 9 ] . tityus serrulatus is the most prevalent species in south america and accounts for most of the fatal stings on that continent , especially in brazil [ 10 , 11 ] . intensive care admission following scorpion stings in tunisia generally arises from either androctonus australis or buthus occitanus species envenomation . about 130 species , falling into four families buthidae , scorpionidae , bothriuridae and ischnuridae , are seen in southern africa . hemiscorpius lepturus accounts for 90% of scorpion - related deaths in iran . in saudi arabia , the commonest species include leirurus quinquestriatus and androctonus crassicauda , while in turkey , euscorpius carpaticus and euscorpius germanus are the most frequent . mesobuthus tamulus and palmoneus gravimanus are the commonest species in india , with the former being among the most toxic of scorpions . volume of venom per sting , protein content and toxicity vary with the individual scorpion . peptide compositions of toxins vary within the same scorpion species depending on the region they inhabit . signs of envenomation depend upon the species , venom components and the physiological response of the person stung . venom is an aqueous mixture of low - molecular - weight ( lmw ) peptides , antimicrobial peptides , mucopolysaccharides , protease inhibitors and high - molecular weight proteins ( hmw ) . lmw ( < 1500 da ) proteins form the largest subgroups ( 1/3rd ) of these proteins . scorpion neurotoxins mainly weigh between 3000 and 9000 da . bradykinin - potentiating peptides ( bpp ) ] account for more than half of the lmw proteins that have been identified . . the kininases act on metabolites of lmw kininogens to form bradykinin ( bk ) . the 30014500 and 61007500 da groups that contain the predominant mass of known scorpion toxins comprise 20% each . the hmw proteins ( > 9000 da ) mainly include phospholipase , hyaluronidase and lyzozymes . scorpion venom lacks enzyme activity or have very low levels of enzyme activity compared to that of snake and hymenoptera venom [ 19 , 23 ] . exceptions are the heterometerus scaber and fulvipes , m. tamulus and c. exilicauda species . venoms are classified based on their mode of action and binding site . based on their molecular size , they are either long - chain or short - chain peptides . chlorotoxin from l. quinquestriatus has binding ability to matrix metalloproteinase ii of glioma cell lines and shows anti - cancer activity . scorpion venom toxins are divided into four classes [ 23 , 24]sodium channel toxins ( natx ) , potassium channel toxins ( ktx ) , chloride channel toxins ( cltx ) and calcium channel toxins ( catx ) . natx are mainly anti - mammal and anti - insect toxins ( e.g. butal t from buthus tumulus ) [ 20 , 21 ] . the -natx inactivates sodium channels by binding to site 3 of the receptor and prolongs nerve action potential . the -toxin produces a more negative membrane potential by binding to site 4 of the receptor . the -toxins group either act exclusively on mammalian systems or on both insects and mammals [ 14 , 23 ] . the -toxins include cssiv from centruroides suffussus and ts1 from t. serrulatus [ 5 , 26 ] . the ktx affect voltage - gated k channels , outward delayed rectifier k channels and the maxi - ca - activated k channels . the k channels are physically occluded ; blockage of these channels prolongs action potential durations . ktx are involved in immune responsiveness : blockage of these channels causes decrease in t - cell activation and subsequent delayed - type hypersensitivity . the amino acid and its alignment ( especially cysteine ) on the external surface of the toxin decide the specificity and affinity of the k toxin . the indian red scorpion ( m. tamulus ) venom contains ktx like iberiotoxin , tamulustoxin and tamapin . the k channel blockers have short - chain peptides and belong to the 30004500 da group , whereas the na channel modulators are long chained with molecular weights of 60017500 da . short - chain peptides ( 3040 amino acids ) mainly recognize k channels , while long - chain peptides ( 5864 amino acids ) recognize na channels [ 2 , 28 ] . the cholinergic activity is either due to reduced destruction of acetylcholine ( ach ) or excessive ach release , acting on post - ganglionic nerve endings [ 26 , 29 ] . studies in rats ( a. australis hector scorpions ) , mice ( centruroides noxius scorpions ) and rabbits ( l. quinquestriatus scorpions ) have shown that venom also induces release of both pro- and anti - inflammatory cytokines . venom also causes an inflammatory reaction in the lungs , kidneys , heart and intestine of experimental rats . an initial phase of hyperadrenergism ( inotropic phase ) followed by a phase of hypotension and depressed myocardial contractility is seen in experimental rabbits . the late phase has been hypothesized to be due to cholinergic effects , fluid loss or vasodilatory substance release by venom or its peptides . inflammatory cytokines like interleukin ( il)-1 , il-6 , il-8 , il-10 , tnf- and nitric oxide ( no ) have been observed in children with scorpion envenomation . tnf- , il-1 or il-6 have all been postulated to play a role in leucocyte activation and leucocytosis . leucocyte activation may lead to production of reactive oxygen species and subsequent multi - organ failure . immediate pain and , in children , screaming and irritability , are observed after a scorpion sting . local reactions are generally uncommon in stings of the indian red scorpion ( m. tamulus ) . pain and sweating with faintness constitute stage i of envenomation . local pain and parasthesias apart , signs and symptoms of envenomation arising from exaggerated autonomic activity ( parasympathetic and sympathetic ) include cool extremities , vomiting , diarrhoea , hyperthermia , abdominal colic , priapism , hypotension or hypertension , sinus tachycardia , congestive heart failure , myocarditis , dysrhythmias , heart blocks and pulmonary oedema . these features occur within 30 min to 4 h and can last for up to 72 h ( especially tachycardia ) . dawley rats caused down - regulation of na k - atpase in the basolateral membrane of alveolar epithelial cells and reduces alveolar oedema clearance . toxin of t. serrulatus causes abnormal breathing patterns like cheyne stokes and biot breathing in mice . stage ii envenomation is comprised mainly of muscarinic signs and symptoms , while stage iii is associated with pulmonary oedema , arrhythmias and cardiovascular collapse . the nervous system manifestations are infrequent and they include encephalopathy , seizures , restlessness , areflexia , psychosis , strokes and muscle cramps . pancreatitis ( systemic inflammatory response related ) in israel and trinidad , acute renal failure from the middle east and north africa , and abortions , are the other unusual manifestations that have been described [ 26 , 32 ] . necropsy studies have revealed haemorrhages in the lung and kidney and thrombi occluding capillaries in the kidney , liver , spleen , intestines , lung and the heart . haematological findings include thrombocytopenia and deficiency in coagulation factors v , vii , viii and xiii . in ventilated rats , scorpion venom infusion has been shown to produce metabolic acidosis , hyperglycaemia , hypermagnesaemia and hyperkalaemia . the kidneys have the largest concentration of venom ( and the brain , the lowest ) , mainly because of the rapid redistribution of the venom from the blood to the tissues coupled with slow removal from the kidney . the liver , lungs and the heart have successively lower venom concentrations when compared to the kidney . even though the scorpion injects venom into the interstitial spaces , 70% blood concentration is achieved within 15 min . radiolabelling of m. tamulus venom in wistar rats revealed a rapid high concentration that reduced to 1/3rd of concentrations within 30 min indicating rapid elimination from blood . renal uptake at 30 min ( 32% ) reduced to only 22% at 3 h indicating slow elimination from the kidneys . during redistribution from blood , toxins disappear faster compared to other venom components and hence local tissue concentration of toxins , especially in the kidney , can cause direct toxicity . venom administration to rats has shown increased vascular resistance , increased perfusion pressure , decreased renal blood flow , decreased glomerular filtration rate ( gfr ) and decreased urinary flow . afferent arteriolar vasoconstriction was probably caused by renal -adrenoreceptor activation or was due to k channel blockade that causes vascular smooth muscle depolarization and contraction . proteinaceous deposits found in the renal tubules was possibly due to high perfusion pressures or action of a yet unrecognized substance similar to the pore - forming peptides of some scorpion venoms ( buthus martensii ) that damage the glomerular basement membrane ( gbm ) . the vasoconstrictor actions were temporary phenomena lasting for 10 min akin to that of an adrenergic vascular escape phenomenon seen in the enteric circulation . in another study , effects of buthus occitanus tunetanus venom plasma creatinine was higher in pregnant rats 30 min after venom exposure when compared to their non - pregnant counterparts ; the plasma creatinine dropped significantly at 4 h only in the non - pregnant rats . haemoconcentration was noted in non - pregnant rats that were partly offset by anaemia in pregnant animals . there was a significant negative correlation of blood urea nitrogen in pregnant rats and a positive correlation of blood urea nitrogen / creatinine ratio in their non - pregnant counterparts . polyuria ( contributing to haemoconcentration ) , metrorrhagia ( possibly due to coagulation defects ) , significant reduction in platelet size ( in non - pregnant animals contributing to coagulation defects ) and haemolysis may predispose to renal insufficiency . in a review from iran , envenomation by h. lepturus in rats led to focal necrosis and haemorrhage in the kidney and other lipid - containing organs like the liver and heart . morphological changes in the glomerular apparatus and the proximal tubular cell - like interstitial oedema , dose - dependent collapse of the proximal and distal tubules , lymphocytic infiltration of the medulla and glomerular destruction were observed . the hyaluronidase affects vessel wall stability and may cause the spread of venom in tissues . loxosceles deserta , the brown recluse spider , also has similar molecular weight enzymes , especially in the 3035 kda band of antigenic components , and the spider venom is known to promote the complement cascade . sphingomyelinase d , an enzyme , is seen in the venom of both h. lepturus and l. deserta and predisposes to skin necrosis , local ischaemia , thrombosis and haemolysis . in a study of 12 dogs with t. serrulatus envenomation by injecting 250 g / kg of venom , no significant electrolyte imbalances , dehydration , nausea or vomiting were observed . urinalysis remained normal in these dogs except for mildly lower urinary ph in envenomed dogs . il-12 is a determinant in glomerulonephritis ( gn ) and absence of il-12 prevented the formation of anti - gbm gn . focal proliferative glomerulonephritis in rats , mice and rabbits is caused when habu snake venom is injected intravenously . in experimental lupus nephritis , reduced glomerular macrophages and t cells were noted especially in il-12-deficient mice with lupus nephritis . reduced il-12 decreased activity of mesangial cell proliferation without affecting the course of mesangial glomerulonephritis . surprisingly , butantoxin from t. serrulatus inhibits t - cell proliferation and il-12 synthesis ; hence other factors in scorpion envenomation may mediate glomerular injury . rat kidney glomeruli secretes prostaglandins ( pg ) pge2 , pgei2 , pgd2 and thromboxane b2 ( txb2 ) . increase in vasodilatory pgs mediates increases in gfr , while pge2 inhibition reduces creatinine clearance . modulatory activity of pgs depends on their interactions with angiotensin ii ( aii ) and arginine vasopressin ( avp ) . aii and avp stimulate pge2 in kidney mesangial cells due to the tight linking of aii and avp receptors to the phopholipase ( pl ) , cox and pge2 isomerase . the peptide fraction of the egyptian scorpion b. occitanus has bpp that may increase urea and creatinine clearance . thus , it is seen that scorpion venom administration in experimental animals predisposes towards renal failure by way of concentration of venom in the kidneys , large molecular weight enzymes , high perfusion pressures , hypotension , reduced renal blood flow , polyuria , haemoconcentration , haemolysis , coagulation defects and systemic inflammatory response also involving complements , vasculitis , fibrin deposition and haemorrhages . this is to be partly sobered by the fact that some venom components like bpp can increase blood flow and creatinine clearance and some toxins can inhibit t - cell proliferation and il-12 production . there are no studies correlating nephropathy with the innumerous ion channel toxins , on a scale of the literature available on the toxin effects on the nervous , musculoskeletal and cardiorespiratory system . k channel toxins may mediate vascular contraction by causing smooth muscle depolarization and hence contribute to reduced renal blood flow . on the other hand , k channel toxins like butantoxin mediate delayed hypersensitivity and reduce il-12 as previously mentioned and hence may play a protective role in kidney injury . in spite of the vulnerability of the kidney due to its ability to concentrate toxins , direct toxin - mediated kidney injury ( except pigmenturia - related tubular damage ) has not been conclusively demonstrated . since anaphylaxis is also rare , it could be partly explained that most of the known scorpion toxins are < 9000 da and may not induce an antigenic response of sufficient magnitude . the hmw ( > 9000 da ) peptides that include enzymes like hyaluronidase and sphingomyelinase which mediate tissue injury and complement activation comprise a very small portion of scorpion venom . direct venom toxicity in general is due to these hmw proteolytic enzymes . also , venom components vary from habitat to habitat . this might also contribute to renal failure being reported from very few countries in the world hence , a combination of other non - toxin - related factors causes renal injury . causes of venom - induced acute kidney injury in asia includes snakes , hymenoptera , spiders , jellyfish and scorpions [ 37 , 42 ] . these stings cause renal involvement by pigment nephropathy , interstitial nephritis , rhabdomyolysis or intravascular haemolysis , vasculitis or direct toxin effect , which is similarly seen in scorpion stings . a review of nephrotoxic insect venom in 2008 did not mention kidney injury due to scorpion venom . about 62 odd cases of renal failure resulting from scorpion envenomation have been reported ( table 1 ) . in a multivariate analysis of cases of scorpion stings from tunisia , urea elevations were associated with mortality and were generally observed in patient subsets with cardiac failure or hypovolaemia . all cases of scorpion sting - related nephropathy have been reported from one of these five countries iran , tunisia , turkey , pakistan and israel ( table 1 ) . reports of scorpion sting nephropathya y , years ; dic , disseminated intravascular coagulation ; lft , liver function tests ; ards , acute respiratory distress syndrome ; ck , creatine kinase ; h , hours . both children and adults ( young and middle - aged ) can develop renal failure , with children having been more commonly reported ( 52/67 ) in literature . patients develop renal insufficiency in the setting of hypovolaemia and cardiac failure - related acute tubular necrosis ( atn ) , pigmenturia , haemoglobin - related tubular damage , hus or interstitial nephritis . delay in seeking hospital care and/or administration of anti - venom increases the risk of renal failure . onset of illness is generally within a few days , although one report described development within 24 h and that particular patient also had hepatitis . haemoglobinuria and haematuria occur between a few hours to 24 h haematuria clears within 15 days . patients present with anaemia , urinary discolouration , ulcer or dermonecrosis , jaundice , oedema or anasarca , oliguria or anuria , breathlessness , hypo- or hypertension and altered sensorium . investigations reveal anaemia , leukocytosis , thrombocytopenia , schistocytes , elevated urea , creatinine , transaminases , creatinine kinase and lactate dehydrogenase , bland or active urinary sediments , urinary haeme and proteinuria , and interstitial nephritis on biopsy . most of these patients require renal replacement therapy along with rbc transfusions , inotropes , antivenom , respiratory support , diuretics , anti - hypertensives and occasional steroids ( in hus and interstitial nephritis ) . diuresis begins by the end of the first week and most improve by 4 weeks if they do not die of disseminated intravascular coagulation or neurological or cardiovascular complications . a review from iran on acute renal failure in 2004 reported six children with atn arising from scorpion sting envenomation . a series of 15 cases of scorpion sting - related acute renal failure was reported in 1978 with all of them having haemoglobinuria and renal failure . mesangial hypercellularity and mild to moderate interstitial inflammation were documented . in vitro studies of scorpion venom on human rbcs possible immune glomerulonephritis had been hypothesized since venom peptides can rarely induce an antigenic response . cytokine release and activation of complement cascade activation are known with some scorpion venoms but glomerulopathy per se has not been described in the literature . our patient only had focal irregularity of the basement membrane and a good quality immunofluorescence staining could not be obtained to detect glomerular deposits . h. lepturus species accounts for 90% of scorpion - related deaths in iran causing haemoglobinuria and renal failure especially in children . in one review , 50% of 141 children admitted had haemoglobinuria alone , 20% had both haemoglobinuria and renal failure and 2.2% died due to renal failure . the hemiscorpius species apart , the buthus and cosmobuthus scorpions ( the three , collectively called the yellow scorpions ) also cause haemolysis and renal failure . haemolysis associated with renal failure and local necrosis was reported once from israel , and the patient had been treated with steroids and had recovered over a course of 4 weeks . reports of hus have been described in three instances two from tunisia and one each from iran and turkey [ 17 , 48 , 51 ] . the first three cases were in children , while the turkish patient was a 28-year - old man . androctonus envenomation led to fatal hus in both infants from tunisia even after treatment with anti - scorpion venom , inotropes and mechanical ventilation . blood pressure was normal in both infants ; both had elevated liver enzymes and creatine kinase levels . steroids had also been helpful in one other case from tunisia wherein a 50-year - old lady stung possibly by a. australis had elevated creatinine , bland sediments , proteinuria ( similar to our patient ) and interstitial nephritis on biopsy . the 60-year - old patient from pakistan had albuminuria , active sediments , haemoglobinuria and developed atn ( post - mortem biopsy ) , disseminated intravascular coagulation and died 15 days after the scorpion sting . it has been hypothesized by some authors that early anti - venom therapy may prevent renal failure . but with a large number of scorpion envenomations worldwide , few people , especially from rural areas , ever manage to receive antivenom even if they are reached in time , due to its unavailability . antivenoms are available in africa , india , turkey , brazil , morocco and saudi arabia . but in india , therapy is mostly with prazosin , dobutamine , nitroglycerine and mechanical ventilation since pharmacies of even large tertiary care teaching hospitals do not stock scorpion anti - venom . anti - venom has been shown only to expedite recovery and lessen duration of hospital stay . renal failure following scorpion stings has been reported mainly in the middle east , eastern mediterranean region and north africa . the kidney is theoretically vulnerable due to higher venom / toxin concentrations , hmw antigenic peptides and enzyme - related direct renal damage and possibly venom - related effects on gfr and perfusion pressure . also , a systemic inflammatory response syndrome , complement cascade activation and other effects like pulmonary oedema , myocarditis and adrenergic excess - related vasoconstriction or parasympathetic stimulation - related hypotension cause reduction in renal blood flow and increase the risk of atn . haemoglobinuria - related pigment nephropathy , atn , hus and interstitial nephritis have all been documented as the aetiology of renal failure in scorpion envenomation . the multitude of known ion channel toxins probably does not play a major role in development of renal insufficiency . effective treatment includes early prazosin therapy , anti - venom , steroids , inotropes , diuretics , treatment of hyper- and hypotension , rbc transfusions and renal replacement therapy .

scorpion envenomations are ubiquitous , but nephropathy is a rare manifestation , reported mainly from the middle east and north africa . rapid venom redistribution from blood , delayed excretion from the kidneys , direct toxicity of venom enzymes , cytokine release and afferent arteriolar constriction have been seen in experimental animals . haemoglobinuria , acute tubular necrosis , interstitial nephritis and haemolytic uraemic syndrome have been documented in human victims of scorpion envenomation . epidemiology , venom components and toxins , effects on the laboratory mammals especially the kidneys and reports of renal failure in humans are reviewed in this article .

aphids ( homoptera : aphididae ) are attacked by many different predatory insects and have evolved an efficient defense behavior . when disturbed , they release cornicle droplets that contain a mixture of triglycerides and alarm pheromones ( nault et al . the alarm pheromones of many aphid species are , in most cases , single terpenes or mixtures of terpenes ( pickett and glinwood 2007 ) . for some species , such as the pea aphid , acyrthosiphon pisum , the sesquiterpene ( e)--farnesene ( ebf ) is the only compound in the alarm pheromone ( francis et al . 2005 ) . the amounts of ebf emitted by individual pea aphids are small , and this may be exacerbated by chemical oxidation or the concentration of the pheromone being diluted rapidly by air movement . few studies have attempted to investigate emission dynamics of aphid alarm pheromone ( schwartzberg et al . , it is unclear whether the emission is confined to the attacked aphid or whether the signal is reinforced by members of the colony . the emission of alarm pheromone by aphids that perceive the chemical , but are not attacked , would amplify the signal and presumably warn more individuals in a colony . however , such amplified emission would also expose inconspicuous aphids to natural enemies or unnecessarily alert aphids that are not at risk . in this paper , we report on a series of experiments in which we use a deuterated ( e)--farnesene derivative ( debf ) , labeled on the geminal dimethyl group , to investigate signal reinforcement in the pea aphid . ( 2004 ) ( fig . 1 ) , epoxidation of ( e)--farnesene ( 1 ) ( 408.70 mg , 2 mmol ) , employing methyltrioxorhenium ( vii ) and urea - hydrogen peroxide adduct in chcl3 at 5c , gave a mixture of mono- and di - oxiranes . the 10,11-epoxy-(e)--farnesene ( 2 ) ( 180.3 mg , 44% , rf 0.48 ) was separated on florisil 100200 ( petroleum ether 4060/ether 40:1 v / v ) . as specified by fielder and rowan ( 1994 ) ( fig . 1 ) , the epoxide 2 was cleaved with periodic acid to give ( e)-4-methyl-8-methylenedeca-4,9-dienal ( 3 ) ( 49.1 mg , 35% , rf 0.27 ) , which was purified on silica 60 ( petroleum ether 4060/ether 40:1 v / v ) . wittig reaction of 3 with d7-isopropyltriphenylphosphonium iodide produced the d6-(e)--farnesene ( 4 ) ( -h6-(e)--farnesene ; debf ) ( 221.60 g , 67% , rf 0.60 ) . purification of 4 was carried out by flash chromatography on silica gel ( silica 60 , merck , darmstadt , germany ) with pentane . the chemical purity of debf after chromatographic purification was 97% as determined by gas chromatography - mass spectrometry ( gc - ms ) . 1synthesis of -h6-(e)--farnesene synthesis of -h6-(e)--farnesene nmr data of deuterated ebf h nmr ( 500 mhz , cdcl3 ) : ( ppm ) = 1.53 ( s , 3h ) , 1.891.94 ( m , 2h ) , 1.972.03 ( m , 2h ) , 2.082.19 ( m , 4h ) , 4.92 ( d , j = 1.3 hz , 1h ) , 4.94 ( d , j = 1.3 hz , 1h ) , 4.98 ( d , j = 10.8 hz , 1h ) , 5.03 ( t , j = 6.8 hz , 1h ) , 5.09 ( tq , j = 6.8 , 1.0 hz , 1h ) , 5.17 ( d , j = 17.7 hz , 1h ) , 6.31 ( dd , j = 17.7 , 10.8 hz , 1h ) . aphid lines we used a red clone ( bp ) of the pea aphid , a. pisum , maintained on vicia faba plants in controlled conditions at 20c , 16l/8d photoperiod , and approximately 75% rh . for experiments , aphid lines descended from a single founder were established ( kunert et al . experiment 1 : debf and aphid behavior three groups of 15 apterous adults of the same line were transferred to three plants and covered with cellophane bags ( 18.8 39 cm , n = 12 lines ) . a piece of filter paper , to which debf , ebf ( both 1 g in 3 l hexane ) , or hexane ( 3 l ) was applied , was placed inside the bags . the solutions were applied three times per day over 5 d. after the first application of each solution , the number of pea aphids walking was counted for 5 min . at the end of the experiment , mothers were counted and removed , and nymphs left for four more days on plants . experiment 2 : debf and ebf release for volatile collection , two groups of 15 third / fourth instars , of the same line , were transferred to two plants ( n = 13 lines ) and placed in glass chambers ( modified 1-l beaker ) . teflon plates were placed around the base of the plant , keeping the soil out of the collection system . two openings ( = 1 cm ) were present on top of the chamber : one provided air ( 2 l min ) filtered through active charcoal , and the other held a filter paper to which either debf ( 1 g in 3 l hexane ) or hexane ( 3 l ) was applied . the chambers had an additional opening 3 cm from the rim holding a super - q filter ( 80/100 mesh ; alltech , deerfield , il , usa ) connected to an air pump ( 1 l min ) . with this set - up , six plants ( three lines ) volatile analysis super q filters were eluted with 140 l of dichloromethane and analyzed by gc - ms on a db-5ms ( j&w ) column . for analysis , , increased to 180c at 5c min , and then increased at 60c l min until 300c . mass spectra from each peak were compared to those in the nist and wiley libraries for tentative peak identification . an internal standard of ( e)--caryophyllene ( 400 ng in 30 l of ch2cl2 ) was added to all samples as an internal standard . statistical analyses the number of aphids walking was analyzed by analysis of variance ( anova ) . wing induction was analyzed by a generalized linear model ( glm ) with a quasi - binomial error structure . the aphid lines and number of nymphs produced in each replicate were included in the model , which was then simplified by removing non - significant variables or interactions , and accepted after an anova ( p > 0.05 ; crawley 2007 ) . the survival of the mothers was analyzed with a glm using a poisson error structure and simplified as described above . data were analyzed with r software 2.6.0 ( 2007 ) and are presented as mean se . in experiment 1 , there was no significant difference among treatments in the mean number of adult pea aphids that survived ( hexane = 13.75 0.52 ; ebf = 17.83 0.44 ; debf = 13.58 0.47 ; t = 1.317 ; p = 0.197 ; n = 36 ) . in both ebf and debf treatments , the proportion of winged morphs among aphid offspring was higher than in the hexane treatment ( fig . 2 ; t = 15.075 ; p < 0.001 , n = 36 ) . the number of aphids that responded to treatment by walking did not differ between ebf and debf ( fig . 2 , t = 1.542 , p = 0.152 , n = 36 ) ; no walking responses were observed in the hexane treatment ( fig . 2 , t = 27.211 , p < 0.001 , n = 36 ) . fig . 2percentage of winged offspring produced by adult pea aphids and number of pea aphid mothers walking after exposure to hexane , ( e)--farnesene ( ebf ) or deuterated ( e)--farnesene ( debf ) . the bars show the mean value se percentage of winged offspring produced by adult pea aphids and number of pea aphid mothers walking after exposure to hexane , ( e)--farnesene ( ebf ) or deuterated ( e)--farnesene ( debf ) . the bars show the mean value se in experiment 2 , aphids started dispersing after debf application , but there was no measurable emission of endogenous alarm pheromone , suggesting that only attacked aphids emit ebf . given the amounts of debf applied and re - collected , we estimated that the minimum amount of ebf detected by our experimental system was about 30 ng , an amount equivalent to that typically released by two to three third / fourth instars ( schwartzberg et al . 2008 ) . therefore , because we used colonies of 30 aphids , no more than 10% of the individuals tested , if any , could have responded by emitting their own ebf . while signal amplification would have the advantage of alerting more aphids in the colony , it also has disadvantages . for example , it is thought that aphids may use the frequency of alarm pheromone perception as a measure of danger . experiments with predators that induce alarm pheromone release and synthetic ebf , respectively , have shown that the proportion of winged offspring is related to the number of aphids consumed and the frequency of application of ebf ( kunert et al . . signal amplification would preclude aphids from employing the frequency of alarm pheromone release as a measure of the severity of an attack . additionally , some natural enemies use ebf to detect their prey ( acar et al . 2006 ) , and amplification of the signal would increase the danger of predator attraction .

when aphids are attacked by natural enemies , they emit alarm pheromone to alert conspecifics . for most aphids tested , ( e)--farnesene ( ebf ) is the main , or only , constituent of the alarm pheromone . in response to alarm pheromone , alerted aphids drop off the plant , walk away , or attempt to elude predators . however , under natural conditions , ebf concentration might be low due to the low amounts emitted , to rapid air movement , or to oxidative degradation . to ensure that conspecifics are warned , aphids might conceivably amplify the alarm signal by emitting ebf in response to ebf emitted by other aphids . to examine whether such amplification occurs , we synthesized deuterated ebf ( debf ) , which allowed us to differentiate between applied and aphid - derived chemical . colonies of acyrthosiphon pisum were treated with debf , and headspace volatiles were collected and analyzed for evidence of aphid - derived ebf . no aphid - derived ebf was detected , suggesting that amplification of the alarm signal does not occur . we discuss the disadvantages of alarm signal reinforcement .

a 71-year - old woman presented with pain of the right knee with 2 year duration . she had been involved in a slip down and sustained right femur shaft fracture 3 years earlier , as well as a left femur shaft fracture 5 years ago . preoperative radiographs of the right knee in the anteroposterior and lateral views showed tricompartment osteoarthritis and a retained intramedullary nail . the femoro - tibial angle that formed between the mechanical axis of the femur and tibia axis was 25 varus ( figs . 1 and 2 ) . the patient underwent a right tka with computer navigation using the stryker 4.0 system ( stryker co. , allendale , nj , usa ) , cemented , and posterior cruciate ligament sacrificing type ( scorpio , stryker co. , ) . a 6.5 mm bicortical self - tapping anchoring screw was inserted 10 cm below the tibiofemoral joint line . another type anchoring pin for the femur was used to avoid a collision with the intramedullary nail . this type of anchoring pin is smaller ( 3.0 mm ) than a anchoring screw , and unicortical anchoring is available ( fig . a femur and tibia resection were performed according to the light - emitting diode tracker of a navigation system and cutting jig . the ligamentous release and balance were also performed based on a trial reduction of the prosthesis and guide in a navigation system . both components were centered to the midline of the joint , and the overall knee alignment was 5 valgus ( fig . after tka , the patient could walk with partial weight bearing and full weight bearing 1 day and 7 days after surgery respectively . a 79-year - old woman presented with pain of the left knee with a 3 year duration . the patient was involved in a traffic accident 15 years earlier and sustained a left distal femur fracture . preoperative radiographs of the left knee in the anteroposterior and lateral views revealed tricompartment osteoarthritis and a containing plate and screws on lateral side of the femur ( fig . 5 ) . the femoro - tibial angle that formed between the mechanical axis of the femur and tibia axis was 15 varus . the patient underwent right tka performed with computer navigation using a stryker 4.0 system , cemented , posterior cruciate ligament sacrificing type ( scorpio ) . a midline skin incision was performed . a 6.5 mm bicortical self - tapping anchoring screw another type of anchoring pin was used on the femur to avoid a collision with the screws . the lateral epicondyle , which was covered with a plate , could not be checked . a multiple checking system , a femur and tibia resection were performed depending on the light - emitting diode tracker of the navigation system and cutting jig . the most distal screw impinged on the box cutting for the posterior cruciate ligament sacrificing type and one screw was removed . ligamentous release and balance were also performed based on a trial reduction of the prosthesis and guide in the navigation system . both components were centered to the midline of the joint , and overall knee alignment was 6 valgus ( figs . 6 and 7 ) . after tka , the patient could walk with partial weight bearing and full weight bearing 1 day and 7 days after surgery respectively . a 71-year - old woman presented with pain of the right knee with 2 year duration . she had been involved in a slip down and sustained right femur shaft fracture 3 years earlier , as well as a left femur shaft fracture 5 years ago . preoperative radiographs of the right knee in the anteroposterior and lateral views showed tricompartment osteoarthritis and a retained intramedullary nail . the femoro - tibial angle that formed between the mechanical axis of the femur and tibia axis was 25 varus ( figs . 1 and 2 ) . the patient underwent a right tka with computer navigation using the stryker 4.0 system ( stryker co. , allendale , nj , usa ) , cemented , and posterior cruciate ligament sacrificing type ( scorpio , stryker co. , ) . a 6.5 mm bicortical self - tapping anchoring screw was inserted 10 cm below the tibiofemoral joint line . another type anchoring pin for the femur was used to avoid a collision with the intramedullary nail . this type of anchoring pin is smaller ( 3.0 mm ) than a anchoring screw , and unicortical anchoring is available ( fig . a femur and tibia resection were performed according to the light - emitting diode tracker of a navigation system and cutting jig . the ligamentous release and balance were also performed based on a trial reduction of the prosthesis and guide in a navigation system . both components were centered to the midline of the joint , and the overall knee alignment was 5 valgus ( fig . after tka , the patient could walk with partial weight bearing and full weight bearing 1 day and 7 days after surgery respectively . a 79-year - old woman presented with pain of the left knee with a 3 year duration . the patient was involved in a traffic accident 15 years earlier and sustained a left distal femur fracture . preoperative radiographs of the left knee in the anteroposterior and lateral views revealed tricompartment osteoarthritis and a containing plate and screws on lateral side of the femur ( fig . 5 ) . the femoro - tibial angle that formed between the mechanical axis of the femur and tibia axis was 15 varus . the patient underwent right tka performed with computer navigation using a stryker 4.0 system , cemented , posterior cruciate ligament sacrificing type ( scorpio ) . a midline skin incision was performed . a 6.5 mm bicortical self - tapping anchoring screw another type of anchoring pin was used on the femur to avoid a collision with the screws . the lateral epicondyle , which was covered with a plate , could not be checked . a multiple checking system , a femur and tibia resection were performed depending on the light - emitting diode tracker of the navigation system and cutting jig . the most distal screw impinged on the box cutting for the posterior cruciate ligament sacrificing type and one screw was removed . ligamentous release and balance were also performed based on a trial reduction of the prosthesis and guide in the navigation system . both components were centered to the midline of the joint , and overall knee alignment was 6 valgus ( figs . 6 and 7 ) . after tka , the patient could walk with partial weight bearing and full weight bearing 1 day and 7 days after surgery respectively . a navigation system has been reported to improve the accuracy of bony cuts and restore the mechanical axis in tka.4 - 6 ) computer - assisted tka using a surgical navigation system provides accurate cuts of the distal femur and proximal tibia without the need for intramedullary instrumentation . intramedullary instruments can not be used in patients with previous trauma and substantial residual bony deformities , or retained hardware that can not be removed . in old age , bone is usually osteoporotic and the fractured bone fixed with an implant may be more osteoporotic . in conventional tka , when removing the implant , the space where the implant had been removed can be stress riser for a given load after tka.7,8 ) early ambulation and early continuous passive motion should be performed more carefully . in these 2 cases , partial weight bearing walking and full weight bearing walking was possible 1 day and 7 days after surgery , respectively . the pins were smaller ( 3.0 mm ) than used at the tibia and could be anchored unicortically . the reasons for changing the anchoring screw to pins were to reduce the anecdotal fracture and collision with retained hardware . however , a unicortical pin at the metaphysis for the tracker often has insecure fixation that can cause poor registration during navigation - assisted tka . two anchoring pins were inserted for the femur but three pins can be inserted for more secure fixation . some cases with an anecdotal fractures have been reported after the placement of navigation tracker pins.9,10 ) there are a few reports that a smaller size and unicortical fixation pin is safer . in one anatomic study , the findings indicated the close proximity of the popliteal vessel to potential injury with the bicortical placement of tracker pins in the anteroposterior direction in a typical total knee approach . navigation - assisted tka is feasible and less invasive for tka of a retained implant of the distal femur after a fracture .

proper ligament balancing , restoration of the mechanical axis and component alignment are essential for the success and longevity of a prosthesis . in conventional total knee arthroplasty ( tka ) , an intramedullary guide is used to improve the alignment . an extramedullary guide can be used in cases of severe femoral bowing or intramedullary nailing but its use is more subjective and relies on the surgeon 's experience . this paper reports two successful cases of navigation - assisted tka for severe right knee osteoarthritis retaining a femoral intrameullary nail , and left knee osteoarthritis retaining a distal femoral plate .

this cross - sectional study included patients with oht or primary open - angle glaucoma ( poag ) older than 40 years of age of both genders , with 20/20 best corrected visual acuity , and any ethnicity . subjects with cataracts or any other ocular disease , and previous incision or laser surgery for glaucoma were not included in the study . institution board review approved the study , and the procedures followed adhered to the principles for medical research involving human subjects of the declaration of helsinki in 1964 ( amended by the 59 wma general assembly , seoul , korea , october 2008 ) . in order to be included in the study , poag patients had typical optic disc damage ( diffuse or localized rim thinning , enlarged cupping , disc hemorrhage , asymmetry in cup - to disc ( c / d ) ratio , 0.2 or greater between eyes ) with corresponding visual field loss on reliable automated perimetry ( at least 3 adjacent points in the expected location of the central 24 - field that have p < 5% on the pattern deviation plot , one of which with p < 1% ; glaucoma hemifield test outside normal limits ; pattern standard deviation [ psd ] with a p < 5% ) , and open angles on gonioscopy . a reliable perimetry was an exam with < 20% fixation loss , and < 33% of both false negative and false positive . oht was defined as any individual with iop higher than 21 mmhg with no glaucoma medication , healthy appearing optic disks and no visual field defect on automated perimetry . seventy eyes of 36 patients with poag or oht enrolled this cross - sectional study . fourteen patients were male ( 38.9% ) and 22 female ( 61.1% ) . as to ethnicity , six were african - brazilian , 10 patients were white , 19 were mixed , and one was asian . after explaining the procedures , all subjects signed an informed consent and underwent a complete eye examination with evaluation of the visual acuity , anterior segment biomicroscopy , tonometry with the goldmann tonometer ( haag - streit ag , switzerland ) after instillation of proparacaine and fluorescein drops , gonioscopy with a three mirror lens og3 m ( ocular instruments , bellevue , washington , usa ) , and optic disk assessment with 78 d volk lens ( volk optical inc . , mentor , oh , usa ) on a tropicamide dilated pupil . the iop was measured after discontinuation of all glaucoma medications for at least 21 days . the c / d ratio assessment was done by two observers , which in common agreement classified the optic disk according to a decimal system . standard automated perimetry ( sita standard 24 - 2 ) was done with the hfa 730 ( carl - zeiss humphrey , dublin , ca , usa ) with appropriate refractive correction . patients with unreliable exams were instructed to do the perimetry a second time in an attempt to get a reliable test , and those who remained with an unreliable test were not included in the study . cct was measured with ultrasonic pachimeter ( 300p pacscan , sonomed - escalon , wayne , pa , usa ) ; the probe was placed at the 5 mm central diameter of the cornea after instillation of proparacaine drops and three measurements where averaged to obtain one single value . pressure - to - cornea index was calculated as the ratio between untreated iop and cct to the power of three ( in mm ) for each subject as proposed by iliev et al . pearson 's product moment correlation coefficients with 95% confidence intervals ( 95% ci ) were calculated to quantify the linear relationship between the pci and a structural measure ( c / d ratio ) and the pci with two functional measures mean deviation ( md and psd values from automated perimetry ) . statistical significance was set at p < 0.05 , and all the analyses were done with medcalc software , version 9.3.7.0 ( medcalc software bvba , belgium ) . this cross - sectional study included patients with oht or primary open - angle glaucoma ( poag ) older than 40 years of age of both genders , with 20/20 best corrected visual acuity , and any ethnicity . subjects with cataracts or any other ocular disease , and previous incision or laser surgery for glaucoma were not included in the study . institution board review approved the study , and the procedures followed adhered to the principles for medical research involving human subjects of the declaration of helsinki in 1964 ( amended by the 59 wma general assembly , seoul , korea , october 2008 ) . in order to be included in the study , poag patients had typical optic disc damage ( diffuse or localized rim thinning , enlarged cupping , disc hemorrhage , asymmetry in cup - to disc ( c / d ) ratio , 0.2 or greater between eyes ) with corresponding visual field loss on reliable automated perimetry ( at least 3 adjacent points in the expected location of the central 24 - field that have p < 5% on the pattern deviation plot , one of which with p < 1% ; glaucoma hemifield test outside normal limits ; pattern standard deviation [ psd ] with a p < 5% ) , and open angles on gonioscopy . a reliable perimetry was an exam with < 20% fixation loss , and < 33% of both false negative and false positive . oht was defined as any individual with iop higher than 21 mmhg with no glaucoma medication , healthy appearing optic disks and no visual field defect on automated perimetry . seventy eyes of 36 patients with poag or oht enrolled this cross - sectional study . fourteen patients were male ( 38.9% ) and 22 female ( 61.1% ) . as to ethnicity , six were african - brazilian , 10 patients were white , 19 were mixed , and one was asian . after explaining the procedures , all subjects signed an informed consent and underwent a complete eye examination with evaluation of the visual acuity , anterior segment biomicroscopy , tonometry with the goldmann tonometer ( haag - streit ag , switzerland ) after instillation of proparacaine and fluorescein drops , gonioscopy with a three mirror lens og3 m ( ocular instruments , bellevue , washington , usa ) , and optic disk assessment with 78 d volk lens ( volk optical inc . , mentor , oh , usa ) on a tropicamide dilated pupil . the iop was measured after discontinuation of all glaucoma medications for at least 21 days . the c / d ratio assessment was done by two observers , which in common agreement classified the optic disk according to a decimal system . standard automated perimetry ( sita standard 24 - 2 ) was done with the hfa 730 ( carl - zeiss humphrey , dublin , ca , usa ) with appropriate refractive correction . patients with unreliable exams were instructed to do the perimetry a second time in an attempt to get a reliable test , and those who remained with an unreliable test were not included in the study . cct was measured with ultrasonic pachimeter ( 300p pacscan , sonomed - escalon , wayne , pa , usa ) ; the probe was placed at the 5 mm central diameter of the cornea after instillation of proparacaine drops and three measurements where averaged to obtain one single value . pressure - to - cornea index was calculated as the ratio between untreated iop and cct to the power of three ( in mm ) for each subject as proposed by iliev et al . pearson 's product moment correlation coefficients with 95% confidence intervals ( 95% ci ) were calculated to quantify the linear relationship between the pci and a structural measure ( c / d ratio ) and the pci with two functional measures mean deviation ( md and psd values from automated perimetry ) . statistical significance was set at p < 0.05 , and all the analyses were done with medcalc software , version 9.3.7.0 ( medcalc software bvba , belgium ) . the mean values of the md and psd from automated perimetry of the 70 eyes were - 9.9 7.6 db and 5.4 3.1 db , respectively . the mean ccc was 533.0 40.4 , mean iop was 19.7 5.1 mmhg , and mean pci was 132.9 51.4 . the pci showed a statistically significant negative correlation with md ( r = 0.356 , 95% ci , 0.549 to 0.126 ; p = 0.003 ) [ fig . 1 ] ; the correlation between pci and psd values of automated perimetry , however , failed to reach statistical significance ( r = 0.215 , 95% ci , 0.433 to 0.025 ; p = 0.07 ) [ fig . 2 ] . good correlation was found between pci and c / d ratio ( r = 0.329 , 95% ci , 0.09 to 0.526 ; p = 0.006 ) [ fig . 3 ] . correlation between the pressure - to - cornea index ( pci ) and mean deviation ( md ) correlation between the pressure - to - cornea index ( pci ) and pattern standard deviation ( psd ) correlation between the pressure - to - cornea index ( pci ) and cup - to - disk ratio ( cd ) in an attempt to integrate iop and cct into a unified risk factor , rather than simply attempting to correct for iop measurement inaccuracy , iliev et al . have proposed a new glaucoma index , the pci . the authors believed that pci could better reflect the individual susceptibility to glaucomatous damage than either iop alone or cct by itself . in a group of 220 normal controls , 53 patients with normal - tension glaucoma ( ntg ) , 76 with oht , and 89 with poag , the authors have assessed the ability of pci to discriminate between glaucoma ( ntg + poag ) , and non - glaucoma ( controls + oht ) and compared with that of three published formulae for correcting iop for cct . mean pci value for normal control was 92 24.8 and for glaucoma patients 173.6 40.9 . pci demonstrated a larger area under the receiver operating characteristic curve ( area under the curve [ auc ] ) and significantly higher sensitivity at fixed 80% and 90% specificities compared with each of the correction formulas . the authors proposed the range of 120140 as the upper limit of normality , and concluded that pci may reflect individual susceptibility to a given iop level , and thus represent a glaucoma risk factor . analyzed cct and iop in the cameroonian non - glaucomatous population and the pci values for this population was close to the cutoff proposed by iliev et al . to the best of our knowledge , this is the second study that evaluated the usefulness of pci in glaucoma patients . in our study , we have evaluated the utility of pci as an index of glaucoma severity , and our results have shown a correlation between the index and the md value of automated perimetry . visual field sensitivity is expressed on a logarithmic ( decibel ) scale ; hence , one might expect a curvilinear relationship between pci and md . the relationship between pci and md depends solely and directly from the formula that defines the relationship between them , and does not depend on the logarithmic nature of retinal sensitivity with respect to luminous magnitudes . the md value of automated perimetry is a weighted average decibel deviation from age normal database ; the lower the md value , the more damaged the visual function is . nevertheless , the md can be affected by media opacity such as cataract and uncorrected refractive error . in our study , only patients with 20/20 vision and no cataract were included , and the automated perimetry was done with the patient 's appropriate refractive correction , so that md values were very representative of glaucoma - related visual dysfunction . the psd value is the standard deviation of the difference between the threshold value at each test location and expected value and as an indicator of localized defects it reflects the roughness of the visual field . it is calculated by summing the absolute value of the difference between the threshold value for each point and the average visual field sensitivity at each point . as higher psd indicates more damaged visual fields , and assuming that psd has a positive correlation with pci , one would expect that the higher the pci value , the higher the psd . in this study , however , the correlation between pci and psd revealed a trend toward a negative correlation , not statistical significant , though . we are unsure if these results are due to the sample size or any selection bias . in general , automated perimetry , as a psychophysical test , is subject to patient cooperation and individual cognitive function causing imprecision of the measurements . we have tried to minimize this imprecision by selecting only automated perimetry exams with good reliable indices . our results revealed good linear correlation between the pci and the c / d ratio . the c / d ratio is a subjective , qualitative method to assess the optic nerve head in glaucoma patients . it is widely used in clinical practice , and it gives an appraisal of the cup diameter in relation to the optic disk size ; on a decimal scale , it ranges from zero ( no cupping ) to one ( optic nerve head completely excavated ) . however , it does not take into account localized defects of the neural rim , disk hemorrhages or the posterior bowing of the lamina cribrosa . besides , glaucoma patients with small optic discs will have proportionally small c / d ratios , giving a falsely impression of healthy looking optic disk . conversely , normal subjects with macrodisks will present with large c / d ratios giving a false impression of damaged optic disk . hence , the c / d ratio is not a precise surrogate of glaucomatous optic disc damage without consideration of the relative disc size , area , and the quantitative assessment of neural rim width and area . using this structural measure is a major shortcoming and quantitative measures of the optic disk structure as provided by new technologies should have been a better choice for correlation studies . another shortcoming of the study is the use of both eyes of the same individual . doing so for the measurement of an attribute or variable , rather than selecting one eye at random or the more severe affected eye for analysis tend to overestimate variability , artifactually influencing p value and decreasing chances of observing a significant effect , decreasing statistical power and increasing chances of type ii error . besides , we have not used any regression models to appropriately treat the eye as the unit of analysis , which tends to bias observations . however , incomplete data collection or selection of eyes for inclusion in a study on clinical grounds has the potential to introduce bias , and we decided to use both eyes of the same patient to avoid waste of data . the results of the study seem to dependent on a single outlier case with a pci value greater than 400 , which is above the maximum range described in a much larger population in the paper by iliev et al . this was a patient with untreated iop of 38 mmhg and cct of 0.46 mm . a post - hoc analysis eliminating this data point revealed that the value of r is reduced , but p still is significant ( except for pci and psd ) . however , we have decided to keep this patient in the study because an estimate indicated that increasing the number of cases could result in statistical significance . more recently , leung et al . have proposed a new pressure - cornea - vascular index ( pcvi ) . the index is derived from the pci and extended with risk factors identified as associated with field - progression in a prospective cohort of 415 patients with ntg followed for 3 years . the authors concluded that pcvi might be useful for predicting progression in ntg with a satisfactory auc comparable to established scoring systems in neurovascular medicine . in our study , we had not assessed any vascular parameters and did not evaluate the pcvi . in summary , the results of our study have revealed that pci has a correlation with both the md value of automated perimetry and the c / d ratio . these observations concur with the hypothesis that pci can be used to stage disease severity .

purpose : the pressure - to - cornea index ( pci ) was proposed in order to integrate intraocular pressure and central cornea thickness as a single - risk factor for glaucoma . the purpose of this study was to correlate the pci with a structural and two functional measures of glaucoma.setting:university hospital in south america.materials and methods : pressure - to - cornea index was calculated for 70 eyes of 36 subjects ( glaucoma and suspects ) . cup - to - disc ( c / d ) ratio , mean deviation ( md ) and pattern standard deviation ( psd ) as recorded by humphrey automated perimetry ( sita 24 - 2 ) were correlated with pci ( pearson 's correlation coefficient).results : pearson 's correlation coefficient between pci and c / d was 0.329 ( 95% confidence interval [ 95% ci ] , 0.090.526 ; p = 0.006 ) ; between pci and md was 0.356 md ( 95% ci , 0.549 to 0.126 ; p = 0.003 ) ; and between pci and psd was 0.215 ( 95% ci , 0.433 to 0.025 ; p = 0.07).conclusion : in addition to serve as a single - risk factor , pci can be used to stage glaucoma severity as well .

the pattern of drinking in india has undergone a change from occasional and ritualistic use to being a social event . these developments have raised concerns about the health and the social consequences of excessive drinking leading to dependence . in the last decade there is a rapid increase in the number of city bars and nightclubs in india and as a result an undocumented rise in alcohol abuse amongst all the sections of the society . the percentage of the drinking population aged less than 21 years has increased from 2% to more than 14% in the past 15 years , according to studies in the southern state of kerala by alcohol and drugs information center india , a non - governmental organization . what is of particular concern and an important indicator of health risks is that the signature pattern of alcohol consumption in india is frequent and heavy drinking . more than half of all drinkers fall into the criteria for hazardous drinking , which is characterized by bingeing and solitary consumption to the point of intoxication . the present study is carried out to look for the demographic factors associated with alcohol dependence syndrome so that the problems of alcohol related co morbidities can be prevented with appropriate preventive measures . the study was conducted in de - addiction clinic of the department of psychiatry , mamata medical college and associated mamata general hospital , khammam , andhra pradesh during the period from july 2008 to february 2009 . all the subjects fulfilling the inclusion and exclusion criteria during the study period were included in this study . all male subjects presenting to the de - addiction clinic of the mamata general hospital with alcohol related problems were potential candidates for this study and they are enrolled into the study if they fulfill the following inclusion criteria : patient of age 18 years and abovepatient who fulfills criteria for alcohol dependence , according to diagnostic and statistical manual of mental disorders , fourth edition ( dsm - iv ) . patient of age 18 years and above patient who fulfills criteria for alcohol dependence , according to diagnostic and statistical manual of mental disorders , fourth edition ( dsm - iv ) . subjects with any of the following will not be included in the study : concurrent presence of other substance dependence , according to dsm - iv , other than nicotinepresence of any significant illness requiring intensive medical / surgical management . concurrent presence of other substance dependence , according to dsm - iv , other than nicotine presence of any significant illness requiring intensive medical / surgical management . patients fulfilling the selection criteria were admitted after obtaining informed consent and demographic details , history , general physical examination and mental status examination were recorded on a semi - structured proforma designed for the study . all male subjects presenting to the de - addiction clinic of the mamata general hospital with alcohol related problems were potential candidates for this study and they are enrolled into the study if they fulfill the following inclusion criteria : patient of age 18 years and abovepatient who fulfills criteria for alcohol dependence , according to diagnostic and statistical manual of mental disorders , fourth edition ( dsm - iv ) . patient of age 18 years and above patient who fulfills criteria for alcohol dependence , according to diagnostic and statistical manual of mental disorders , fourth edition ( dsm - iv ) . subjects with any of the following will not be included in the study : concurrent presence of other substance dependence , according to dsm - iv , other than nicotinepresence of any significant illness requiring intensive medical / surgical management . concurrent presence of other substance dependence , according to dsm - iv , other than nicotine presence of any significant illness requiring intensive medical / surgical management . patients fulfilling the selection criteria were admitted after obtaining informed consent and demographic details , history , general physical examination and mental status examination were recorded on a semi - structured proforma designed for the study . a total of 68 male patients were registered at the de - addiction clinic , at the department of psychiatry , mmc and mamata general hospital , khammam between the period july 1 , 2008 and february 28 2009 . whereas a total of 63 patients met the dsm - iv criteria for alcohol dependence , 7 were excluded from the study as they met criteria for another drug dependence . the remaining 56 patients were each offered admission for further management , but 16 patients refused / did not come for admission . the mean age at presentation in the study was 37.2 ( 8.51 ) years . mean age at presentation in various studies with similar design this study found that 62% of patients were married at time of presentation [ figure 1 ] . in most western studies , the marital status of the patient has been most commonly found to be being separated or divorced , ranging between 43% and 60% respectively . one large study in 2713 alcohol dependent patients however , reports higher rate of married patients than controls ( 57% ) . the differences are possibly due to cultural differences . sample in the current study came from a predominantly rural population , mostly from nuclear families . this reflects the population being catered to by the hospital where this study took place ; an urban center with huge rural catchment area 32 ( 80% ) [ table 1 ] patients had education less than high school level . most of the studies have shown that most of the patients in similar clinical settings had education less than high school [ table 2 ] . showing the marital status of the individuals type and background of family the educational status of the individuals rate for various forms of employment was 85% , involving mostly unskilled or skilled work . previous studies have reported an equal percentage of patients who are unemployed or are gainfully employed . the current study had higher rates of patients who were employed , but mostly involved in work that did not require much education and hence , comparison becomes difficult [ table 3 ] . the majority of patients in the study belonged to the lower middle and middle socio - economic class . other studies too have reported their patient population as belonging to lower middle to lower class [ table 4 ] . mean age at which first drink of alcohol ( in any form or amount ) was taken is 18.9 years [ table 5 ] . the mean age at getting intoxicated by the alcohol for the first time ( i.e. , feeling the effects of alcohol , or taking an equivalent of 80 - 90 ml or more of alcohol in one setting or within a brief period of approximately 1 - 2 h ) was 19.8 years . a study among in - patients reported the mean age at first consumption to be 15.4 years and at regular consumption to be 23.1 years . the collaborative study of the genetics alcoholism ( coga ) group found the mean age at onset of alcoholism was 25 years [ table 5 ] . the mean age at onset alcohol abuse was 24 years and at onset of alcohol dependence ( according to dsm - iv ) was 28.3 years . a study by powell et al . , had found mean age at onset of drinking was 17.3 years , while age at problem - level drinking was 30.4 years . majority of patients had presence of withdrawal symptoms ( 95% ) and tolerance ( 95.5% ) [ table 6 ] . dsm - iv indicates that diagnoses of substance dependence should be further characterized with regard to the presence of a physiological component . coga study found that such a distinction predicated more physiological complications and more alcohol - related emotional / psychiatric symptoms such as depression and anxiety . withdrawal symptoms and tolerance in this study , the patients and key informants were asked to enumerate reasons for seeking current treatment [ tables 7 and 8 ] . based on the compilation of various reasons , these could be grouped into certain categories . on analysis , financial strain due to alcohol use was most commonly attributed for current treatment seeking by both patients ( 70% ) and key informants ( 92.5% ) , though the patients mostly denied it as their first reason . this was followed by presence of family conflicts and concern about physical health ( 65% each ) , followed by seeking treatment due to social pressure ( 35% ) , experiencing withdrawal symptoms ( 25% ) , or psychological reasons ( 22.5% feelings of guilt , low mood , etc . ) . most of the patients had more than 2 reasons for seeking treatment and this was comparable to reasons provided by the key informats . this was a unique effort in this study to incorporate such clinical parameters into the assessment , as it seems to yield similar severity status . studies reported similarly reasons for admission to current treatment in which decision by the patient was the most common reason ( 73 - 76% of patients ) , while other reasons included medical condition ( 12% ) and employer pressure ( 8% ) but stopped short of further analysis . reasons for seeking treatment informants reasons for seeking treatment mean age at first drink was 18.9 years and age at onset of alcohol dependence was 28.3 years . the alcohol dependence syndrome was more common in low socio - economic class and people with education up to high school level . measures to improve the education and socio - economic standards in the rural areas are urgently needed to control the alcohol abuse and related co morbidities .

aims : to study the demographic factors associated with alcohol dependence syndrome so that the problems of alcohol related co morbidities can be prevented with appropriate preventive measures.materials and methods : the study was conducted in de - addiction clinic of the department of psychiatry , mamata medical college , khammam , andhra pradesh from july 2008 to february 2009 . patient who fulfills criteria for alcohol dependence , according to diagnostic and statistical manual of mental disorders , fourth edition were included.results:mean age ( standard deviation ) at first drink was 18.93 ( 3.81 ) years and at onset of alcohol dependence was 28.28 ( 6.55 ) years . the most common reason being given by the patients was financial strain ( 70% of the patients ) due to alcohol use and its consequences . educational qualification of 12th standard or above was seen only in 7.5% . alcohol dependence syndrome was more common in unemployed , unskilled and semi - skilled patients . majority of patients ( 80% ) belonged to lower socio - economic class.conclusion:alcohol dependence syndrome and its related co morbidities can be minimized to a great extent if the educational and socio - economic standards are improved in countries like india where there is increase in alcohol consumption as a life style choice .

high - risk human papillomavirus ( hr - hpv ) infection is the most common sexually transmitted infection ( sti ) among young adult women and plays a critical role in the development of genital cancer . recently , 15 hr oncological viral strains have been identified , including the hpv 16 group ( alpha-9 ) of the alpha - papillomavirus genus ( hpv 31 , hpv33 , hpv 35 , hpv 52 , and hpv 58 ) and the hpv 18 group ( alpha-7 ; hpv39 , hpv 45 , hpv 59 , and hpv 68 ) [ 1 , 2 ] . biological susceptibility to hr - hpv acquisition and reduced immune competence for clearance of hr - hpv infection could result from common treatable vaginal infections that disrupt the intricately balanced vaginal ecosystem and its innate protective mechanisms against infection and disease . earlier reports have suggested a link between genital tract inflammation and cervical cancer , only a few studies have controlled for hr - hpv genotypes , and no study has examined this infection in female sex workers ( fsws ) . the association between self - reported abnormal vaginal discharge and cervical intraepithelial neoplasia ( cin ) in hr - hpv - infected women further suggests a link between genital tract anomalies and cervical cancer [ 4 , 5 ] . bacterial vaginosis ( bv ) and trichomoniasis are associated with high levels of anaerobic microorganisms and their byproducts , which can damage the vaginal epithelium , degrade cervical mucus , and cleave immunoglobulin a [ 68 ] , and the evidence suggests an important association between hr - hpv and alterations in the vaginal microbiome [ 9 , 10 ] . bv and trichomoniasis are categorized , along with vulvovaginal candidiasis , under the slight misnomer of vaginitis . candida albicans is the most prevalent species in the majority of cases of asymptomatic colonization and vulvovaginal candidiasis . however , certain species of candida are more pathogenic than others , induce hyphal and pseudohyphal formation , and enhance proteolytic activity and antigen modulation . these properties enable candida to penetrate the mucosal surface and induce mucosal swelling , erythema , and exfoliation of cells . in addition , some studies have reported that cervical cytologic abnormalities occur more often in women who have abnormal vaginal microbiota than in women without this condition . are more prone to acquiring cervical cytologic abnormalities over time than women who have a known , disturbed , bacterial vaginal microbiota [ 9 , 10 ] . therefore , the purpose of the current study was to determine the prevalence of vulvovaginitis caused by trichomonas vaginalis , candida spp . , or bv in a group of fsws with hr - hpv infection . approval for the study was obtained from the ethics committee of galician ( spain ) human research . hiv - negative fsws ( age , 1849 years ) who visited an sti information and prevention center in the north - eastern region of spain between january 2010 and december 2011 were included in the study . the sample size was calculated to allow for a prevalence of 50% or higher for hr - hpv , with a confidence interval of 95% and an error of 6.5% . the test was used for statistical analysis , while the odds ratio ( or ) was used to measure the strength of the association between vaginal infections and high - risk human papillomavirus types . logistic regression analysis was used to assess the simultaneous effect of more than 1 variable on the risk of hpv infection and to identify possible confounding factors . we determined the or by using nonconditional logistic regression but using the correct definition ; we could not estimate the prevalence ratio ( pr ) because we did not have any patients who were not exposed to the infection . the software used for the statistical analysis was the statistical package for the social sciences ( spss ) , version 18.0.1 . cervical samples were collected with a cervical brush , and all cervical swabs were placed a 1 ml a tube containing of the digene specimen transport medium ( digene corporation , gaithersburg , md , usa ) and were stored at 20c until testing . the presence of hr - hpv infection was determined through the digene hr - hpv test hybrid capture ii ( hc2)by using the hr - hpv probe b cocktail , which identified types 16 , 18 , 31 , 33 , 35 , 39 , 45 , 51 , 52 , 56 , 58 , 59 , and 68 , in accordance with the manufacturer 's instructions . to minimize the cost of testing , an hc2 hpv dna test was performed for all samples with only the high - risk hpv probe mixture . positive specimens were detected by binding of hybridization complexes onto the surface of the microplate wells coated with monoclonal antibodies specific to rna - dna hybrids . immobilized hybrids were detected by the addition of an alkaline phosphatase - conjugated antibody to rna - dna hybrids , followed by the addition of a chemiluminescent substrate . the presence of bv was evaluated microscopically in samples collected from the posterior vaginal fornix . bv was diagnosed on the basis of the presence of 3 of the following clinical and microscopic findings : vaginal ph greater than 4.5 , presence of clue cells , grey homogenous vaginal discharge , and positive whiff test , in which a fishy odor is released after the addition of 10% potassium hydroxide solution to the vaginal fluid . the vaginal swab was placed in 0.2 ml of sterile physiologic saline for wet mount evaluation and examined microscopically ( 400x ) for motile t. vaginalis within 15 min . sample collection was performed using calcium alginate swabs , which were transported in the stuart amies transport medium ( copan innovation ) , kept at room temperature , and sent to the laboratory within 6 h of collection for culture on roiron medium ( maim s.l . ) . t. vaginalis was first detected by direct microscopic visualization of the fresh sample , followed by culture of the vaginal discharge on the roiron medium and aerobic incubation at 37c . the broth was examined microscopically for motile trichomonads on days 1 , 3 , and 5 of culture . samples were collected using calcium alginate swabs and transported in the stuart amies transport medium . the samples were kept in a t environment and then sent to the laboratory within 6 h , as recommended by the manufacturer . confirmatory microbiological culture of vaginal discharge was carried out in sabouraud chromogenic medium ( candida id2 ( can2 ) ; biomrieux ) in the case of women for whom a positive diagnosis was suspected but for whom a negative result was obtained by direct visualization . for samples that did not test positive following microbiological culture , complementary diagnosis analysis using the vitek id - yst system ( biomrieux ) was also performed . this cross - sectional study involved fsws who visited an sti information and prevention center in vigo , spain , between january 2010 and december 2011 . all fsws who visited the center were invited to participate in the study , and the refusal rate was very low . finally , 208 hiv - negative fsws aged 1849 years were involved in this study . the mean age of the study population was 27 years , whilst 60% of participants belonged to the target age group of 1825 years . hr - hpv prevalence peaked in younger women aged 1825 years . as expected , the prevalence of t. vaginalis in fsws was high ( 17% ) . hr - hpv infection was present in 7.535.5% of these women , and the peak prevalence of this infection occurred 20 years earlier than that for t. vaginalis . data for hr - hpv - positive samples were grouped as belonging to hpv16-related genotypes ( 31 , 33 , 35 , 52 , and 58 ) , hpv18-related genotypes ( 39 , 45 , 59 , and 68 ) , or other genotypes , including hpv51 and hpv66 . in the study group of 208 fsws , 81 women had positive results for hpv16 infection and 48 for hpv18 infection . data from univariate analysis showed that bv was strongly associated with hr - hpv , as was the presence of t. vaginalis . we found no relationship between candida spp . and hr - hpv , with the exception of hpv18 ( table 2 ) . among women with vaginal t. vaginalis , candida spp . was associated with hpv16 infection ( or = 0.10 , 95% ci : 0.0020.048 ) . moreover , among women with bv , vaginal candida spp.colonization was associated with hpv18 infections ( mantel and haenszel or = 0.030 , 95% ci : 0.0110.083 ) . when both vaginal candida spp . and t. vaginalis were removed from the model , bv was still significantly associated with hr - hpv in all the fsws . in contrast , when bv was removed from the model , vaginal candida spp.colonization was not associated with hr - hpv , except for hpv18 ( or = 0.448 , 95% ci : 0.2320.866 ) . the presence of vaginal candida spp.colonization alone was not significantly associated with all the hr - hpv genotypes , except for hpv18 ( or = 0.448 , 95% ci : 0.2320.866 ) . however , colonization was present concurrently with bv or trichomoniasis , it negatively affected the outcomes for certain hpv types . the distribution of bv in women with hr - hpv was statistically significant in hpv16 and hpv16-related genotypes ( hpv31 , 33 , 35 , and 52 ) . thus , we expect that , among the 16 hr - hpv - negative participants , 88% did not have bv . this equates to 66% of hpv31-negative , 82% of hpv33-negative , 75% of hpv35-negative , and 72% of hpv52-negative participants . in contrast , the presence of t. vaginalis was statistically significant in women with hpv18 , hpv45 , hpv66 , or hpv68 ; the presence of candida spp . this result was further associated with both candida spp . and t. vaginalis ( table 3 ) . according to cheng et al . and muoz et al . , hpv types may be organized according to ( 1 ) hpv16-related types ( 31 , 33 , 35 , 52 , and 58 ) or hpv types belonging to the alpha - papillomavirus-9 species , ( 2 ) hpv18-related types ( 39 , 45 , 59 , 68 ) or hpv types belonging to the alpha - papillomavirus-7 species , or ( 3 ) others , including hpv51 and hpv66 . it is characterized by replacement of lactobacilli by a mixture of anaerobic and aerobic bacteria ( common agents include gardnerella vaginalis , mycoplasma hominis , and ureaplasma urealyticum ) . in women without bv , hydrogen peroxide - producing lactobacilli dominate vaginal microbiota . the microorganisms responsible for bv increase the levels of mucin - degrading enzymes , which may play a role in the degradation of the gel layer that coats the vaginal and cervical epithelium and endocervical mucus [ 19 , 20 ] . bv - associated microorganisms also produce cytokines and inflammatory mediators , which have been linked to pregnancy complications and may increase susceptibility to stis . such enzymes may promote virulence by destroying the protective mucosal barrier and may hence increase susceptibility to cervical hr - hpv infection by facilitating adherence , invasion , and , eventually , incorporation of hpv - activated oncogenes into the genome of cells in the transformation zone . abnormal vaginal microbiota may also be implicated in the maintenance of subclinical hr - hpv infection . enzymes that are produced by anaerobic bacteria and are involved in the pathogenesis of bv can potentially alter immune signals and promote degradation of protective host factors , rendering women more susceptible to acquiring hr - hpv [ 2225 ] . however , it is still not known whether bv and cervical hr - hpv infections are related because there is a biological symbiotic relationship between them or because both occur frequently in sexually active women . the role of sexual transmission in causing or promoting bv continues to be a topic of debate , as studies have shown that lesbians have a high prevalence of bv . infection is the most prevalent mycosis in the majority of cases of asymptomatic colonization and vulvovaginal candidiasis . however , certain species of candida are more pathogenic than the other species and induce hyphal and pseudohyphal formation and enhance proteolytic activity and antigen modulation . these features enable candida spp . to penetrate the mucosal surface and induce mucosal swelling , erythema , and cell exfoliation . the role of t. vaginalis and candida spp . in hr - hpv pathogenesis has been evaluated by some authors , including engberts et al . who established that the presence of candida spp . is not associated with an increased risk of acquiring hr - hpv . in a study involving cytological analysis of samples obtained from 17,391 outpatients during january 1997august 1998 in brazil , 390 samples ( 2.24% ) had alterations that were consistent with hpv infection and were sometimes associated with cin i. the results showed that g. vaginalis was the most frequent bacterial agent found in women with hr - hpv infection ( 23.6% versus 17.4% ; p < 0.05 ) , while in the control group the most frequent agent was candida spp . this result is in accordance with the findings of wang and lin and chakrabarti et al . , who also reported that there was no obvious association between the presence of candida spp . and cytologic changes in the vagina is more frequently found in women without cervical intraepithelial neoplasia ( cin ) . as reported by watts et al . in contrast to the finding for bv and t. vaginalis , it appears that the presence of candida spp . in the vagina this is consistent with the hypothesis that the local cervicovaginal milieu plays a role in susceptibility to hr - hpv infection since women who carry candida spp . are likely to possess a healthy lactobacillus - predominated vaginal microbiota , in contrast to women with dysbacteriosis . only a limited number of studies have recognized an association between the presence of t. vaginalis and cin or an association between t. vaginalis and hr - hpv infection . these findings may be explained by common etiological factors and , in particular , by the number of sex partners , which is naturally difficult to prove with a high degree of certainty . one hypothesis is that the parasite can act as a potential catalyst in the development of secondary infections , including hr - hpv , by producing a wide array of enzymes such as cysteine proteinase enzymes that are linked with cytotoxicity and the degradation of basement membrane components . furthermore , some studies have shown that the double - stranded rna of t. vaginalis is also associated with differential expression of enzymes and may therefore affect virulence [ 3539 ] . therefore , it is also possible that t. vaginalis could alter the natural history of various sexually transmitted diseases and , in particular , hr - hpv , by increasing virulence . this study clearly shows that women with positive cytological results for t. vaginalis are at significant risk of acquiring hr - hpv infection [ 40 , 41 ] . in addition to the conventional stis caused by neisseria gonorrhoeae , chlamydia trachomatis , and t. vaginalis , hr - hpv infection has been found to be common ( 36% ) , and oncogenic genotypes have been found in 44% of hr - hpv - positive patients . in another study , of these 80 patients , 57 were available for hr - hpv testing ( age : 8 patients < 30 years , 42 patients 3050 years , and 7 patients 50 years ) . as controls , high - risk hr - hpv was tested for in 150 patients with normal cytological features , from each of these 3 age categories . women with t. vaginalis had a significantly higher prevalence of hr - hpv than women with normal results for cervical smear tests , irrespective of age ( p < 0.01 ) . nol stated that these data suggest a potential association between these 2 infectious agents because of sexual intercourse and probably because of biochemical or immunological reasons . concluded that the prevalence of t. vaginalis in flanders is low , with only 3.7/1,000 women infected . hr - hpv is present in 15% of these women and the peak of its prevalence occurs 20 years earlier than that of detection of t. vaginalis . the present study design does not allow for a definitive answer for this paradoxical increase in t. vaginalis prevalence in the absence of hr - hpv in the age group of 4050 years . in our study , bv and t. vaginalis infection was common , with oncogenic genotypes ( hpv18 and hpv18-related types 59 and 68 ) being present in 61% , 45.5% , and 69% of positive samples . these findings emphasize the importance of a cervical cancer prevention strategy , such as a cervical cytology screening program for hr - hpv , which is to be introduced soon . this study suggests a positive association between bv and hr - hpvs ( hpv 16 , 31 , 33 , 35 , and 52 ) . presence of t. vaginalis was statistically significant , in women with hpv 18 , 45 , 66 , and 68 . were not significantly associated with hr - hpv ; they were more frequently among hr - hpv - negative women . these data confirm that screening for genital infections may reveal the simultaneous presence of different sexually transmitted microorganisms . these results suggest and emphasize the value of screening for genital infections in hr - hpv - positive patients in order to decrease the presence of other microorganisms and to reduce the probable synergistic effects of the vaginal microbiota . prevention is important not only to avoid other stis and their sequelae but also to reduce the influence of concomitant infection with other microorganisms on hr - hpv infection .

background . infection with and persistence of high - risk human papillomavirus ( hr - hpv ) are the strongest risk factors for cervical cancer . in addition , other genital microorganisms may also be involved in the progression of hpv - associated lesions . objetive . to evaluate the association of the vaginal microbiota ( candida spp . , trichomonas vaginalis , and bacterial vaginosis ) with hr - hpv infection in spanish female sex workers ( fsws ) . methods . this cross - sectional study involved 208 ( fsws ; age , 1849 years ) who visited a sexually transmitted infection ( sti ) information and prevention center ( sergas ) between january 2010 and december 2011 . face - to - face interviews were carried out . cervical and vaginal samples were examined for human papillomavirus ( hpv ) , trichomonas vaginalis , candida spp . , and microorganisms related to bacterial vaginosis ( bv ) . results . hr - hpv was found to be significantly associated with bv in fsws with positive results for hpv16-related types ( 31 , 33 , 35 , and 52 ) . t. vaginalis was isolated in fsws with the following hr - hpvs : 18 , 45 , 66 , and 68 . candida spp . were isolated only in fsws with hpv 18-positive infection . conclusion . we demonstrate a significant prevalence of hr - hpvs in fsws with disturbances in the vaginal microbiota .

we defined a notified case as any patient whose illness was considered compatible with denv by a health professional ; samples from these cases were sent to provincial laboratories or directly to the national reference laboratory . case definitions used for probable and confirmed cases of dengue fever , dengue hemorrhagic fever , and dengue shock syndrome were those proposed by paho ( 4 ) . because denv transmission was not endemic in argentina , laboratories checked continuously for the further introduction of the virus . because the introduction of denv was relatively recent , the total population infected relatively small , and no continuous transmission was detected , all positive serologic samples obtained at the provincial levels were sent to inevh to be confirmed by other techniques . if a bigger , confirmed outbreak occurs in the future , we anticipate that a small number of positive samples would be evaluated at inevh . during interepidemic periods , we conducted laboratory surveillance of febrile syndromes of undetermined etiology . samples obtained from our surveillance program for diseases that are clinically similar to denv ( e.g. , rubella , measles , argentine hemorrhagic fever , leptospirosis and other illnesses fulfilling criteria for viral hemorrhagic fevers ) were also tested for denv . local - level physicians in argentina were familiarized with the case definitions for denv syndromes from published guidelines ( 4 ) . after the discovery of the first occurrence of the virus and the organization of the national denv surveillance system , the training of local healthcare personnel , including physicians and epidemiologists , was intensified through periodic courses , meetings , and seminars . in small localities , such as those in the north of the country , only a few hospitals exist in each locality , and most of them are government - owned ; therefore , all personnel were informed and were willing to participate . in bigger provinces , such as buenos aires , the healthcare system is organized into regions , each of which has several public hospitals , but all of these hospitals are supported by a provincial department of epidemiology . the laboratory method used in argentina has been described in previous reports ( 2,3,7,8 ) . briefly , serum samples taken from patients with suspected denv infection 5 days after onset of symptoms ( late acute ) were tested weekly during the summer and fall and monthly during the rest of the year at provincial laboratories . the laboratories used a commercial kit ( ultramicro enzyme - linked immunosorbent assay capture immunoglobulin [ ig ] m dengue test , instituto pedro kouri , havana , cuba ) or igm antibody capture enzyme - linked immunosorbent assay ( mac - elisa ) . all positive samples and at least 10% of negative samples were sent to inevh for quality control and confirmation . when the surveillance system was first initiated , all negative samples were tested at the national reference center . at inevh , samples were tested by mac - elisa , plaque reduction neutralization test ( prnt ) , hemagglutination inhibition ( hi ) test , viral isolation in c6/36 cells , and immunofluorescence detection ( using monoclonal antibodies obtained from paho ) , or by polymerase chain reaction ( pcr ) techniques , according to the date of onset . in addition , the capsid / premembrane and envelope / nonstructural protein 1 regions of viral isolates were sequenced to determine the genotypes . acute - phase serum samples ( < 5 days after onset of symptoms ) were sent directly to inevh and assayed for viral isolation or pcr ; samples > 5 days of onset were tested by mac - elisa for igm antibodies . during the convalescent - phase period , a second sample was obtained from patients with positive results for denv by mac - elisa and from most patients with acute - phase samples ( with results either positive or negative by viral isolation or pcr ) ; these samples were tested along with the acute - phase samples by using prnt or hi tests . serial dilutions of each patient s serum were tested against the four denv serotypes for 80% identity by prnt or hi tests . the denv-1 haw , denv-2 ngc , denv-3 h87 , and denv-4 h241 strains were obtained from the dengue branch , centers for disease control and prevention , san juan , puerto rico . antigens from mice brains infected with each denv were prepared at inevh by using the sucrose - acetone method and used in mac - elisa and hi tests . according to the igg titers by prnt or hi tests primary cases were indicated by titers < 160 in the acute - phase sample ( < 5 days after onset of symptoms ) , titers < 1,280 in the late acute- or convalescent - phase sample , and low or null cross - reactivity among the different denv serotypes . secondary cases were indicated by titers > 160 in the acute - phase sample , titers > 2,560 in the late acute- or convalescent - phase sample , and high cross - reactivity among the different denv serotypes . a laboratory network including all provinces at risk for denv ( according to ae . the national reference laboratory , which is self - sufficient for production of key reagents ( such as antigens and antiserum ) , participates in the proficiency tests organized under paho / who and maintains country - proficiency tests on a continuing basis . commercial kits were evaluated at the national reference center before being used in national programs . louis encephalitis , west nile virus , and other flaviviruses were also incorporated into denv diagnostic protocols . dengue laboratory network , argentina . thirty laboratories were designated by the national ministry of health , the provincial ministries of health , and the local municipalities to integrate the laboratory network ( figure 1 ) . staff persons from 15 of those regional laboratories were trained on denv diagnosis at inevh , and 12 were actively working on denv serologic surveillance . courses and rotations were part of the training ongoing since the network of laboratories was organized . the remaining laboratories had difficulties in obtaining reagents , keeping staff because of lack of funding , and other operational problems , so they could not sustain the work through the entire surveillance period . training was emphasized , and inevh focused on the problems of these laboratories to maintain high quality control . staff from all 30 laboratories attended annual meetings at which the results and problems concerning the organization of the laboratory , clinical , and epidemiologic denv surveillance were discussed . laboratories sent the samples directly to inevh when they were unable to carry out sample testing on their own . during the epidemiologic surveillance of cases compatible with denv from december 1995 to december 2001 , our laboratory received 493 serum samples from travelers returning to argentina with suspected denv ( table 1 ) and from case - patients with no epidemiologic data . cases were classified as imported or indigenous as a result of epidemiologic analysis , considering travel histories in the 3 weeks before onset of illness . of 226 positive case - patients , 150 reported travel histories to paraguay ( 127 cases ) , brazil ( 11 cases ) , honduras ( 3 cases ) , venezuela ( 3 cases ) , ecuador ( 1 case ) , mexico ( 2 cases ) , dominican republic ( 1 case ) , puerto rico ( 1 case ) , and the virgin islands ( 1 case ) . seven other cases were imported to other provinces inside the country during the denv outbreaks of 1998 and 2000 . no epidemiologic data were available for the remaining 69 cases . during the denv outbreak in salta province in 1998 ( 4 ) , we could not determine whether infection occurred in bolivia or argentina , so these cases were considered as probably imported but were included in the total number of cases ( 378 ) for the outbreak ( table 2 ) . imported cases were detected in different provinces throughout the country in different years : salta ( 1996 , 2000 ) , buenos aires ( 19972000 ) , santa fe ( 1997 , 1999 , 2000 ) , misiones ( 1998 , 2000 ) , jujuy ( 1998 , 2000 ) , cordoba ( 1998 , 2000 ) , rio negro ( 1998 ) , tucuman ( 1999 ) , chaco ( 2000 ) , corrientes ( 2000 ) , and formosa ( 2000 ) ; these cases involved either denv-1 , denv-2 , or denv-3 serotypes ( table 1 ) . denv , dengue fever virus ; mac - elisa , immunoglobulin m antibody capture enzyme - linked immunosorbent assay ; prnt , plaque reduction neutralization test ; hi , hemagglutination inhibition test ; nd , not done . the serotype was determined only by serologic testing unless we indicate otherwise that virus isolation or polymerase chain reaction was also used . in many of these cases , the serotype could not be determined because of cross - reactions in serologic tests . some samples were tested by one technique , and others were tested by two or more techniques ; therefore , these numbers do not represent the total obtained by adding the rows . denv , dengue fever virus ; mac - elisa , immunoglobulin m antibody capture enzyme - linked immunosorbent assay ; prnt , plaque reduction neutralization test ; hi , hemagglutination inhibition test ; nd , not done . some samples were tested by only one technique , others were tested by two or more techniques ; thus , these numbers do not represent the total obtained by adding the rows . from january 1997 to december 2001 , a total of 2,987 serum samples were tested for suspected denv from provinces in the subtropical ( north ) and the temperate ( central ) areas of the country ; 696 samples were positive ( table 2 , figure 2 ) . of these cases , 378 occurred during an outbreak in salta province that occurred from january 3 to may 31 , 1998 , and was caused by denv-2 ( 3,7 ) ( table 2 , figure 2 ) . during the salta outbreak , prevalence in adults 1579 years of age ( 82.5% ) , was higher compared to that in children < 14 years of age ( 17.5% ) . because most samples came from adults , the disease may have been subclinical or undetected in most children . the most affected locality in this region was tartagal ( 22s , 63w ) , which had an incidence rate of 67.5 cases per 10,000 inhabitants . serologic testing by prnt or hi for all four denv serotypes in 154 cases from this outbreak resulted in 38 ( 24.7% ) that showed primary responses and 84 ( 54.5% ) that showed secondary responses . the secondary cases were considered to be indigenous because none of these patients had traveled in the previous 3 weeks . we did not gather any information about traveling before those 3 weeks , so whether the patients were infected by another flavivirus in argentina or in any other country before that is unknown . the remaining 32 cases ( 20.7% ) with borderline igg titers could not be classified . louis encephalitis or yellow fever likely due to vaccination ) explained 83% of the secondary serologic patterns ; the rest remained unexplained , suggesting the unrecognized occurrence of previous infection with other denv serotypes or flaviviruses other than st . louis encephalitis or yellow fever ( 7 ) . the second more important cluster of denv cases occurred during a denv-1 outbreak in 2000 that affected misiones , formosa , and jujuy provinces ( table 2 , figure 2 ) , causing 294 cases from february 15 to april 22 . most affected localities were clorinda ( formosa province , 25s , 57w ) , with an incidence of 12.3 cases per 10,000 inhabitants and iguazu ( misiones province , 25s , 54w ) , with an incidence of 51.7 cases per 10,000 inhabitants . denv-2 isolated in salta in 1997 and 1998 showed 98% to 99% nucleotide identity with denv-2 isolated from brazil in 1990 and jamaica in 1983 ( avils et al . denv-1 isolated in 2000 from misiones , formosa , and jujuy provinces showed 95% identity with a denv-1 strain isolated from french guiana in 1989 and 91% identity with a denv-1 strain isolated from the caribbean ( jamaica ) in 1977 ( 8) . laboratory surveillance in argentina enabled detection of an early circulation of denv in 1997 and confirmation of two denv outbreaks that occurred from 1998 to 2000 . this surveillance system identified 922 laboratory - diagnosed cases of denv ( 696 indigenous , 157 imported , and 69 unknown ) during the period of study . we obtained 38 denv-1 , 6 denv-2 , and 1 denv-3 isolates or pcr amplicons . imported cases were detected in a wide subtropical and temperate area of the country , while indigenous circulation of denv occurred only in the subtropical area of the country . the first outbreak caused by denv-2 occurred in northwestern argentina in 1998 ; the second outbreak caused by denv-1 occurred in the north - northeastern part of the country in 2000 . a serosurvey conducted in bolivia in 1997 in a neighborhood of santa cruz city ( 10,000 inhabitants ) showed that > 5% of the populace was infected by denv-2 ( 5 ) . paraguay s ministry of health reported an infection rate of 49.3 cases per 10,000 inhabitants ( including laboratory - diagnosed and reported cases ) during a denv-1 outbreak in 1999 and 2000 ( 6 ) . brazil reported incidence rates in different municipalities in 2000 that varied from 0.037 to 866.9 cases per 10,000 inhabitants ( 9 ) . the geographic and climatologic situation in argentina is favorable because most of the country has a temperate climate . nevertheless , the introduction of different denv serotypes increases the risk for dengue hemorrhagic fever because of the increased number of secondary denv infections . the sequence of serotypes in secondary infections seems to be an important risk factor for dengue hemorrhagic fever because denv-2 secondary infections account for most of those cases . viral virulence related to specific genotypes all denv cases that have occurred in argentina to date have been considered clinically to be dengue fever . denv-1 that circulated in argentina during the period of study belongs to an american genotype closely related to viruses circulating previously in other american countries that have been associated only with mild disease ( 8) . denv-2 found in argentina belongs to an asian genotype that has been associated with severe disease ( 11 ) . in argentina , dengue hemorrhagic fever has not been reported , possibly because the size of the affected population was small and this complication occurs in a small proportion of secondary denv infections ( 18125/1,000 ) ( 10 ) , and in an even smaller proportion of primary infections .

local transmission of dengue fever virus in argentina is increased by the presence of aedes aegypti mosquitoes and dengue outbreaks in neighboring countries . from 1995 to 2001 , a laboratory - based active surveillance program detected 922 dengue cases . indigenous transmission involving dengue-1 and -2 serotypes was confirmed only in subtropical areas in northern argentina .

fe(ii)-oxidizing bacteria ( feob ) gain energy from the chemical oxidation of fe(ii ) coupled to reduction of oxygen or nitrate or using light energy coupled to reduction of co2 , e.g. , anoxygenic photosynthesis . at the near neutral ph of many surface waters , the oxidation of fe(ii ) for that reason , cyanobacteria , which generate oxygen as a result of oxygenic photosynthesis , can act as indirect fe(ii)-oxidizing bacteria where anoxic and fe(ii)-containing waters encounter to sunlit surface environments . the contribution of cyanobacteria to fe(ii ) oxidation has been quantitatively addressed in fe(ii)-rich hot spring environments and in benthic photosynthetic communities living at the sediment water interface . although the modern oceans are predominantly oxygenated to great depths , promoting the speciation of iron as ferric [ fe(iii ) ] rather than ferrous [ fe(ii ) ] , fe(ii ) may be increasingly mobilized out of sediments and stabilized in the marine water column due to expanding low - oxygen conditions in so - called oxygen minimum zones ( omz ) . where omz intersect with the photic zone , fe(ii ) oxidation by planktonic oxygen - producing cyanobacteria could contribute to the marine iron cycle . furthermore , anoxic and fe(ii)-rich bottom waters are a pervasive feature of oceans in the precambrian era [ before about 500 million years ( my ) ago ] at a time when oxygen was building up in the surface oceans as a result of cyanobacteria and other oxygenic phototrophs . therefore , redox interfaces between anoxic and fe(ii)-containing waters and photosynthetically produced oxygen were likely common throughout much of earth s history . iron redox processes fractionate the naturally occurring isotopes of iron dependent on their mass ( e.g. , fe , fe , fe , and fe ) , such that the quantitative contribution of biotic and abiotic iron cycling at the earth s surface may be recorded in sediments composed of iron - rich minerals . due to the large fractionations between fe(ii ) and fe(iii ) species , fe(ii ) oxidation generally produces a solid iron phase that is enriched in heavy isotopes of iron relative to aqueous fe(ii ) , regardless of the mechanism of oxidation . this makes it difficult to parse the contribution of enzymatic fe(ii)-oxidizing bacteria from abiotic fe(ii ) oxidation , not to mention indirect fe(ii ) oxidation by oxygen - producing cyanobacteria by using iron isotopes . however , subtle differences in the mechanism of oxidation and precipitation , and in the characteristics of the iron minerals or phases ( e.g. , mineralogy , particle size , or presence of impurities ) formed , can influence the overall fractionation between aqueous fe(ii ) and iron minerals . furthermore , the role of cyanobacteria in direct or indirect redox cycling of iron at the cell surface is increasingly recognized and may be associated with distinct isotope fractionation . therefore , detailed mechanistic studies of iron isotope fractionation during different pathways of fe(ii ) oxidation are warranted and may help to define isotopic , mineralogical , or microscopic signatures associated with certain biological processes . furthermore , the isotopic composition of iron minerals is known to be modified by electron and atom exchange between aqueous fe(ii ) and fe(iii ) ( oxyhdr)oxide minerals . these processes have been most effectively characterized under reducing conditions , when a supply of aqueous fe(ii ) is produced by , for instance , microbial fe(iii ) reduction . however , at fe(ii)o2 interfaces with a flux of aqueous fe(ii ) , electron and atom - exchange could also occur on newly formed fe(iii ) ( oxyhydr)oxide minerals . although the effect of some organics as well as si and low ph on blocking electron and atom exchange have been investigated , the effect of cell surfaces and microbially produced organics on this reaction and via blocking sites on fe(iii ) minerals , particularly in an oxidizing system , are not known . in this contribution , we tracked the iron isotope composition of different pools of iron during fe(ii ) oxidation by the marine planktonic cyanobacterium synechococcus pcc 7002 . several prior studies have characterized the interaction of this oxygen - producing strain with fe(ii ) , which gives us a body of work to aid in interpreting the nature of different iron phases in the system , and their mechanism of transformation . additional microscopy and mineral characterization in this study are used to build the picture of how iron is processed during indirect fe(ii ) oxidation resulting from oxygenic photosynthesis . the results have implications for understanding the reactivity of iron minerals as well as identifying isotopic signatures associated with biological activity . synechococcus pcc 7002 was routinely cultivated on ph 6.8 marine phototroph ( mp ) medium containing 6 mg l ferric ammonium citrate as the fe(iii ) source at 24 c under an irradiance of 12.8 mol photons m s from a standard 40w tungsten light bulb as measured by a li-250a light probe ( li - cor , inc . ) . for fe(ii ) fe(ii ) amendments were added from a sterile , anoxic fecl2 stock solution , and the medium was filtered twice through a 0.22 m filter in an anoxic glovebox ( 100% n2 ) , separated by 48 h incubations at 4 c to ensure that all fe(ii ) precipitated as carbonate and phosphate minerals with growth media components were removed . the final fe(ii ) concentration in the medium after filtration was 2 mm as measured by the spectrophotometric ferrozine assay . a log - phase culture of synechococcus pcc 7002 grown with ferric ammonium citrate was degassed for 5 min with sterile n2/co2 ( 90%:10% ) and inoculated into the 2 mm fe(ii)-containing medium to a final concentration of 5 10 cells ml . glass media bottles were acid washed in 1 m hcl for 24 h and then soaked in fresh ultrapure water ( resistivity of 18.2 m cm ) for two successive 24 h treatments before use . all anoxic bottles were closed with butyl rubber stoppers that had been washed in 1 n hcl for 24 h and then thrice boiled in ultrapure water . this concentration of 2 mm fe(ii ) was chosen for experiments because a freshly inoculated culture of synechococcus pcc 7002 took about 10 days to oxidize this , during which time we could sample sufficiently often to have resolution on the evolution of the isotopic composition of different iron pools . despite the fact that this strain grows more slowly at 2 mm fe(ii ) than at lower fe(ii ) concentrations , due to fe(ii ) toxicity , sufficient growth did occur to fully oxidize all fe(ii ) . although this concentration is at the upper end of fe(ii ) concentrations in modern sunlit environments , it is within the range documented for environments where cyanobacteria have been documented as having a role in fe(ii ) oxidation . during fe(ii ) oxidation , which lasted about 10 days , volumes of 2 ml were repeatedly removed with a syringe from the bottles of two contemporaneous replicate experiments ( bottles 1 and 2 ) in an anoxic glovebox . before extracting , the bottles were shaken to yield a homogeneous slurry of iron precipitates . the aliquots were subsequently centrifuged for 10 min at 16 000 g , and the supernatants were filtered through a nylon 0.22 m centrifuge tube filter ( costar spin - x , corning , international ) to yield particle - free aqueous fe(ii ) , henceforth fe(ii)aq . the solids were washed with anoxic ultrapure water to remove any loosely bound iron . a second wash utilized anoxic 0.5 m sodium acetate ( adjusted to ph 4.85 using acetic acid ) to recover sorbed iron ( fenaac ) from the solids ( 24 h incubation in the dark ) . the concentrations of fe(ii ) and total iron in the four different iron fractions were measured with the ferrozine assay . the fe(ii ) in the fe(ii)aq , water wash , and fenaac was stabilized in a final concentration of anoxic 1 m hcl prior to measurements . fe(iii ) was determined as the difference between fe(ii ) measurement and total iron measurements ( after reduction of iron by hydroxylamine hydrochloride ) . purification of the fe(ii)aq , fenaac , and feppt fractions was performed in positively pressured clean laboratories of the isotope geochemistry group at the university of tuebingen under conditions and with reagents that have previously been described . the concentrations of iron in the water washes of feppt were below the detection limit of the ferrozine assay ( < 0.01 mm , 0.56 g ml ) , and so , these samples were not purified . sample aliquots of the separated iron fractions containing 5 g of iron were purified for iron isotope measurements using anion exchange chromatography according to prior methodology . an adequate amount of fe fe double spike was added to the samples prior to fe purification to ensure accurate correction of the instrumental mass bias and possible fe isotope fractionation during anion chromatography caused by the organic matrix of the samples . iron isotope analyses were performed on the thermofisher scientific neptuneplus multicollector inductively coupled plasma mass spectrometer ( mc - icp - ms ) of the isotope geochemistry group of the university of tuebingen . polyatomic interferences , such as arn on fe or aro on fe were resolved using the high mass - resolution mode ( 16 m slit ) . the four iron isotope beams were simultaneously detected with 90 integration cycles at 8 s each during the runs . background corrections for sample signals were based on on - peak - zero measurements on the pure analyte solution ( 0.3 m hno3 ) run before and after each sample . iron isotope data are reported relative to the isotopically certified international reference material irmm-014 ( institute for reference materials and measurements in gent , belgium ) using the -notation : the results are reported in units of per mil ( ) . the reproducibility of the double - spike measuring method as determined by repeated fe measurements of the irmm-014 reference material in between sample runs was 0.00 0.032 ( 2sd ; n = 18 ) . interspersed measurements of our in - house iron standard , hanfe , yielded fe = 0.282 0.039 ( 2sd ; n = 12 ) , which is in excellent agreement with previously published values of 0.28 0.05 ( 2sd ; n = 19 ) and 0.279 0.030 ( 2sd ; ( f ) of fe(ii ) remaining were utilized to determine the isotopic enrichment factor ( fe ) using the following equations:12 fe(ii)aq-0 indicates the fe(ii)aq at the beginning of the experiment . the isotopic fractionation fe ( ) is related to fe by the equation:3 the fitting parameters were determined by minimizing the sum of values . data were also fit by linear regression , with slope and intercept determined by minimizing the sum of values . high energy synchrotron x - ray scattering experiments were performed on the solid , dry products of fe(ii ) oxidation by synechococcus pcc 7002 at the advanced photon source at argonne national laboratory , beamline 11-id - b . 5 mm fe(ii ) , freeze - dried , and washed three times with millipore water to remove excess salt . cells of synechococcus pcc 7002 were grown under similar conditions as described above until the initial ca . mineral aggregates were imaged by confocal laser scanning microscopy ( clsm ; leica spe , mannheim , germany ) . a 635 nm laser was used for excitation autofluorescence of synechococcus pcc 7002 , with a maximum emission peak at 660 nm ( detected range of emission , 640700 nm ) . the fe(iii ) minerals were visualized using the reflection signal of the 488 nm laser . alexa dye conjugates were screened in order to optimize visualization of exopolysaccharides ( eps ) without overlap with the autofluorescence emission maximum of the pigments of synechococcus pcc 7002 ( 660 nm ) . alexa 488 ( maximum emission peak at 520 nm ) was chosen because sba bound to the eps in higher amounts , resulting in brighter fluorescence than the other lectins screened [ wheat germ agglutinin alexa fluor 555 conjugate ( wga-555 ) and lectin pna from arachis hypogaea ( peanut ) , alex fluor 568 conjugate ( pna-568 ) ] . brighter fluorescence at lower laser power was observed with sba-488 , which binds terminal - and -n - acetylgalactosamine and galactopyranosyl residues , compared to wga-555 and pna-568 , which are specific to sialic acid and n - acetylglucosaminyl residues and terminal -galactose , respectively . a turn - on type selective probe for fluorescent labeling of dissolved , sorbed , or ligand - bound fe(iii ) was previously used to visualize the relationship of fe(iii ) synechococcus pcc 7002 cells and minerals from this same incubation . because of spectral overlap , the lectin and fe(iii)-binding probe could not be combined in a single experiment here , and therefore , we compare new results to prior data . the auto - quant deconvolution algorithm implemented in the leica las af software was applied to blind deconvolute the 3d image stacks . the spatial relationships of species detected using fluorescence dyes and cell autofluorescence in clsm image stacks were analyzed using scatterj , a plugin for correlation analysis of species - specific maps for use in imagej and fiji . the fe(ii)aq values from two replicated experiments evolved from an initial value near 0 to lighter values during oxidation ( table 1 , figure 1 ) . the fe(ii)aq fraction , measured after the sample was centrifuged and filtered , consisted of only fe(ii ) . all iron concentration and speciation data ( measured by ferrozine ) the first feppt samples analyzed , at about 40% fe(ii ) oxidized , had feppt of about 2 , trending toward 0 at 100% fe(ii ) oxidized . the speciation of feppt , which was measured after washing with water and sodium acetate , consisted of predominantly fe(iii ) , with generally < 10% fe(ii ) . iron in the water wash of the precipitates was below the detection limit of the ferrozine assay ( < 0.01 mm , 0.56 g ml ) . the sodium acetate wash removed sorbed iron , which contained both fe(ii ) and fe(iii ) . the fenaac fraction represented 1020% of total iron in the system after feppt began to form . the fenaac had an intermediate isotopic composition between fe(ii)aq and feppt but was variable and generally lighter than 0. ( a ) bottle 1 and ( b ) bottle 2 are biological replicates of the fe(ii ) oxidation experiment with synechococcus pcc 7002 . green circles are fe(ii)aq data ; orange squares are feppt data ; blue diamonds are fenaac data . the solid green lines are the rayleigh fits of the fe(ii)aq data , with an fe for fe(ii)aq of 1.79 ( a ) to 2.15 ( b ) . the solid orange lines are the rayleigh fits of the feppt data , with fe for feppt of 2.44 ( a ) and 2.98 ( b ) . samples from b2 and e2 had anomalous values for fenaac and feaq , respectively . these samples were most likely lost after drying due to electrostatic charging of the teflon beakers . the fast reaction between fe(ii ) and oxygen favors the heavy isotopes of iron in the resulting fe(iii ) minerals that precipitate . abiotic and biotic fe(ii ) oxidation both follow this trend , resulting in fe of 24 between aqueous fe(ii ) and fe(iii ) minerals , with minerals enriched in heavy isotopes . our fe for fe(ii)aq ( 1.792.15 for bottles 1 and 2 , respectively ; table 1 ) , determined from a rayleigh fit of the fe(ii)aq data , is on the low end of this range , similar to what was previously documented for fe(ii ) oxidation by anoxygenic phototrophs . the rayleigh fit of the feppt data from both replicates resulted in fe of 2.44 and 2.98 , larger than that attained for the fe(ii)aq data ( table 1 ) and within the literature range . prior explanation for the offset in fe between these two fractions is that , following precipitation , the feppt underwent partial equilibration with another phase of iron in the system , possibly a ligand - bound or sorbed fe(iii ) phase or fe(ii)aq . below , we use our mineralogical and microscopic characterizations of the experiment to explore possible exchange processes in this system . the third , quantitatively significant fraction of iron in the system in addition to fe(ii)aq and feppt was fenaac ( up to 18% of total fe ) . the fenaac data had an intermediate isotopic composition between fe(ii)aq and feppt , from 0.10 to 0.73 throughout the experiment , and contained both fe(ii ) and fe(iii ) ( supplementary table 1 ) . the presence of fe(iii ) in fenaac has previously been observed in fe(ii ) oxidation experiments with anoxygenic phototrophs but not with nitrate - dependent fe(ii)-oxidizing bacteria . the fenaac must have been sorbed onto one of the surfaces , either feppt or cells , on the basis of its extraction with sodium acetate . in order to infer whether equilibration processes were occurring between fenaac and feppt , it is necessary to know ( 1 ) where fenaac was in our experiments and ( 2 ) what type of iron species [ i.e. , fe(ii ) or fe(iii ) ] that it was . our use of a lectin - binding dye in confocal microscopy documents that eps was also forming during fe(ii ) oxidation with synechococcus pcc 7002 ( figure 3 ) . we can use this data set to first determine whether eps was important in binding / sorbing iron extracted as fenaac and then to determine whether iron was associated with the surface of cells and/or feppt . an overlay of figure 3a c , which show the location of cells , eps , and feppt , indicates that eps is colocalized with feppt ( figure 3d ) . the correlation analysis in figure 3e implies there is no spatial overlap of eps with cells . in previous work with synechococcus pcc 7002 under identical growth conditions as in figure 3 , a fluorescent sensor for soluble or ligand - bound fe(iii ) was used in clsm , and fluorescence was localized directly at the synechococccus pcc 7002 cell surfaces . while spectral interferences prevented us from simultaneously labeling eps and fe(iii ) in our current clsm experiments , we can infer from comparing our data set with the previously published one that there was fe(iii ) sorbed to the surface of cells but not eps or feppt . in support of this , eps is expected to stay with the aqueous phase during filtration through a 0.2 m filter or be washed off of feppt in the water wash . we did not detect any fe(iii ) in the fe(ii)aq fraction or measure any detectable iron in the water wash . from these results , we exclude eps as having a major role in binding soluble fe(iii ) in the current system . previous experiments with synechococcus pcc 7002 cells demonstrated that sorption to cells is a major fate for aqueous iron , although the oxidation state of sorbed iron was not determined in those experiments , so we can not rule out that some fe(ii ) was also sorbed to cells . however , sorption onto cells has previously been documented as a fate for aqueous iron with diverse cyanobacteria , with fe(iii ) more commonly detected at the cell surface than fe(ii ) , via attachment of fe o fe polymers to phosphoryl groups , strengthening the inferences made from clsm that synechococcus pcc 7002 cells sorbed fe(iii ) . ( a ) x - ray diffraction ( xrd ) pattern obtained from x - ray total scattering data of the feppt phase after complete fe(ii ) oxidation , freeze - drying , and water washing . the indexed reflections for lepidocrocite ( lp ) and goethite ( gt ) are shown . ( b ) a 3-component linear combination fit of 58% ferrihydrite , 22% goethite , and 20% lepidocrocite ( supplementary table 4 ) . ( a ) autofluorescent cells , ( b ) stained with the lectin - binding dye sba-488 , ( c ) the reflection signal from fe(iii ) minerals , and ( d ) an overlay of ( a c ) . correlation plot of the fluorescence intensity in individual pixels from ( e ) autofluorescence ( a ) vs sba-488 ( b ) and ( f ) sba-488 ( b ) vs fe(iii ) minerals ( c ) . this analysis demonstrates that eps , which is bound by sba-488 , is coating fe(iii ) minerals but is not spatially associated with cells . the other surface in our experiments that could have sorbed iron extracted as fenaac was feppt . the three techniques we used to address mineralogy indicate that our feppt was a mixture of 58% ferrihydrite , 22% goethite , and 20% lepidocrocite ( figure 2 ) , and ferrihydrite was likely the predominant mineral present during the experiments ( see supporting information ) . minerals such as ferrihydrite and goethite , similar to what was present in our experiments , can sorb fe(ii ) . both fe(ii ) and fe(iii ) were detected in the fenaac ( supplementary table 1 ) , raising the possibility that fe(iii ) was extracted from the mineral . however , we verified that no fe(iii ) was extracted from synthetic ferrihydrite with our 0.5 m sodium acetate solution prior to beginning experiments ( data not shown ) , consistent with previous reports that used a 1 m sodium acetate solution . a further inference in support of sorbed fe(ii ) being extracted from feppt by sodium acetate is that feppt still contained some fe(ii ) after extraction , as measured by ferrozine ( supplementary table 1 ) . we take this as evidence that sorbed iron associated with the mineral was predominantly fe(ii ) , although we can not exclude that some fe(iii ) may also be sorbed to the mineral surface . we observed evidence for three reactions in our experiments that are essential for understanding the observed fractionations of iron isotopes , and these are summarized in figure 4 . they are ( 1 ) fe(ii ) oxidation to fe(iii ) , which forms feppt , ( 2 ) sorption of fe(iii ) to cells , and ( 3 ) sorption of fe(ii ) to feppt . these observations fit a two step - model of fe(ii ) oxidation , where fe(ii ) is oxidized and undergoes rapid isotopic equilibration with a pool of fe(iii ) , which then precipitates as fe(iii ) minerals . we suggest , however , that in our experiments , feppt undergoes subsequent partial equilibration with fe(ii)aq . controls on the overall iron isotope fractionation in the system are ( 1 ) fe(ii ) oxidation and precipitation of fe(iii ) as feppt ; ( 2 ) sorption of fe(iii ) to cells ; ( 3 ) partial equilibrium atom and electron exchange after sorption of fe(ii)aq to feppt . ( 1 ) generates feppt ( solid orange line ) that is 23 heavier than fe(ii)aq ( solid green line ) . ( 2 ) produces sorbed fe(iii ) on cells with an estimated equilibrium fefenaac - fe(ii)aq of 1.84. ( 3 ) produces fe(ii ) sorbed on goethite with an estimated fefenaac - fe(ii)aq of 0.8. the resulting fenaac predicted from ( 2 ) and ( 3 ) are denoted by the light blue diamonds . the fitting of our fe(ii)aq and feppt with rayleigh equations representing isolation of the feppt pool from fe(ii)aq after precipitation and a linear equation representing complete isotopic equilibrium are helpful in interpreting the fractionation mechanisms taking place . the larger values for linear fits of all data as compared to rayleigh fits indicate that complete isotopic equilibrium between fe(ii)aq and feppt is not occurring during fe(ii ) oxidation and precipitation ( table 1 ) . the smaller fe values for rayleigh fits of the fe(ii)aq ( 1.79 and 2.15 as compared to 2.44 and 2.98 for feppt ) are on the order of fractionation observed in other biological fe(ii ) oxidation experiments in batch at circumneutral ph : 1.5 , 1.52 , and 12. such small net fractionations have been noted when the presence of significant quantities of sorbed or ligand - bound fe(iii ) has been detected or observed . up to a few percent of total iron was found as fe(iii ) in the fenaac fraction ( supplementary table 1 ) . on the basis of detection of iron sorbed to cells with a dye that is specific for an aqueous or ligand - bound fe(iii ) , we suggest that this fe(iii ) could equilibrate with fe(ii)aq . in experiments with synechococcus sp . cells at ph 6 , added fe(ii ) [ which was adsorbed as fe(iii ) ] was 1.84 heavier than aqueous fe(ii ) , and equilibrium with fe(ii)aq was inferred from the data . this fractionation is very similar to our fe values derived from rayleigh fits of fe(ii)aq ( 1.79 and 2.15 ) . because of the similar type of organism that we used , it is likely that equilibrium fractionation between fe(ii)aq and fe(iii ) sorbed to cells is a relevant process in our experiments . while the feppt data are not well fit by a linear model representing complete equilibrium exchange with fe(ii)aq , the range of the fe determined from rayleigh fits of the two feppt data sets ( 2.44 and 2.98 ) is of the same magnitude expected for equilibrium between feppt and fe(ii)aq . wu et al . inferred a feferrihydrite - fe(ii)aq ( where feferrihydrite - fe(ii)aq = feferrihydrite fefe(ii)aq ) of 3.2 , while beard et al . and frierdich et al . reported a fegoethite - fe(ii)aq of 1.1. considering the 58% ferrihydrite , 22% goethite , and 20% lepidocrocite in our precipitates as determined from x - ray scattering ( figure 2 ) , the equilibrium fefeppt - fe(ii)aq for our minerals could range from 2.3 to 2.7 , depending on whether the assumed fractionation for lepidocrocite is the same as for goethite or ferrihydrite , respectively . the larger fe we calculate for our second data set ( 2.98 ) may reflect that ferrihydrite , with a larger fe value , was likely the mineral present during active fe(ii ) oxidation ( see supporting information ) . in our batch experiments , fe(ii)aq may continue to react with feppt , given their proximity and the time frame of experiments ( 10 days ) . sorption of fe(ii ) on feppt provides a likely mechanism for partial isotope equilibrium and is supported by our detection of fe(ii ) in feppt ( supplementary table 1 ) . sorption of fe(ii ) is an important pathway in recrystallization of fe(iii ) ( oxyhydr)oxide minerals , particularly ferrihydrite . during this process , equilibrium atom and electron exchange occur between sorbed fe(ii ) and fe(iii ) minerals , with complete equilibrium attained within 2 weeks for goethite , for instance ( a similar time frame as our 10 day experiment ) . in this model , fe(ii ) sorbs to fe(iii ) minerals and donates an electron into the bulk mineral structure , adding to the fe(iii ) mineral , and causing the desorption of a newly produced fe(ii ) from the mineral . this is also consistent with the shifts in mineralogy we see during the course of oxidation ( see supporting information ) . we do not see evidence for complete equilibrium between fe(ii)aq and feppt , given the poor linear fit of feppt ( table 1 ) . atom and electron exchange between feppt and sorbed fe(ii ) is expected to be diminished in the presence of organic compounds . our clsm data indicate that eps is colocalized to minerals ( figure 2 ) . it is possible that atom and electron exchange can still occur when fe(iii ) minerals are coprecipitated with organics , as retardation of this process seems to result from blockage of surface sites when organics coat already formed fe(iii ) minerals , or if long chain carbon molecules are present . therefore , we suggest that only partial atom and electron exchange occurred in our system as a result of the eps coating the feppt . during atom exchange , the fractionation when fe(ii)aq sorbs onto goethite varies among experiments . one study reports sorbed fe(ii ) is 0.73 heavier than fe(ii)aq , and another reported sorbed fe(ii ) was 1.24 heavier . crosby et al . directly measured sorbed fe(ii ) extracted with sodium acetate during microbial fe(iii ) reduction experiments , which was just 0.3 heavier than fe(ii)aq for experiments using hematite and up to 0.8 heavier for experiments using goethite as the sorbing surface . our fefenaac - fe(ii)aq ranged from 0.45 to 2.66 , which is much larger and more variable than the experiments of crosby et al . , which may be in part because our fenaac includes both fe(ii ) and fe(iii ) . another factor is that , in our experiments , the less crystalline mineral ferrihydrite was likely the sorbing surface present during experiments ( see supplementary figures 1 and 2 and supporting information ) . several studies have noted a trend of larger fractionations during sorption to less crystalline minerals or higher surface area minerals . the feppt in our experiments had a surface area of 122.1 m g. we calculated fenaac considering that the fe(iii ) fraction of fenaac should be 1.84 heavier than fe(ii)aq due to adsorption of fe(iii ) at cell surfaces , and the sorbed fe(ii ) fraction of fenaac should be at least 0.8 heavier than fe(ii)aq . the calculated fenaac is a good model of our actual fenaac values ( figure 4 ) . this calculation supports the model presented here , in which fe(ii ) is oxidized to fe(iii ) , which is sorbed onto cells and equilibrates with fe(ii)aq , and full equilibration of fe(ii)aq with feppt via atom and electron exchange is hindered by the presence of eps on feppt . our experiments provide evidence that iron isotope fractionation during microbially influenced fe(ii ) oxidation by cyanobacteria is not a simple reaction , controlled only by the abiotic oxidation of fe(ii ) with oxygen and rapid precipitation of fe(iii ) at circumneutral ph . multiple secondary processes generate a significant fraction of sorbed iron that is isotopically distinct from either residual fe(ii)aq or feppt , and subsequent equilibration between the iron pools can further modify the isotopic composition of these phases . our data indicate that sorption of fe(iii ) at cell surfaces likely further fractionates the fe(ii)aq pool . in addition , abiotic sorption of fe(ii ) to fe(iii ) mineral surfaces can also fractionate fe(ii)aq , through equilibrium atom and electron exchange subsequent to fe(ii ) sorption , despite the presence of eps . follow - up experiments could investigate atom and electron exchange in this system . specifically , isotopically enriched fe(ii)aq solutions mixed with preformed cells and minerals would be useful for monitoring atom exchange between the fe(ii)aq and feppt . the processes and phases described here can overprint the anticipated fractionations and compositions of fe(iii ) minerals and organic - associated iron present in the environment and can challenge interpretation of the genesis of fe(iii ) minerals in the geological record , where the residual fe(ii)aq pool is no longer present . although iron atom and electron exchange has received the most attention as a process relevant to fe(iii)-reducing systems where fe(ii ) is in contact with fe(iii ) minerals , our data suggest this process could also be relevant in environments where fe(ii ) is abundant during fe(ii ) oxidation and fe(iii ) mineral formation . this includes oxidizing environments with high enough fluxes of fe(ii ) for fe(ii ) to persist even in the face of rapid oxidation , such as fe(ii)-rich springs or seeps and marine upwelling zones that tap ferruginous bottom waters , past or present . furthermore , our work documents atom and electron exchange in the presence of iron minerals whose formation pathways are biologically induced and when organic phases coat iron minerals . finally , iron redox cycling and sorption of iron at the surface of cyanobacteria may be an important component of modern and ancient aquatic iron cycling , and our work highlights the effect of such processes on iron isotope systematics .

in this study , we couple iron isotope analysis to microscopic and mineralogical investigation of iron speciation during circumneutral fe(ii ) oxidation and fe(iii ) precipitation with photosynthetically produced oxygen . in the presence of the cyanobacterium synechococcus pcc 7002 , aqueous fe(ii ) ( fe(ii)aq ) is oxidized and precipitated as amorphous fe(iii ) oxyhydroxide minerals ( iron precipitates , feppt ) , with distinct isotopic fractionation ( 56fe ) values determined from fitting the 56fe(ii)aq ( 1.79 and 2.15 ) and the 56feppt ( 2.44 and 2.98 ) data trends from two replicate experiments . additional fe(ii ) and fe(iii ) phases were detected using microscopy and chemical extractions and likely represent fe(ii ) and fe(iii ) sorbed to minerals and cells . the iron desorbed with sodium acetate ( fenaac ) yielded heavier 56fe compositions than fe(ii)aq . modeling of the fractionation during fe(iii ) sorption to cells and fe(ii ) sorption to feppt , combined with equilibration of sorbed iron and with fe(ii)aq using published fractionation factors , is consistent with our resulting 56fenaac . the 56feppt data trend is inconsistent with complete equilibrium exchange with fe(ii)aq . because of this and our detection of microbially excreted organics ( e.g. , exopolysaccharides ) coating feppt in our microscopic analysis , we suggest that electron and atom exchange is partially suppressed in this system by biologically produced organics . these results indicate that cyanobacteria influence the fate and composition of iron in sunlit environments via their role in fe(ii ) oxidation through o2 production , the capacity of their cell surfaces to sorb iron , and the interaction of secreted organics with fe(iii ) minerals .

the online version of this article ( doi:10.1007/s13353 - 014 - 0199 - 8 ) contains supplementary material , which is available to authorized users . salmonella is a widespread foodborne pathogen with the ability to adapt to different environments , consequently creating significant challenges to food - producing industries in controlling this pathogen in food chain products . swine ( sus scrofa ) are an important reservoir of salmonella because colonization and shedding of this bacterium occurs within asymptomatic pigs , imposing elevated risks to public and animal health . thus , diverse intervention strategies are needed to control the transmission of salmonella from pig products to humans and to the environment . in bacterial infections , the severity of infection is impacted by the pathogenicity of the microorganism and its interaction with the host immune defense system ( zanella et al . toll - like receptor 4 ( tlr4 ) is a well - characterized gram - negative bacterial lipopolysaccharide ( lps ) recognition receptor and a host inflammatory response activator well conserved among animal species ( noreen et al . 2012 ; yang et al . 2012 ) . schrder and schumann ( schrder and schumann 2005 ) suggested that mutations in the tlr4 regions involved with pathogen recognition and transduction signaling may affect host susceptibility to infection . polymorphisms in the tlr4 gene have been associated with different infectious diseases in humans , such as meningitis and tuberculosis , as well as some types of cancers ( noreen et al . 2012 ) and with infection and disease in cattle , chicken and pigs ( yang et al . tlr4 is located on sus scrofa 1 ( ssc1 ) v10.2 ( 289,776,058 bp to 289,785,087 bp ) . ( 2006 ) identified the genomic structure of porcine tlr4 , and shinkai et al . specifically for tlr4 , 13 snps were widely distributed in 11 pig breeds , and of those , seven were non - synonymous . thirty four snps were identified in tlr4 using pigs representing european commercial breeds and some traditional breeds ( n = 259 ) , and of these , 17 snps were located in the non - coding region and 17 snps were found in the coding region ( palermo et al . furthermore , polymorphisms in the tlr4 gene have been identified as potential genetic markers for disease susceptibility in pigs ( uenishi and shinkay 2009 ) . our collaborative group has reported up - regulation of tlr4 and its target genes in pigs challenged with salmonella enterica serovar typhimurium ( huang et al . 2011 ) . therefore , to determine if tlr4 is a possible candidate gene associated with salmonella shedding , we first , identified snps in the tlr4 gene of our previously described low and persistent shedder pig populations ( huang et al . 2011 ; uthe et al . second , we investigated associations of the tlr4 snps with salmonella shedding status . selecting for pigs with reduced salmonella fecal shedding would decrease environmental contamination and lower pathogen transmission to other animals and humans ; thus , identification of loci in tlr4 associated with salmonella fecal shedding is the focus of this study . all procedures involving animals in the nadc-40 and nadc-77 populations were approved by the usda , ars , nadc animal care and use committee . briefly , all the pigs used in this study were intranasally challenged at 7 weeks of age with 1 10 cfu of s. typhimurium 4232 as previously described ( huang et al . 2011 ; uthe et al . 2009 ) . at days 2 , 7 , 14 , and 20/21 post - inoculation ( pi ) , salmonella fecal shedding was quantified using a standard bacteriological test previously described ( uthe et al . 2009 ) . of the initial 117 animals , 40 ( n = 40 ) pigs were chosen based on their extreme fecal culture status ; quantitative classification of the phenotype was scored based on cumulative salmonella fecal shedding on days 2 , 7 , 14 , and 20/21 pi ( huang et al . 2011 ) . genomic dna was extracted from blood samples and purified as previously described ( uthe et al . nine sets of primers were designed based on the ensembl gene sequence for enssscg00000005503 using beacon designer ( table 1 ) . primers were selected to cover all exons ( n = 3 ) of tlr4 including a 713 bp upstream region and a 1225 bp downstream region ( ssc1 : 289,775,345 bp289,786,312 bp ensembl genome build 10.2 ) . the pcr mix for each reaction contained 16.75 l dh2o , 1.25 l each primer ( 10 m ) , 2 l of dntps , 2.5 l 10x buffer with mgcl2 , 0.25 l platinum taq dna polymerase high fidelity ( invitrogen grand island , ny , usa ) and 1 l of 10 ng/l pig dna . dna samples were amplified using the mj research ptc-200 pcr thermal cycler ( biorad laboratories , hercules , ca ) . the pcr reaction was performed as follows : 94 c for 2 min , 30 cycles of 94 c for 30 s , 58 c for 30 s , 72 c for 1 min and a final step of 72 c for 7 min . pcr products were visualized by agarose gel electrophoresis to confirm a single correct size product and purified using minelute 96 uf pcr purification kit ( qiagen ) prior to dna sequencing using an ab 3730xl dna analyzer ( applied biosystems ) at iowa state university , ames , ia.table 1identified snps and position in the tlr4 gene of salmonella low and persistent shedder pigssingle marker association ( p - value)genbank accession number for the snplocation in sus scrofa genome ( bp)amino acidnlocationprimerssnp designationqualitativequantitativesiteamino acid15upstream5gaaccatgcagtagaacagg3ctggaagtctgtagtcaagg 5u : a-1082g#0.0330.064nossc1:289,774,9832 5u : t-1019c#0.0330.064nossc1:289,775,0463 5u : c-984t#0.0330.064nossc1:289,775,08145cacaagaaggaagagatagc3 caccaagggaagctctagg 5u : c-522t#0.1330.244nossc1:289,775,5435 5u : g-400a0.3630.550rs80830544ssc1:289,775,6656 5u : g-75c#0.0250.056nossc1:289,775,9797intron 25acagaagattggatggaagga3 gagataagaaagctgagacc 2i : a232c0.0040.029rs80881287ssc1:289,780,2268 2i : c298t**0.0020.013rs80787918ssc1:289,780,2929intron 25cctcacttgatatgtttgcc3gttcctccaggacagatttg 2i : c2567t#0.0010.025nossc1:289,782,76110exon 3 c318a0.0030.037rs80923358ssc1:289,782,83411 g417a0.0030.037rs80951861ssc1:289,782,93312 t611a*0.0070.054rs80811682ssc1:289,783,127204l / h13exon 35attcaaggtctggctggttc3 tgaagacatcaggaagcaag g826a*0.2850.514shinkai et al . ( 2006)ssc1:289,783,342276v / i14 g960a0.0640.105rs80981701ssc1:289,783,47615 g962a*0.0340.046rs80955017ssc1:289,783,478321r / h16 c1027a*0.1760.231rs80894552ssc1:289,783,543343q / kexon 35acatccacgttgtcttccg3cagttcattcctcacccag17exon 35cttcctcctggtatctgtgg3ggcagtcctgtgtatctcg g2397a0.0250.056rs80834103ssc1:289,784,913183downstream5actcccaacgtgtcccttg3ccaagaagtgccactttcaac 3d : c208t**0.0020.011rs80907449ssc1:289,785,250 to first codon of exon 1 ; position in intron 2 ; position in coding region ; position in 3utr downstream of last codon ; * non - synonymous snps ; * * haplotype components ; # novel snps identified snps and position in the tlr4 gene of salmonella low and persistent shedder pigs to first codon of exon 1 ; position in intron 2 ; position in coding region ; position in 3utr downstream of last codon ; * non - synonymous snps ; * * haplotype components ; # novel snps sequences were analyzed and polymorphisms were identified using phred / phrap / consed / polyphred software ( nickerson et al . snps were removed if the minor allele frequency ( maf ) was less than 10 % , if the snps failed to genotype in more than 10 % of the samples , or if the snps failed the hardy weinberg equilibrium ( p < 0.001 ) . statistical analyses were conducted within plink and r statistical environment ( version 1.07 , ( purcell et al . 2007 ) ) . a chi - squared test ( ) was used to test associations of snps located in tlr4 and the qualitative measurement of salmonella shedding ( persistent versus low ) . the wald test was used to verify associations with salmonella shedding as a quantitative trait . a significance threshold for the association analysis was set to p 0.05 . following the single marker association test , a haplotype test was conducted within plink to identify if a haplotype was more informative than a single snp . first , an omnibus association test was performed to identify the overall association of the haplotype with the qualitative measurement of salmonella shedding . if an association was identified , a haplotype - specific test was performed to identify which combination of the alleles provided the strongest evidence for an association with salmonella shedding in swine . following haplotype construction , a stepwise regression using a backward - elimination process was performed to identify the effect of each associated snp in relationship to the haplotype ; in this test , all associated snps were included and excluded individually from the analysis , and the association of the haplotype was tested each time using plink . huang et al . ( 2011 ) identified the tlr4-dependent set of genes ( tlr4 regulon ) as a major inducer of the transcriptional response in salmonella persistently shedding pigs , and this tlr4 regulon was not significantly affected in the low shedding pigs . thus , tlr4 is considered a potential candidate to analyze the association of genetic polymorphisms with the diverse phenotypic patterns of salmonella shedding in swine . in this study , two swine populations were investigated , nadc-40 and nadc-77 ( uthe et al . 2011 ) , each population with 10 low and 10 persistent salmonella shedding animals ( fig . 1 ) . for the quantitative measurement of salmonella shedding per pig , a cumulative measurement taken within days 2 , 7 , 14 , and 20/21 pi was calculated ( huang et al . sequencing analysis of those 40 ( n = 40 ) animals identified 18 snps ; 12 were previously described in the literature and/or annotated in genbank and six are novel snps ( table 1 ) . five of the novel snps are located within the 5untranslated region ( utr ) and one is within intron 2.fig . 1area under the log curve illustrating the log of cumulative colony forming units ( cfu ) . quantitative bacteriology of salmonella shedding in swine fecal samples was performed at day 2 , 7 , 14 , and 20/21 days post - challenge with salmonella enterica serovar typhimurium , and cfu were determined area under the log curve illustrating the log of cumulative colony forming units ( cfu ) . quantitative bacteriology of salmonella shedding in swine fecal samples was performed at day 2 , 7 , 14 , and 20/21 days post - challenge with salmonella enterica serovar typhimurium , and cfu were determined the swine tlr4 gene ( 9030 bp / sgsc sscrofa10.2/susscr3 ) is composed of three exons ( 93 , 167 , and 2266 bp ) . taking together these results and the literature , 50 snps have been identified in tlr4 , with 22 snps located in the coding regions ( thomas et al . our investigation identified four of those nine non - synonymous snps in exon 3 segregating in the nadc-40 and nadc-77 pig populations . of the 18 snps identified in the two pig populations , 13 ( n = 13 ) snps were associated ( p 0.05 ) with salmonella shedding as a qualitative phenotype using a chi - squared test ; of those 13 snps , seven were also associated with salmonella shedding as a quantitative phenotype using a wald statistical test ( table 1 ) . using a haplotype construction and the backward - elimination process , the most significant haplotype for both measurements of salmonella shedding , qualitative ( p 7.9 10 ) and quantitative ( p 4.0 10 ) ( table 2 ) comprised a region of 4.9 kb composed of snps , rs80787918 ( snp8 ) and rs80907449 ( snp18 ) ( r = 0.902 ) located at ssc1:289,780,292 bp and ssc1:289,785,250 bp , respectively ( table 1).table 2haplotypes frequency ( snps rs80787918 and rs80907449 ) and associations with qualitative and quantitative phenotypes of salmonella sheddinghaplotype frequencyqualitative p valuequantitative p valuehaplotypepersistent shedderslow shedderscc0.6750.30.000790.004201tc00.0250.31430.1054ct00.0250.31430.1445tt0.3250.650.003340.02912 haplotypes frequency ( snps rs80787918 and rs80907449 ) and associations with qualitative and quantitative phenotypes of salmonella shedding four snps , rs80811682 ( snp12 ) , snp13 , rs80955017 ( snp15 ) , and rs80894552 ( snp16 ) , located on exon 3 of tlr4 gene are non - synonymous mutations and they are positioned between markers rs80787918 ( snp8 ) and rs80907449 ( snp18 ) . when the additive effect of those markers was tested within the haplotype constructed with markers rs80787918 ( snp8 ) and rs80907449 ( snp18 ) , we did not observe any improvement in the association test . however , analyzing together the markers rs80787918 ( snp8 ) , rs80811682 ( snp12 ) , snp13 , rs80955017 ( snp15 ) , rs80894552 ( snp16 ) and rs80907449 ( snp18 ) , the haplotype composed of alleles ( ctggcc ) was found in higher frequency ( 65 % ) in persistent shedders than 31 % in low shedders pigs ( p < 0.003 ) . possibly , the addition of more markers in the haplotype is being penalized by the increased number of degrees of freedom and reduced number of samples per each class affecting the significance of our association results . when markers snp13 and rs80894552 ( snp16 ) , which were not significant in the single marker association test , were removed from the haplotype , the significance improved to p < 0.001 . the haplotype composed of alleles ctgc was found in 65 % of the high shedders and 30 % of low shedders and the haplotype ( taat ) was found in 20 % of the high shedders and 41 % of the low shedders ( p < 0.04 ) . finally , a specific haplotype ( ctc ) constructed with markers rs80787918 ( snp8 ) , rs80811682 ( snp12 ) and rs80907449 ( snp18 ) , was observed in 65 % of the persistent shedders and 30 % on the low shedders pigs ( p < 0.001 ) . the opposite haplotype ( tat ) was observed in 27.5 % of the persistent shedders and in 59.4 % of the low shedders pigs ( p < 0.003 ) . a trend was observed between haplotypes constructed with markers : rs80787918 ( snp8 ) , snp13 and rs80907449 ( snp18 ) ( cgc ) ; rs80787918 ( snp8 ) , rs80955017 ( snp15 ) and rs80907449 ( snp18 ) ( cgc ) ; rs80787918 ( snp8 ) , rs80894552 ( snp16 ) and rs80907449 ( snp18 ) ( ccc ) , where they were observed in 67.5 % of the persistent shedders and 30 % of low shedder pigs . haplotype cc of snps rs80787918 ( snp8 ) and rs80907449 ( snp18 ) was identified in higher frequency in persistent shedding pigs ( 67.5 % : n = 14 ) compared to low shedding pigs ( 30 % ; n = 6 ) ; furthermore , the frequency of haplotype tt in low shedding pigs ( 65 % ; n = 13 ) was greater when compared to persistent shedding pigs ( 32.5 % ; n = 6 ) . no animals from the persistent shedding group were identified with the haplotype tc or ct , while it was observed in low frequency in the low shedding group ( 2.5 % ) . together , these results suggest that the region located between markers rs80787918 and rs80907449 , more specifically on exon 3 , is possibly harboring the causative mutation for salmonella colonization and shedding variation in swine . the results from this study support the concept that tlr4 is an important modulator associated with the porcine response to salmonella infection in swine . particularly interesting is that the haplotype with the highest significant association to the shedding phenotypes was found most often ( 65 % ) in the persistent shedder pigs than in low shedder pigs . genetic variation in molecular functional regions , such as a ligand recognition site , can alter host resistance / susceptibility to specific pathogens ( uenishi et al . furthermore , synonymous mutations in a gene can play a significant role in transcriptional regulation ( sauna and kimchi - sarfaty 2011 ; sato et al . ( 2011 ) who demonstrated polymorphisms in tlr5 and tlr2 alter the cellular response to s. choleraesuis , our results highlight the importance of linking genetic variations that may influence the molecular function of a key transcriptional regulator ( tlr4 ) with salmonella shedding in swine . mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendations or endorsement by the u.s .

toll - like receptor 4 ( tlr4 ) is a key factor in the innate immune recognition of lipopolysaccharide ( lps ) from gram - negative bacteria . previous studies from our group identified differences in the expression profile of tlr4 and genes affected by the tlr4 signaling pathway among pigs that shed varying levels of salmonella , a gram - negative bacterium . therefore , genetic variation in this gene may be involved with the host s immune response to bacterial infections . the current study screened for single nucleotide polymorphisms ( snps ) in the tlr4 gene and tested their association with salmonella fecal shedding . pigs ( n = 117 ) were intranasally challenged at 7 weeks of age with 1 109 cfu of s. typhimurium 4232 and were classified as low or persistent salmonella shedders based on the levels of salmonella being excreted in fecal material . salmonella fecal shedding was determined by quantitative bacteriology on days 2 , 7 , 14 , and 20/21 post exposure , and the cumulative levels of salmonella were calculated to identify the low ( n = 20 ) and persistent ( n = 20 ) salmonella shedder pigs . from those 40 animals , the tlr4 region was sequenced , and 18 single nucleotide polymorphisms ( snps ) in tlr4 were identified . twelve snps have been previously described and six are novel snps of which five are in the 5 untranslated region and one is in intron 2 . single marker association test identified 13 snps associated with the qualitative trait of salmonella fecal shedding , and seven of those snps were also associated with a quantitative measurement of fecal shedding ( p < 0.05 ) . using a stepwise regression process , a haplotype composed of snps rs80787918 and rs80907449 ( p 4.0 103 ) spanning a region of 4.9 kb was identified , thereby providing additional information of the influence of those snps on salmonella fecal shedding in pigs.electronic supplementary materialthe online version of this article ( doi:10.1007/s13353 - 014 - 0199 - 8 ) contains supplementary material , which is available to authorized users .

we conducted a cross - sectional seroprevalence survey at znbts from september 20 to december 10 , 2011 . the estimated prevalence was set at 50% because data were not available regarding the true prevalence of the infection in the area . considering a population of 1,000,000 inhabitants and a confidence level of 95% , the sample size was set at 384 donors . sample size was then increased to 500 donors to account for those lost to follow - up . during the study period , all consecutive adult donors attending znbts , who had been screened and selected for blood donation , donors were screened by serologic tests for hepatitis b virus , hepatitis c virus , hiv , and trepomena pallidum ; they were selected for blood donation if results of all screening tests were negative . a structured interview was conducted by using a close - ended questionnaire after the donor signed the informed consent form and before the screening . from each enrolled person , 10 ml of venous blood was collected . after the screening tests , the remaining serum was divided into 2 aliquots : 1 was stored at 20c at the sample processing site for performance of the igg elisa at mnazi mmoja hospital in unguja , zanzibar , and 1 was dispatched to the l. spallanzani national institute for infectious diseases in rome , italy , for testing by immunofluorescence assay ( ifa ) for igg . at the end of the collection phase , samples were tested by panbio dengue igg indirect elisa kit ( inverness medical innovations australia pty ltd , sinnamon park , queensland , australia ) according to the manufacturer s instructions . a positive elisa result was defined as having an index value > 1.1 . to compensate for the low specificity of the elisa , we tested samples by ifa with homemade slides and a mix of uninfected and denv-2 ( new guinea c strain)infected vero e6 cells . donors were considered positive for igg against denv if results of both tests were positive . univariate association between denv igg positivity and donor characteristics was assessed by means of odds ratios ( ors ) and 95% cis , by for categorical values , and student t - test for continuous variables . all variables were entered in the backward selection model , and a cutoff level of p = 0.10 was used for subsequent selections . data management and analysis were performed by using stata version 11 ( statacorp , college station , tx , usa ) . five hundred persons consecutively attending znbts were selected for blood donation and , therefore , were eligible to be enrolled in the study . most donors had a water storage container ( 71.2% ) and/or resting water near their home ( 88.0% ) . bed nets were used by 59.4% of the donors , but only 40% reported insecticide spraying at home . * or , odds ratio . age , 5-y increase is defined as the odds ratio for every 5 years of increase . resting water is defined as any presence of water resting around the house ( i.e .. ponds , puddles ) . water storage is defined as any container used to collect rain water with no mention of the size and type of container . of the 500 blood samples , 253 ( 50.6% ) were positive by both tests , 77 ( 15.4% ) were positive by elisa and negative by ifa , and 170 ( 34.0% ) were negative by both tests . considering ifa as the reference standard , elisa sensitivity and specificity were 100% and 68.8% , respectively . according to univariate analysis , positive donors were significantly more likely to be older ( or 1.32 , 95% ci 1.191.47 ) and live in urban districts ( or 3.18 95% ci 2.214.69 ) compared with denv igg - negative donors ( table 1 ) . according to multivariate analysis , older age ( adjusted or [ aor ] 1.42 , 95% ci 1.271.61 ) and living in an urban district ( aor 4.09 , 95% ci 2.726.17 ) borderline evidence indicated an association of positivity with the presence of resting water near the home ( aor 1.66 , 95% ci 0.992.76 ) . * or , odds ratio . age , for 5-y increase is defined as the or for every 5 years of increase . water storage is defined as any container used to collect rain water with no mention of the size and type of container . we found high denv igg seroprevalence ( 50.6% ) in adult blood donors residing in the urban district . our results , compared with the previous low prevalence rate ( 7.7% ) detected on pemba island and the zanzibar archipelagos in 2007 ( 7 ) , suggest an endemic pattern of transmission of denv infection in zanzibar , similar to the situation in other african countries ( 10 ) . considering the progressive reduction of laboratory - confirmed malaria cases in zanzibar and the nonspecific influenza - like symptoms of denv primary infections , this wide denv circulation in zanzibar appears to be largely underdiagnosed . patients with primary denv infection are likely to be mistakenly treated with antimalarial drugs on the basis of clinical symptoms , as we observed on pemba ( 11 ) . the strong association with age could be explained by the progressively longer exposure of the older donors to the risk for infection . this association could be also explained by the successful malaria vector control initiative ( use of long - lasting insecticidal nets , indoor residual spraying and biolarviciding at mosquito breeding sites , and environmental management ) , which could account for the lower denv prevalence in the younger population ( 12 ) . of note , in our study the proportion of persons who used bed nets and those who did not keep water storage containers near home was quite low . this may have been because of the low proportion of women who donate blood , a consequence of the cultural belief that women are weaker because of blood losses during menstrual periods and pregnancies . this explanation was reported by the workers at znbts and is in accordance with results from a previous study on african immigrants ( 13 ) . our sample population was thus less representative of the general population and could have affected the results , either by underestimating or overestimating the inferred prevalence . second , we did not attempt to detect denv circulating serotypes in this study ; nevertheless , there is evidence of denv-3 circulation in previous reports about imported denv cases in europe and japan ( 46 ) . third , no antibody neutralizing assay has been performed to rule out cross - reactions with other circulating flaviviruses , but epidemiologic data from zanzibar do not indicate outbreaks of other flavivirus infections . our data constitute the first step toward better defining the circulation of denv in the archipelago and toward building up a preparedness and response plan to fight denv infection .

we conducted a seroprevalence survey among 500 healthy adult donors at zanzibar national blood transfusion services . dengue virus igg seroprevalence was 50.6% and independently associated with age and urban residence . these data will aid in building a surveillance , preparedness , and response plan for dengue virus infections in the zanzibar archipelago .

cerebral palsy ( cp ) is a term to describe a permanent , mutable motor development disorders stemming from a primary brain lesion that cause secondary musculoskeletal problems leading to limitations in activities of daily living1 . motor impairment is the main manifestation of cerebral palsy ( cp ) , and it has a consequent effect on the biomechanics of the body . children with cp may also exhibit cognitive , visual and hearing impairments , which , along with motor impairment , task restrictions and environmental restrictions , have a negative effect on functional performance2 , 3 . neuromotor impairment in this disease can involve different parts of the body , has led to development of specific topographic classifications , such as quadriplegia , hemiplegia and diplegia4 . however , children with cp are currently classified based on their degree of functional independence , which encompasses the functions of the body , activities and social participation . the gross motor function classification system ( gmcfs ) for cerebral palsy5 classifies children according to age ( 02 , 24 , 46 and 612 years ) and respective functional levels . three - dimensional gait analysis is used to assist in the functional characterization of children with cp , allowing a detailed evaluation of kinetic and kinematic aspects of each phase of the gait cycle . this form of analysis is an important tool in the evaluation of the results of clinical interventions for cp sufferers , who have functional limitations due to excessive muscle weakness and abnormalities in both joint kinematics and postural reactions6 . different therapeutic interventions have been employed in an attempt to favor selective muscle control and coordination in children with cp . 7 reported that the use of an ankle - foot orthosis assists with gait improvement . in a systematic review of the influence of rigid and articulated orthoses , pasini neto et al . however , this type of orthosis is directed at children with accentuated motor impairment , spasticity and contractures . on the other hand , articulated orthoses offer the benefits of stability and freedom during gait , raising the functional potential of children with cp . the aim of postural insoles is to reorganize the tonus of the muscle chains and influence body posture through correction reflexes . these insoles affect muscle proprioception and lead to changes in the ascending proprioceptive chains9 . according to gagey and weber9 , the stimulation of specific regions of the soles of the feet causes a change in postural tonus , and repositions of the leveling of the pelvis and muscle asymmetries along the spinal column . postural reprogramming occurs when the mechanoreceptors of the plantar region are activated by a deformation in the skin caused by the bars , wedges , half - moons and shims incorporated into postural insoles10 . in a study of such postural insoles used by children with cp , the kinetic , kinematic and electromyographic analyses revealed a reduction in plantar flexion as well as better coordination between the tibialis and gastrocnemius muscles and improved force distribution during the support phase11 . the hypothesis guiding the present study was that postural insoles would generate a change in sensory afference , stimulating a postural reaction which improves a gait performance . therefore , the aim of this study was to assess the effect of postural insoles on gait performance of children with cp using the gait variables of cadence and velocity as the primary outcomes . the present study was a preliminary randomized , controlled , double - blind , clinical trial . the study method was conducted , conformed to the principles of the declaration of helsinki and the regulating norms and guidelines for research involving human subjects formulated by the brazilian national health council , ministry of health , established in october 1996 . the study received approval from the ethics committee of the universidade nove de julho ( sao paulo , brazil ) under protocol number 436960/2011 . all parents / guardians agreed to the participation of the children by signing a statement of informed consent . twenty - five children were recruited and ten were selected based on the eligibility criteria . the inclusion criteria were a diagnosis of spastic diplegic cp and classification on levels of i or ii of the gross motor function classification system ( gmfcs ) . the following were the exclusion criteria : surgical procedures or the administration of phenol in the previous 12 months ; neurolytic block in the previous six months ; cognitive or visual impairment that could have interfered with the performance of the procedures ; and ankle deformities that were non - reducible to neutral . the control group ( cg ) made use of an insole without correction elements ( placebo insole ) and the experimental group ( eg ) made use of an insole with correction elements ( postural insole ) . neither the children nor their guardians were aware of the group to which the participants had been allocated ; thus conditions for a blind study for the placebo effect of the insole in the cg were satisfied . during the randomization procedure , a set of sealed , each envelop contained a card stipulating to which group the child would be allocated . the middle portion is made up of ethylene vinyl acetate measuring 3 mm in thickness . the lower portion is composed of material formed by a weave of cotton fibers and resin measuring 1 mm in thickness and contains wedges and shims made of ethylene vinyl acetate10 . the pieces used in the present study were half - moon and anti - valgus ( fig . 1.representation figure of the elements used in postural insoles a- half - moon ; b- anti - valgus ( podaly ) . ) . 2.the representation of placebo insole used by the control group ( podaly ) . ) . representation figure of the elements used in postural insoles a- half - moon ; b- anti - valgus ( podaly ) . the representation of placebo insole used by the control group ( podaly ) . following the positioning of the pieces , the evaluation process was performed under three different conditions : 1 ) barefoot ; 2 ) wearing shoes without insoles ; and 3 ) wearing shoes with placebo insole ( cg ) or postural insoles ( eg ) . the test order under the different conditions was randomly determined by lots to avoid standardization of the behavior of the sample . the children were first shown the equipment and instructed on how to the procedures would be carried out . a training session was then performed , simulating a regular gait exam , but without data collection . in this , the children were instructed to walk normally on a track demarcated on the floor measuring four meters in length and 90 centimeters in width that was marked on the floor . the markers were then fixed to the children in the standing position , as suggested by davis et al12 . the markers were enveloped in adhesive tape lined with microscopic glass spheres and attached to a plastic base with double - sided adhesive tape to the skin allow better visualization by the infrared cameras . the equipment used for the gait evaluation was the smart - d 140 system ( bts engineering ) , with eight cameras sensitive to the infrared spectrum . the gait cycle was determined from the moment of initial contact of one foot ( foot strike ) through to the foot strike of the second foot and toe - off of the first foot until the second foot strike of the first foot ( one complete stride ) . the children were instructed to walk along the demarcated track six times for the data collection . this procedure was performed under the three different conditions ( barefoot , wearing shoes without insoles and wearing shoes with postural insoles or placebo insoles ) . the researcher in charge of this phase of the study was unaware of the group to which each child belonged ( double - blind trial ) . for data involving the analysis of the right and left legs ( step length and stride length ) , the data were first submitted to the kolmogorov - smirnov test to determine if they were normally distributed . as data were parametric , the results were expressed as means and standard deviations or 95% confidence intervals . the effect size was calculated considering the mean difference of between the results obtained for the participants wearing shoes with postural insole . repeated - measure anova was used for the intra - group analysis under each condition . the data were organized and tabulated using the statistical package for the social sciences ( spss v.19.0 ) . among the 25 children recruited for the present study , thirteen children did not meet the eligibility criteria and two refused to participate . thus , the sample was made up of 10 children with cp , five of whom were randomly allocated to the cg and five were randomly allocated to the eg ( fig . 3fig . no statistically significant differences between groups were found for the anthropometric data ( table 1table 1 . the anthropometric characteristics of the sampleanthropometric dataage ( years)height ( cm)body mass ( kg)mean8123 20standard deviation2.9355.2mean sd of anthropometric data of participants ) . mean sd of anthropometric data of participants regarding the temporal gait variables , the intra - group analysis revealed a significant improvement in cadence ( number of steps per minute ) ( p = 0.03 ) and velocity ( p = 0.02 ) in the eg when wearing the postural insoles in comparison to walking barefoot and with shoes alone . moreover , no significant differences were found in temporal gait variables between the latter two conditions ( barefoot and shoes alone ) ( table 2table 2 . gait variables when barefoot , wearing shoes without insoles and wearing shoes with postural insoles ( eg ) or shoes with placebo insole ( cg)barefootshoes without insoleshoes with insolesegcgegcgegcgsupport phase ( % ) 55.3 ( 5.4)55.3 ( 2.3)46.0 ( 9.6)48.6 ( 11.2)53.8 ( 4.8)51.4 ( 5.3)swing phase ( % ) 41.6 ( 3.0)42.0 ( 3.7)37.6 ( 9.1)41.9 ( 4.3)40.0 ( 2.1)43.1 ( 6.7)double support phase ( % ) 5.1 ( 1.6)6.2 ( 1.6)6.7 ( 9.1)6.4 ( 1.6)10.9 ( 4.7)20.0 ( 28.6)step time ( s)0.83 ( 0.12)0.96 ( 0.18)0.91 ( 0.17)1.06 ( 0.23)0.90 ( 0.20)0.98 ( 0.17)cadence ( step / min)111.5 ( 18.7)111.0 ( 20.7)87.7 ( 25.1)99.9 ( 27.4)124.9 ( 7.0)*99.2 ( 25.9)step length ( m)0.34 ( 0.10)0.33 ( 0.06)0.34 ( 0.14)0.33 ( 0.06)0.32 ( 0.91)0.33 ( 0.05)stride length ( m)0.78 ( 0.21)0.75 ( 0.15)0.67 ( 0.19)0.71 ( 0.12)0.79 ( 0.19)0.71 ( 0.13)velocity ( m / s)0.89 ( 0.10)0.81 ( 0.17)0.83 ( 0.13)0.82 ( 0.09)0.98 ( 0.13)*#0.84 ( 0.17)*p 0.05 ( intra - group analysis repeated - measure anova ) ; # p 0.05 ( inter - group analysis independent t - test ) ) . in the intergroup analysis , a significant increase in gait velocity ( p = 0.01 ) was found in the eg in comparison to the cg . moreover , no significant differences were found between groups under the conditions of barefoot and smooth insoles without corrective pieces , demonstrating the homogeneity of the sample ( table 2 ) . * p 0.05 ( intra - group analysis repeated - measure anova ) ; # p 0.05 ( inter - group analysis independent t - test ) in the analysis of the effect of the postural insoles in comparison to the placebo insole , a tendency toward a positive effect was seen with the use of the postural insoles for the majority of gait variables analyzed . however , significant differences were only found for to cadence ( p = 0.019 ) and velocity ( p = 0.021 ) ( table 3table 3 . effect of treatment on all outcome measuresshoes with postural insolesegcgeffectsupport phase ( % ) 7.8 ( 7.322.9)2.7 ( 5.811.3 ) 5.05swing phase ( % ) 2.4 ( 11.015.0)1.1 ( 4.06.4)1.21double support phase ( % ) 13.6 ( 21.148.3)4.1 ( 1.69.9)9.46step time ( s)0.04 ( 0.10.1)0.07 ( 0.10.3)0.07cadence ( step / min)37.1 ( 11.063.2)0.78 ( 16.915.3)37.92*step length ( m)0.06 ( 0.000.02)0.1 ( 0.10.08)0.02stride length ( m)0.06 ( 0.0080.02)0.01 ( 0.010.08)0.12velocity ( m / s)0.14 ( 0.030.32)0.08 ( 0.020.04)0.22**p 0.05 ( inter - group analysis independent t - test ) ) . * p 0.05 ( inter - group analysis independent t - test ) the findings of the present study evidence a tendency toward an immediate positive effect on temporal gait variables when the use of postural insoles were worn by children with cp . however , the inter - group analysis revealed significant differences only with regard to cadence and velocity . a number of authors have demonstrated the importance of analyzing temporal gait variables in children with cp13 , 14 . redekop , andrysek and wright15 assessed computerized gait analysis with regard to functional level on relation to the gmfcs and found adequate to excellent reliability considering temporal , spatial and kinematic variables of the pelvis , hip , knee and ankle . according to abel and damiano13 , thus , the positive results in the analysis of the effect demonstrated in table 3 , although individually not statistically significant , reflect the significant increases in gait velocity and cadence when taken together . according to morita et al.16 , enhanced gait efficiency is directly related to an increase in velocity , and children with cp used an increase in cadence as their main strategy for increasing velocity . this observation may explain the findings of our present study , in which significant changes were only found with regard to cadence and velocity . healthy children show greater spatiotemporal variables are found thant those with cp . according to holt et al . 17 , healthy four - year - olds have mean cadence and velocity values of 152 steps / min and 0.99 m / s , respectively . in the present study , mean cadence when barefoot and wearing shoes without insoles was 111 and 87 steps / min , respectively , and immediately increased to 124 steps / min when using the postural insoles were worn , allowing the children with cp achieve a cadence closer to the cadence demonstrated by healthy children . likewise , mean velocity when barefoot and wearing shoes without insoles was 0.89 and 0.83 m / s , respectively , and increased significantly to 0.98 m / s when using the postural insoles , allowing the children with cp to nearly reach the reference value for velocity of healthy children described by holt et al.17 ( 0.99 m / s ) . it should be stressed that the present study offers preliminary findings on the effects of postural insoles on the gait of children with cp . considering the mean velocity of 0.98 m / s ( standard deviation : 0.13 m / s ) in the eg and 0.84 m / s ( standard deviation : 0.17 m / s ) in the cg , giving for a bi - directional alpha of 0.05 and an 80% test power , 12 children per group would be needed to determine the effects of postural insoles more specifically in this population . thus , our research group is currently preparing a study with an adequate sample size , three - dimensional gait analysis , and an assessment of function in children with cp . the use of postural insoles led to improvements in gait performance ( velocity and cadence ) in children with cerebral palsy classified as levels i or ii of the gmfcs . the present investigation is a preliminary study and did not perform kinetic or kinematic analyses of the gait cycle and did not compare the effect of postural insoles with different types of orthosis during gait .

[ purpose ] improved gait efficiency is one of the goals of therapy for children with cerebral palsy ( cp ) . postural insoles can allow more efficient gait by improving biomechanical alignment . the aim of the present study was to assess the effect of postural insoles on gait performance of children with cp classified as levels i or ii of the gross motor function classification system ( gmfcs ) . [ subjects and methods ] the study was a randomized controlled double - blind clinical trial . after meeting the legal aspects and the eligibility criteria , 10 children between four and 12 years old were randomly divided into a two groups : a control group ( n=5 ) , and an experimental group ( n=5 ) . children in the control group used a placebo insoles , and children in the experimental group used postural insoles . evaluation consisted of three - dimensional gait analysis under three conditions : barefoot , shoes without insoles and shoes with postural insoles or shoes with placebo insoles . [ results ] regarding the immediate effects of insole use , significant improvements in gait velocity and cadence were observed in the experimental group in comparison to the control group . [ conclusion ] the use of postural insoles led to improvements in gait velocity and cadence of the children with cerebral palsy classified as levels i or ii of the gmfcs .

the online version of this article ( doi:10.1007/s40119 - 014 - 0033 - 8 ) contains supplementary material , which is available to authorized users . due to an ageing population , percutaneous coronary intervention ( pci ) is being increasingly undertaken in patients with multiple comorbidities and complex lesions . as a result , pci is increasingly performed in patients considered to be at prohibitive risk for coronary artery bypass grafting ( cabg ) [ 1 , 2 ] . many of these patients also have significant left ventricular ( lv ) impairment , and pci in this setting is associated with an increased risk of peri - procedural complications [ 3 , 4 ] . mechanical - assist devices have been frequently employed to support high - risk pci in these situations in the hope of reducing this risk . such mechanical assistance has been conventionally provided by intra - aortic balloon counterpulsation ( iabp ) , although randomised trials have failed to show benefit of elective iabp both in high - risk pci and cardiogenic shock complicating acute myocardial infarction . this may be due to the limited support that counter - pulsation provides in augmenting cardiac output and in reducing left ventricular afterload . percutaneous left ventricular assist devices ( lvad ) such as the tandemheart ( cardiac assist inc . , pittsburg , usa ) have been shown to be safe and feasible in this setting , and also provide superior haemodynamic support as compared to iabp [ 7 , 8 ] . however , the tandemheart is complex to use and requires a trans - septal puncture . the impella ( abiomed , danvers , usa ) device is a percutaneous catheter - based impeller - driven lvad which aspirates blood from the lv cavity expelling the removed blood into the aorta . it has been shown to provide superior cardiac support compared with iabp in both animal [ 9 , 10 ] and human studies , reducing lv end diastolic pressure , wall stress , myocardial oxygen consumption and improving coronary perfusion and cardiac output [ 1214 ] . the impella device has gained increasing popularity in acute cardiac care , most commonly in high - risk pci . large registries like uspella and europella have demonstrated the feasibility and safety of this device in this setting . more recently , the protect - ii trial is the first randomised controlled trial demonstrating the haemodynamic benefit of this device over iabp in elective patients undergoing high - risk pci and a trend to improved clinical outcomes at 30 days . in addition to high - risk pci , impella has also been used successfully in cardiogenic shock [ 1720 ] , acute cardiac transplant rejection [ 21 , 22 ] and refractory heart failure as a bridge to transplantation [ 23 , 24 ] . historically , iabp has been the mechanical assist device of choice in the uk , although recent trials from the uk have shown no early benefit of prophylactic iabp in elective high - risk pci , with a recent analysis showing a beneficial effect on 5-year mortality . we therefore , present our experience with the use of the impella device in a uk quaternary cardiac centre . the queen elizabeth hospital , birmingham , uk , is a large quaternary cardiac centre and provides regional cardiac transplantation . we retrospectively analysed our interventional procedural database and identified all patients undergoing impella implantation since the start of the programme in october 2008 until january 2014 on an intention to treat basis . clinical and procedural data was procured from electronic patient records , the procedural database and procedure logs in the cardiac catheter laboratory . all patients were included in an intention to treat manner ; there were no exclusions . the 5l impella was inserted via the subclavian artery following surgical exposure and application of a dacron graft , as previously described . for trans - femoral access , arterial puncture was performed after fluoroscopic localisation of the femoral head , with or without ultrasound guidance and , more recently , with the use of a 4f ( french ) micropuncture kit ( micropuncture introducer set silhouette transitionless , cook medical inc . , usa ) to minimise vascular complications . a femoral angiogram was then performed to ensure adequate vessel calibre ( > 4 mm ) and to assess tortuosity and calcification . pre - closure was achieved using two sutures ( perclose proglide 6f suture - mediated closure ( smc ) system , abbott vascular , illinois , usa ) following which a 13f or 14f sheath was inserted for the 2.5l and 3.8l devices , respectively . a judkins right or amplatz catheter was used to cross the aortic valve following which the 0.018 impella guide wire was positioned in the aortic apex . the impella device was then positioned carefully in the lv apex over the 0.018 wire and set to maximal output . special manoeuvres were required for insertion of the device in the five patients who had concomitant severe aortic stenosis ( as ) , as described recently by our group . outcome data for mortality was obtained from electronic patient records linked to the office of national statistics database . peri - procedural myocardial infarction ( pmi ) was defined as a total creatinine kinase level greater than three times the upper limit of normal on the morning after the procedure [ 28 , 29 ] . data are presented as mean standard deviation ( sd ) for continuous variables and percentages for discrete variables . the analysis in this article is based on previously conducted data , and does not involve any new studies of human or animal subjects performed by any of the authors . impella implantation was attempted in a total of 49 patients during the study period : of these , 45 patients underwent high - risk pci , 3 patients required emergency haemodynamic support as a bridge to cardiac transplantation and one patient with severe as underwent balloon valvuloplasty ( bav ) with impella support . implantation of a 2.5l impella failed in one patient undergoing high - risk pci due to extreme calcific iliofemoral disease . of these , the 2.5l impella device was used in 36 ( 75% ) patients , the 3.8l device in 11 ( 23% ) patients , and the 5l device in one patient ( 2% ) . the baseline clinical characteristics of these patients are shown in table 1.table 1baseline characteristicsclinical characteristicstotalhigh - risk pcibavbridge to transplant n = 49 n = 45 n = 1 n = 3age ( years ) ( mean*)72 13 ( 3792)74 11 ( 4792)6748 11(3763)male39 ( 80%)35 ( 78%)13 ( 100%)bmi ( kg / m ) ( mean)27 4 ( 1840)27 4 ( 2040)2522 3 ( 1824)hypertension40 ( 82%)37 ( 82%)03 ( 100%)diabetes17 ( 35%)16 ( 36%)01 ( 33%)smoking28 ( 57%)26 ( 58%)02 ( 66%)dyslipidemia32 ( 65%)31 ( 69%)01 ( 33%)renal function egfr ( ml / min ) ( mean)51 6 ( 11117)54 27(16117)4315 4 ( 1120 ) egfr 306022 ( 45%)21 ( 47%)13 ( 100% ) egfr < 3012 ( 30%)9 ( 14%)03 ( 100%)pvd4 ( 8%)4 ( 9%)00cvd3 ( 6%)3 ( 7%)00previous mi24 ( 49%)22 ( 49%)02 ( 67%)previous cabg3 ( 6%)3 ( 7%)00previous pci9 ( 18%)7 ( 16%)02 ( 67%)lvef < 35%39 ( 80%)36 ( 80%)13 ( 100%)lvef ( % ) ( mean)28 14 ( 1060)28 14 ( 1060)1015 4 ( 1020)impella characteristics successful implant48 ( 98%)44 ( 98%)13 ( 100% ) 2.5l device36 ( 76%)34 ( 77%)02 ( 67% ) 3.8l device11 ( 23%)10 ( 23%)10 5.0l device1 ( 2%)001 ( 33% ) bav balloon valvuloplasty , bmi body mass index , cabg coronary artery bypass grafting , cvd cerebrovascular disease , ecmo extracorporeal membrane oxygenation , egfr estimated glomerular filtration rate , lvef left ventricular ejection fraction , mi myocardial infarction , pci percutaneous coronary intervention , pvd peripheral vascular disease , sd standard deviation mean sd ( range ) baseline characteristics bav balloon valvuloplasty , bmi body mass index , cabg coronary artery bypass grafting , cvd cerebrovascular disease , ecmo extracorporeal membrane oxygenation , egfr estimated glomerular filtration rate , lvef left ventricular ejection fraction , mi myocardial infarction , pci percutaneous coronary intervention , pvd peripheral vascular disease , sd standard deviation 45 patients , including five patients with concomitant severe as , underwent high - risk pci . impella was successfully implanted in all but one patient ( 44/45 [ 98% ] ) . of these , 10 patients were given the 13f 3.8l device , while 34 patients were given the 12f 2.5l device . impella was removed following pci before the patient left the catheter laboratory in all but two patients and in these patients , successful vascular closure was achieved with pre - closure use of one or two proglide devices . pci was performed via the radial approach in 11 patients ( 25% ) and the femoral approach in 33 ( 75% ) . the peri - procedural variables are shown in table 2.table 2procedural characteristics in patients undergoing pcipre - procedural characteristics n = 45urgency of procedure elective pci ( angina)17 ( 38% ) urgent pci ( nstemi)28 ( 62% ) cardiogenic shock ( nstemi)3 ( 7% ) pulmonary oedema ( nstemi)2 ( 4%)high - risk pci features unprotected left main stem24 ( 53% ) last remaining vessel9 ( 20% ) multi - vessel18 ( 40% ) severe lv impairment ( lvef < 35%)36 ( 80%)decision for pci refused cabg ( mdt)28 ( 62% ) patient preference2 ( 4% ) physician s decision ( no mdt)8 ( 18% ) haemodynamic compromise ( no mdt)5 ( 11% ) other2 ( 4%)logistic euroscore8 3 ( 115)nwqip pci risk score6 11 ( 0.471)peri - procedural characteristicsn = 45number of lesions treated2.0 1.0number of stents2.7 1.7 ( 18)rotational atherectomy14 ( 31%)glycoprotein inhibitor use1 ( 2%)complete revascularisation45 ( 100%)impella characteristics successful insertion44 ( 98% ) 2.5l device34 ( 77% ) 3.8l device10 ( 23% ) removal on table42 ( 95% ) cabg coronary artery bypass grafting , lvef left ventricular ejection fraction , mdt multi - disciplinary team , nstemi non - st elevation myocardial infarction , nwqip north west quality improvement programme , pci percutaneous coronary intervention procedural characteristics in patients undergoing pci cabg coronary artery bypass grafting , lvef left ventricular ejection fraction , mdt multi - disciplinary team , nstemi non - st elevation myocardial infarction , nwqip north west quality improvement programme , pci percutaneous coronary intervention the majority of patients ( 80% ) had severe lv impairment ( lvef < 35% ) with 53% ( 24/45 ) of patients undergoing pci to an unprotected left main stem . five patients had severe as with coronary artery disease and pci was performed in preparation for transcatheter aortic valve replacement ( tavr ) or balloon valvuloplasty for clinical stabilisation . pci was performed electively in 17 ( 38% ) patients : 11 of these patients were discussed at a heart multi - disciplinary team ( mdt ) meeting and were thought to be at higher risk for cabg . 28 ( 62% ) patients had a non - st elevation myocardial infarction ( nstemi ) and underwent in patient revascularisation . these 28 patients11 elective and 17 urgent were discussed by the heart mdt and refused cabg due to co - morbidities . five other patients developed ischaemic haemodynamic compromise , three of which had cardiogenic shock and two pulmonary oedema , while awaiting mdt discussion . one patient with severe lv dysfunction due to non - compaction and a single coronary ostium had impella - assisted pci to a severely diseased right coronary artery ( rca ) . finally , 18 patients ( 40% ) underwent multi - vessel pci ; 117 stents ( mean sd 2.7 1.7 lesions ) were used to treat 89 lesions ( mean sd 2.0 1.0 stents ) in 45 patients . complete revascularisation , defined as revascularisation of all intended targets was achieved in all patients . one patient with severe as and intractable cardiogenic shock underwent successful bail - out balloon valvuloplasty with impella ( 3.8l ) support . two of these patients had acute cardiogenic shock following st - elevated myocardial infarction ( stemi ) , despite successful primary angioplasty and conventional management including iabp . one patient had a 2.5l impella implanted via the femoral approach , which allowed sufficient haemodynamic recovery and made the patient suitable for transplantation with an excellent outcome . the other patient had a 5l impella ( 21f ) inserted via surgical subclavian access , but unfortunately , passed away due to gastro - intestinal bleeding and multiple organ system failure complications unrelated to impella . the third patient with acute decompensated heart ( and renal ) failure due to dilated cardiomyopathy had a 2.5l impella implanted femorally but needed early extracorporeal membrane oxygenation ( ecmo ) due to insufficient haemodynamic response ; he eventually underwent successful cardiac and renal transplantation . one patient in whom impella could not be implanted due to vessel tortuosity underwent high - risk pci but died due to ischaemic cardiogenic shock immediately following the procedure . two further patients in this group died due to cardiogenic shock , while one died of pre - existing severe sepsis , having also had a stroke with a dense neurodeficit in the setting of significant bilateral carotid artery stenoses . two patients were given blood transfusions but only as an aid to clinical recovery in the setting of pre - existing anaemia with no evidence of bleeding or a fall in haemoglobin levels . one patient who underwent rotablation and pci to an unprotected left main stem and left - anterior descending ( lad ) artery unfortunately developed coronary perforation and tamponade . impella provided excellent support while this was managed and the device was removed successfully at the end of the procedure . another patient who underwent bav had a suspected retroperitoneal haematoma which could not be radiologically confirmed , as he passed away as soon as the impella device was removed . there were no other major vascular complications.table 3outcomesoutcomestotal ( n = 49)high - risk pci ( n = 45)bav ( n = 1)transplant ( n = 3)30-day mortality10 ( 20%)8 ( 18%)1 ( 100%)1 ( 33%)blood transfusion3 ( 6%)2 ( 5%)01 ( 33%)vascular complications1 ( 2%)01 ( 100%)0stroke1 ( 2%)1 ( 2%)00pmi1 ( 2%)1 ( 2%)1 ( 100%)0hospital stay ( days)5 6 ( 122)5 6 ( 122) bav balloon aortic valvuloplasty , pci percutaneous coronary intervention , pmi peri - procedural myocardial infarction bav balloon aortic valvuloplasty , pci percutaneous coronary intervention , pmi peri - procedural myocardial infarction within the group of patients studied , 30-day survival was 80% ( 39/49 ) , while overall survival after a median follow - up of 29 months ( range 171 months ) was 65% ( 32/49 ) , as shown in fig . 1kaplan meier curve showing survival over a median follow - up period of 29 months ( range 171 ) kaplan meier curve showing survival over a median follow - up period of 29 months ( range 171 ) 45 patients , including five patients with concomitant severe as , underwent high - risk pci . impella was successfully implanted in all but one patient ( 44/45 [ 98% ] ) . of these , 10 patients were given the 13f 3.8l device , while 34 patients were given the 12f 2.5l device . impella was removed following pci before the patient left the catheter laboratory in all but two patients and in these patients , successful vascular closure was achieved with pre - closure use of one or two proglide devices . pci was performed via the radial approach in 11 patients ( 25% ) and the femoral approach in 33 ( 75% ) . the peri - procedural variables are shown in table 2.table 2procedural characteristics in patients undergoing pcipre - procedural characteristics n = 45urgency of procedure elective pci ( angina)17 ( 38% ) urgent pci ( nstemi)28 ( 62% ) cardiogenic shock ( nstemi)3 ( 7% ) pulmonary oedema ( nstemi)2 ( 4%)high - risk pci features unprotected left main stem24 ( 53% ) last remaining vessel9 ( 20% ) multi - vessel18 ( 40% ) severe lv impairment ( lvef < 35%)36 ( 80%)decision for pci refused cabg ( mdt)28 ( 62% ) patient preference2 ( 4% ) physician s decision ( no mdt)8 ( 18% ) haemodynamic compromise ( no mdt)5 ( 11% ) other2 ( 4%)logistic euroscore8 3 ( 115)nwqip pci risk score6 11 ( 0.471)peri - procedural characteristicsn = 45number of lesions treated2.0 ( 31%)glycoprotein inhibitor use1 ( 2%)complete revascularisation45 ( 100%)impella characteristics successful insertion44 ( 98% ) 2.5l device34 ( 77% ) 3.8l device10 ( 23% ) removal on table42 ( 95% ) cabg coronary artery bypass grafting , lvef left ventricular ejection fraction , mdt multi - disciplinary team , nstemi non - st elevation myocardial infarction , nwqip north west quality improvement programme , pci percutaneous coronary intervention procedural characteristics in patients undergoing pci cabg coronary artery bypass grafting , lvef left ventricular ejection fraction , mdt multi - disciplinary team , nstemi non - st elevation myocardial infarction , nwqip north west quality improvement programme , pci percutaneous coronary intervention the majority of patients ( 80% ) had severe lv impairment ( lvef < 35% ) with 53% ( 24/45 ) of patients undergoing pci to an unprotected left main stem . five patients had severe as with coronary artery disease and pci was performed in preparation for transcatheter aortic valve replacement ( tavr ) or balloon valvuloplasty for clinical stabilisation . pci was performed electively in 17 ( 38% ) patients : 11 of these patients were discussed at a heart multi - disciplinary team ( mdt ) meeting and were thought to be at higher risk for cabg . 28 ( 62% ) patients had a non - st elevation myocardial infarction ( nstemi ) and underwent in patient revascularisation . these 28 patients11 elective and 17 urgent were discussed by the heart mdt and refused cabg due to co - morbidities . five other patients developed ischaemic haemodynamic compromise , three of which had cardiogenic shock and two pulmonary oedema , while awaiting mdt discussion . one patient with severe lv dysfunction due to non - compaction and a single coronary ostium had impella - assisted pci to a severely diseased right coronary artery ( rca ) . finally , 18 patients ( 40% ) underwent multi - vessel pci ; 117 stents ( mean sd 2.7 1.7 lesions ) were used to treat 89 lesions ( mean sd 2.0 1.0 stents ) in 45 patients . complete revascularisation , defined as revascularisation of all intended targets was achieved in all patients . one patient with severe as and intractable cardiogenic shock underwent successful bail - out balloon valvuloplasty with impella ( 3.8l ) support . two of these patients had acute cardiogenic shock following st - elevated myocardial infarction ( stemi ) , despite successful primary angioplasty and conventional management including iabp . one patient had a 2.5l impella implanted via the femoral approach , which allowed sufficient haemodynamic recovery and made the patient suitable for transplantation with an excellent outcome . the other patient had a 5l impella ( 21f ) inserted via surgical subclavian access , but unfortunately , passed away due to gastro - intestinal bleeding and multiple organ system failure complications unrelated to impella . the third patient with acute decompensated heart ( and renal ) failure due to dilated cardiomyopathy had a 2.5l impella implanted femorally but needed early extracorporeal membrane oxygenation ( ecmo ) due to insufficient haemodynamic response ; he eventually underwent successful cardiac and renal transplantation . one patient in whom impella could not be implanted due to vessel tortuosity underwent high - risk pci but died due to ischaemic cardiogenic shock immediately following the procedure . two further patients in this group died due to cardiogenic shock , while one died of pre - existing severe sepsis , having also had a stroke with a dense neurodeficit in the setting of significant bilateral carotid artery stenoses . two patients were given blood transfusions but only as an aid to clinical recovery in the setting of pre - existing anaemia with no evidence of bleeding or a fall in haemoglobin levels . one patient who underwent rotablation and pci to an unprotected left main stem and left - anterior descending ( lad ) artery unfortunately developed coronary perforation and tamponade . impella provided excellent support while this was managed and the device was removed successfully at the end of the procedure . another patient who underwent bav had a suspected retroperitoneal haematoma which could not be radiologically confirmed , as he passed away as soon as the impella device was removed . there were no other major vascular complications.table 3outcomesoutcomestotal ( n = 49)high - risk pci ( n = 45)bav ( n = 1)transplant ( n = 3)30-day mortality10 ( 20%)8 ( 18%)1 ( 100%)1 ( 33%)blood transfusion3 ( 6%)2 ( 5%)01 ( 33%)vascular complications1 ( 2%)01 ( 100%)0stroke1 ( 2%)1 ( 2%)00pmi1 ( 2%)1 ( 2%)1 ( 100%)0hospital stay ( days)5 6 ( 122)5 6 ( 122) bav balloon aortic valvuloplasty , pci percutaneous coronary intervention , pmi peri - procedural myocardial infarction bav balloon aortic valvuloplasty , pci percutaneous coronary intervention , pmi peri - procedural myocardial infarction within the group of patients studied , 30-day survival was 80% ( 39/49 ) , while overall survival after a median follow - up of 29 months ( range 171 months ) was 65% ( 32/49 ) , as shown in fig . 1.fig . 1kaplan meier curve showing survival over a median follow - up period of 29 months ( range 171 ) kaplan meier curve showing survival over a median follow - up period of 29 months ( range 171 ) we have reported the first real - world experience of the impella percutaneous left ventricular assist device including the 3.8l device across a range of indications . we have demonstrated its safety , feasibility and efficacy in high - risk pci , to support bav and as a bridge to transplantation in acute decompensated heart failure . the bcis-1 investigators reported no reduction in 30-day major adverse cardiac events ( mace ) with the use of iabp prophylactically in elective high - risk pci . however , on longer follow - up of 5 years , there was a statistically significant reduction in allcause mortality in the iabp group , although the mechanism of this benefit is unclear . while the role of iabp in the elective setting remains controversial , current guidelines and recent trials have also questioned the efficacy of iabp in cardiogenic shock complicating acute myocardial infarction . the impella ventricular assist device ( vad ) system , however , has been shown to provide superior haemodynamic support than iabp [ 16 , 17 ] . the randomised protect - ii study demonstrated that in the setting of high - risk elective pci , the impella device not only provided superior haemodynamic support than iabp , but also resulted in a statistically significant 22% reduction in major adverse events at 90 days largely driven by a reduction in repeat revascularisation . impella facilitated more aggressive and complete revascularisation with a higher use of rotational atherectomy ( 14% vs. 9% ) . similarly , we have also shown a high proportion of rotational atherectomy ( 31% ) in our high - risk pci cohort . at present , there is no agreed definition of what constitutes a high - risk pci , although large registries such as europella and uspella have included patients undergoing pci to unprotected left main stem , pci to the last remaining vessel and multivessel pci in the context of impaired left ventricular function . patients not suitable for cabg due to high risk are increasingly undergoing pci which has been shown to be a safe and feasible strategy in these patients . 62% of our patients were discussed by the mdt and were deemed to be at too high risk for cabg due to co - morbidities . several risk scoring models such as the mayo pci risk score have been developed to anticipate the in - hospital major event rate in pci . we have used the north west quality improvement programme ( nwqip ) scoring system , which had been standardised to a uk population and validated against the established international risk models . population - based risk models , however , may not capture all the high - risk characteristics of an individual patient , and the definition of high - risk pci and the need for mechanical lv support remains the discretion of the operator . indeed our mean nwqip predicted major event rate was 6 11% ( range 0.471 ) , which is comparable to the mean mayo pci score in the protect ii trial ( 8.8 3.4% in the impella group ) , although our in - hospital mortality was significantly higher at 20% . the overall 30-day major arrhythmic events ( mae ) rate in the uspella registry was 8% with 4% in - hospital mortality . in the europella registry , 30-day mortality was 5.5% with a 30-day mae rate of 12.3% ( including major vascular complications ) . our in - hospital mortality and mae of 20% is substantially higher than both these registries . this may either be a reflection of smaller patient numbers in this study , or the higher - risk profile of the population , as discussed earlier . indeed in the smaller cohort of the protect i trial ( n = 20 ) , the 30-day mae rate was 20% , while in the larger protect ii trial ( n = 225 ) it was 7% . interestingly , we report only one major vascular complication in our cohort as compared to 4% in uspella and 5.5% in the europella registry . while this could be a reflection of smaller patient numbers , it also suggests increasing experience with larger arterial access in the era of percutaneous valvular interventions ( tavi ) and the use of newer techniques such as micropuncture and pre - closure with percutaneous suture devices . this is important in a cohort of patients who can ill - afford any further haemodynamic setbacks . in this cohort , 52% had pci to unprotected left main stem , 40% underwent multi - vessel pci and 20% had pci to the last remaining vessel essentially comparable to the uspella and europella registries . however , our patients were older ( mean age 75 vs. 70 years in the uspella and europella registries ) and had a greater incidence of severe lv impairment ( 80% vs. 69% ) . although the distribution of elective and urgent pci was comparable to the uspella registry ( the europella registry only included elective cases ) , we have used impella in 3 patients with cardiogenic shock , 2 with ischaemic pulmonary oedema and , importantly , in 5 patients with coexisting severe as ; patients with these conditions were not included in the larger registries . overall , our patients were a substantially higher - risk cohort when compared to these registries . this reflects a growing confidence in the use of impella and its expanding indications in acute cardiac care . severe as is reported to be a contraindication to impella use due to theoretical concerns of either reducing effective valve orifice or inducing aortic incompetence . however , with increasing experience there have been anecdotal reports [ 33 , 34 ] and a recent series describing the use of impella in this setting . the use of impella in our patients with severe as is an example of the expanding indication for this device . the methods used for implanting the device in these patients have been described by our group in a previous report . one patient with severe as and lv dysfunction underwent emergency bav with impella support for intractable cardiogenic shock . the rapid deterioration and death that followed removal of the device in this patient for suspected retroperitoneal bleeding highlighted the significant haemodynamic benefit provided by impella . impella was used with success in one patient with acute post - infarct cardiogenic shock as a bridge to transplantation . there are anecdotal reports of the use of impella in similar situations with both the 2.5l and 5l device , and also in transplanted hearts for acute rejection [ 21 , 22 ] . a recent series has also reported the use of 5l impella support in 9 patients with cardiogenic shock due to end - stage ischaemic cardiomyopathy ( 3 patients ) and post - st elevation myocardial infarction ( 6 patients ) . this less invasive percutaneous vad is therefore , a useful additional tool in advanced heart failure management . we have described the use of the impella cp 3.8l device in 11 patients10 for pci and one bav . to our knowledge , this is the first series reporting the use of the 3.8l device which has only recently received conformit europenne ( ce ) marking . notably , despite the larger lumen vascular access ( 14f vs. 13f for 2.5l ) , there was no increase in the incidence of vascular complications , and the haemodynamic support was reliable and superior to that provided by the 2.5l device . we have confirmed that the indications for the impella device are expanding and that it can be used in acutely unwell patients with a high degree of success . the main limitations of the impella device include the requirement for large lumen vascular access and closure , and the significantly higher cost of the device as compared to mechanical support using iabp . a recent study has also confirmed cost - effectiveness of impella compared with iabp , an issue which is crucial in the current economic environment . moreover , the 2014 european society of cardiology guidelines on myocardial revascularisation no longer recommend routine use of iabp in cardiogenic shock , complicating myocardial infarction ( class iii recommendation ) . however , mechanical support using devices such as impella may now be considered for short - term support ( class iib recommendation ) in this setting . no comparison with iabp use was attempted , as iabp was used in milder degrees of circulatory disturbance whereas impella was often used as a last resort . this retrospective analysis provides an insight into " real - world " experience with impella . severe as is reported to be a contraindication to impella use due to theoretical concerns of either reducing effective valve orifice or inducing aortic incompetence . however , with increasing experience there have been anecdotal reports [ 33 , 34 ] and a recent series describing the use of impella in this setting . the use of impella in our patients with severe as is an example of the expanding indication for this device . the methods used for implanting the device in these patients have been described by our group in a previous report . one patient with severe as and lv dysfunction underwent emergency bav with impella support for intractable cardiogenic shock . the rapid deterioration and death that followed removal of the device in this patient for suspected retroperitoneal impella was used with success in one patient with acute post - infarct cardiogenic shock as a bridge to transplantation . there are anecdotal reports of the use of impella in similar situations with both the 2.5l and 5l device , and also in transplanted hearts for acute rejection [ 21 , 22 ] . a recent series has also reported the use of 5l impella support in 9 patients with cardiogenic shock due to end - stage ischaemic cardiomyopathy ( 3 patients ) and post - st elevation myocardial infarction ( 6 patients ) . this less invasive percutaneous vad is therefore , a useful additional tool in advanced heart failure management . we have described the use of the impella cp 3.8l device in 11 patients10 for pci and one bav . to our knowledge , this is the first series reporting the use of the 3.8l device which has only recently received conformit europenne ( ce ) marking . notably , despite the larger lumen vascular access ( 14f vs. 13f for 2.5l ) , there was no increase in the incidence of vascular complications , and the haemodynamic support was reliable and superior to that provided by the 2.5l device . we have confirmed that the indications for the impella device are expanding and that it can be used in acutely unwell patients with a high degree of success . the main limitations of the impella device include the requirement for large lumen vascular access and closure , and the significantly higher cost of the device as compared to mechanical support using iabp . a recent study has also confirmed cost - effectiveness of impella compared with iabp , an issue which is crucial in the current economic environment . moreover , the 2014 european society of cardiology guidelines on myocardial revascularisation no longer recommend routine use of iabp in cardiogenic shock , complicating myocardial infarction ( class iii recommendation ) . however , mechanical support using devices such as impella may now be considered for short - term support ( class iib recommendation ) in this setting . no comparison with iabp use was attempted , as iabp was used in milder degrees of circulatory disturbance whereas impella was often used as a last resort . this retrospective analysis provides an insight into " real - world " experience with impella . we have demonstrated its feasibility and safety in a cohort of higher risk patients with extended and novel indications , and reported for the first time globally the feasibility and safety of the 3.8l device . with increasing experience in the use of impella , the device may be used to provide invaluable support to increasingly complex patients who would , in turn , have the largest benefit . v. venugopal , j. spiro , a. zaphiriou , s. khan , j. n. townend , p. f. ludman and s. n. doshi declare no conflicts of interest . this article does not contain any new studies with human or animal subjects performed by any of the authors . the analysis in this article is based on previously recorded data , and does not involve any new studies of human or animal subjects performed by any of the authors . 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 the source are credited .

aimsthe impella is a percutaneous ventricular assist device . the majority of published data describes the 2.5l and 5.0l devices , and little data is available for the newer 3.8l device . we examined the indications and outcomes from our single - centre real - world registry at the queen elizabeth hospital , birmingham , uk , using all three pump sizes.methods and resultsrecords from all patients who underwent attempted impella - assisted procedures at our centre were examined retrospectively . impella implantation was attempted in 49 patients ( mean age 72 13 years ; 80% male ) and was successful in 48 ( 98% ) . 45 patients underwent high - risk percutaneous coronary intervention ( pci ) , one patient underwent balloon aortic valvuloplasty and 3 patients had impella as a bridge to cardiac transplantation . the 2.5l and 3.8l devices were used in 36 ( 75% ) and 11 ( 23% ) patients , respectively , while one patient ( 2% ) had the 5l device . vascular complications occurred in only one patient ( 2% ) and stroke and peri - procedural myocardial infarction occurred in one patient ( 2% ) , while in - hospital mortality was 20% ( 10/49).conclusionsin this large real - world registry , we have demonstrated the safety and feasibility of the impella device for a wide range of indications . this includes the first series of the 3.8l device which provides superior support with no increase in vascular complications.electronic supplementary materialthe online version of this article ( doi:10.1007/s40119 - 014 - 0033 - 8 ) contains supplementary material , which is available to authorized users .

low birth weight ( lbw ) is associated with an increased risk for a number of conditions arising in adulthood , such as type 2 diabetes , dyslipidemia , coronary artery disease , essential hypertension , and cerebrovascular accidents . insulin resistance ( i.e. , reduced insulin sensitivity ) is a well - established , early metabolic abnormality in the pathogenesis of these conditions . it has been proposed that a reduced insulin sensitivity in lbw subjects results from the adaptation to adverse in utero conditions during a critical period of development . however , it is well known that lbw newborns are also exposed to postnatal stress , which is reflected in higher neonatal morbidity and mortality . this has led to the hypothesis that postnatal stress may as well contribute to the metabolic modifications in lbw children , independently of the adequacy of their birth weight to gestational age . if early postnatal stress plays a role in long - term metabolic modifications , prematurity may be an important confounding factor . most previous studies linking lbw to the propensity toward disease in adulthood have focused on those who were small for gestational age ( sga ) and born at term . only few studies demonstrate the association of prematurity with a tendency to disease in letter adulthood but data 's are still conflicting , and further evidence is still required to establish a definitive role . moreover , it has been recognized that reduced insulin sensitivity , a hallmark in most lbw - related conditions may be present as early as the 1 year of life . hence , we planned a prospective cohort study to elaborate insulin sensitivity in infants who were lbw and born prematurely . in addition , it has been suggested that besides in utero stress and postnatal stress , other independent variables such as ponderal index ( pi ) , birth weight , catch - up growth for weight and length , and current weight and length may also contribute in insulin sensitivity . our second aim , therefore , was to determine the role of other variables to predict insulin sensitivity in infancy . the present study was a prospective ( cohort ) study , conducted in umaid hospital and dr . sample size was calculated at power 80% , confidence interval ( ci ) 95% , four independent variables and assuming expected multiple regression coefficient 0.4 as observed in a previous study titled as , determinants of insulin sensitivity and secretion in very - low - birth weight children bazaes et al . the resulting value of sample size came out to be 68 . in the beginning phase of the study , total 150 neonates born in this tertiary care center were enrolled to compensate loss during follow - up . those with a birth weight at par or above the 10 percentile were defined as being appropriate for gestational age ( aga ) , and those with a birth weight below 10 percentile as being sga . the study population was divided into four strata ( preterm aga , preterm sga , term aga , and term sga ) according to their gestational age and their birth weight for gestation thereafter sampling was done by stratified random sampling method . the study cohort was formed by 50 preterm aga and control was formed by 50 term aga neonates . another cohort of 50 sga neonates ( 25 preterm sga and 25 term sga ) was also included . this group was added for the sake of comparison and in order to determine other variables to predict insulin sensitivity in infancy . during follow - up ( till 9 months of the age ) numbers of participating infants were decreased , 15 were excluded ( 8 preterm aga , 1 term aga , 5 preterm sga , and 1 term sga ) and 35 did not come in follow - up ( 6 preterm aga , 13 term aga , 9 preterm sga , and 7 term sga ) . finally , 100 infants ( 36 preterm aga , 36 term aga , 11 preterm sga , and 17 term sga ) were gone through anthropometric and hormonal evaluation , with adequate sample size . exclusion criteria included neonates of diabetic mother and those having first degree relative with type-2 diabetes mellitus , chronic illness , or medical therapy known to influence insulin sensitivity , chromosomal syndromes , congenital anomalies , congenital infections and sick neonates ( intracranial hemorrhage , perinatal asphyxia , pyomeningitis , necrotizing enterocolitis ) , neonates requiring positive pressure ventilation , and vasopressors . the nature , purpose , and possible risks of the study were explained to the parents in detail before consent was obtained . complete history including family history and antenatal , natal , and postnatal history was taken in all subjects . general physical examination and systemic examination was done , and accurate assessment of gestational age was done by using the expanded new ballard score . detailed anthropometry ( length , weight , pi , head circumference , chest circumference , and weight - for - length ) was performed according to the world health organization ( who ) guidelines by a trained pediatrician . birth weight , length , and head circumference were converted into standard deviation scores ( sds ) ( z = [ observed value mean value]/sd ) according to the who reference to allow comparison of subjects with different gestational ages , chronological ages , and sexes . the weight - for - length index was used to provide an age - adjusted evaluation of relative obesity . routine blood investigation and fasting blood sugar level of all neonates ( after 24 h fasting ) were performed . however , the upper age cutoff for the enrolled subjects was limited to 9 months instead of 12 months in this study to avoid biases arising out of the transition to childhood from infancy during the last few months . the reason behind not using 6 months of age as cut off is because by that age , the term neonates may not be exposed to complementary feed , whereas preterm neonates would have received both breastfeeding and complementary feeding ( corrected age of 6 months for prematurity ) . both cohorts and controls were followed at corrected age of 3 months , 6 months , and 9 months . all infants were on exclusive breastfeed for 5.56 0.84 months ( preterm aga 5.47 0.78 , preterm sga 5.60 0.85 , term aga 5.60 0.85 , term sga 5.79 0.84 , p 0.05 , range 47 months ) and then started complementary feed according to the indian academy of pediatrics guideline . on each follow - up , anthropometry assessment was performed . at the age of 9 months ( 5 ml ) , blood sample was obtained for fasting plasma glucose and serum insulin ( si ) level ( after 24 h fasting ) . glucose was estimated by glucose oxidase - peroxidase aminophenazone - phenol method using the commercially available kit ( human mbh , weisbaden , germany ) . si was estimated by immunoradiometric assay using commercial kits ( dpc inc . , los angeles , usa ) . the intra- and inter - assay coefficients of variation for the insulin were 5.2 and 7.3% , respectively ; sensitivity was 1.2 u / ml and specificity was 80% . insulin resistance was determined using homeostasis model assessment version 2 ( homa-2 calculator available at http://www.dtu.ox.ac.uk/homa ) . for calculation of homa-2 , glucose values differences in demographic characteristics and clinical measures between control and premature groups were investigated by means of analysis of variance for continuous variables and the chi - square test for proportions . differences in neonatal characteristics were evaluated with the use of fisher 's exact test for proportions and the mann general linear model ( ancova ) was used to investigate differences in glucose - regulation variables among the four groups of subjects . the four groups , divided according to their gestational age ( preterm vs. term ) and weight for gestation ( aga vs. sga ) , were included as factor , whereas pi and weight - for - length at 9 months ( wfl ) were included as covariance in this model . the specific hypotheses tested were the difference in insulin sensitivity between appropriate- and small - for - gestational - age subjects within the premature groups and the difference in insulin sensitivity between the premature groups and the term group . further analysis was performed by multiple regression models using log insulin resistance index ( iri ) as depended variable and gestational age , pi , weight - for - length , and sga status as independent variables . the data on fasting si and homa iri were logarithmically transformed to meet the assumptions of normality . all analysis was carried out by using the spss version 20.0 statistical package ( spss , inc . , differences in demographic characteristics and clinical measures between control and premature groups were investigated by means of analysis of variance for continuous variables and the chi - square test for proportions . differences in neonatal characteristics were evaluated with the use of fisher 's exact test for proportions and the mann general linear model ( ancova ) was used to investigate differences in glucose - regulation variables among the four groups of subjects . the four groups , divided according to their gestational age ( preterm vs. term ) and weight for gestation ( aga vs. sga ) , were included as factor , whereas pi and weight - for - length at 9 months ( wfl ) were included as covariance in this model . the specific hypotheses tested were the difference in insulin sensitivity between appropriate- and small - for - gestational - age subjects within the premature groups and the difference in insulin sensitivity between the premature groups and the term group . further analysis was performed by multiple regression models using log insulin resistance index ( iri ) as depended variable and gestational age , pi , weight - for - length , and sga status as independent variables . the data on fasting si and homa iri were logarithmically transformed to meet the assumptions of normality . all analysis was carried out by using the spss version 20.0 statistical package ( spss , inc . , out of hundred subjects , 36 preterm aga , 11 preterm sga , and 17 term sga , as study cohorts and 36 term aga , as control cohort , were enrolled in this study . as it was expected , gestational age , weight ( sd score ) , length ( sd score ) , head circumference ( sd score ) , and weight - for - length index were significantly different among the groups . preterm sga babies were smaller ( length sds ) ( p = 0.013 ) and thinner than preterm aga ( p = 0.001 post hoc test ) . data about neonatal characteristics were summarized in table 2 , and no significant difference was observed in maternal characteristics and oxygen requirement , antibiotic days , and days until full oral feeding established . iri ( median 1.80 , interquartile range 3.90 [ 0.534.39 ] , skewness 1.60 [ rt skewed ] , kurtosis 2.42 , mean 2.95 , and sd 3.20 ) and si ( median 8.80 , interquartile range 17.55 [ 2.6220.16 ] , skewness 1.67 [ rt skewed ] , kurtosis 2.94 , mean 13.49 , sd 14.29 ) had abnormal distribution and converted to log 10 value to normalize the distribution [ figure 1 ] . generalized linear model ( ancova ) was constructed to adjust gestational age , weight for gestation , pi , and weight - for - length ( r = 0.76 , adjusted r = 0.75 ; means 75% variability explained by this model ) . change in r value for these factors show that gestational age , birth weight , and weight - for - length were good predictors for iri ( p = 0.0001 ) as compared to pi ( p = 0.002 ) . pairwise contrast shows that ( adjustment for multiple comparisons by bonferroni ) , both preterm aga ( mean difference 0.617 , 95% ci ; 0.430.80 , p = 0.0001 ) and preterm sga ( mean difference 0.764 , 95% ci ; 0.441.09 , p = 0.0001 ) similarly , infants who had been born term sga had significantly higher insulin resistance than term aga ( mean difference 0.725 , 95% ci ; 0.490.96 , p = 0.0001 ) . no significant difference was found in between preterm sga group and preterm aga group ( mean difference 0.147 95% ci ; 0.130.42 , p = 0.927 ) . it was noticed that when we combine both preterm cohort , insulin resistance gets increased by 5.2% but not become significantly higher ( mean difference 0.076 , 95% ci ; 0.260.11 , p = 0.95 ) than term sga . baseline characteristics of the study subjects * maternal and neonatal characteristics in two groups of premature neonates indicator of glucose homeostasis * right skewed distribution of insulin resistance index further analysis was done by using multiple regression models summarized in table 4 . iri at 9 months was found negatively correlated with gestational age ( = 0.42 , p = 0.001 ) , birth weight z - score ( = 0.35 , p = 0.001 ) , pi ( = 0.61 , p = 0.001 ) , length at 9 months ( z - score ) ( = 0.413 , p = 0.001 ) and catch - up growth for length ( = 0.311 , p = 0.002 ) and positively correlated with sga status ( = 0.511 , p = 0.001 ) , weight - for - length ( = 0.393 , p = 0.001 ) , and catch - up growth for weight ( = 0.198 , p = 0.048 ) . sequential multiple regression analysis for identifying influence of perinatal stress and anthropometric variables on insulin resistance index ( dependent variable ) multiple regression models were constructed using sequential method including gestational age , pi , weight - for - length as continuous variables , and type according to weight for gestation ( aga vs. sga ) as categorical variables . minimum variables which predict the largest amount of variability ( birth weight and length have collinearity with pi and weight and length at 9 months have collinearity with weight - for - length ) were included in this model to validate and stabilize the model . subsequently , pi , weight - for - length , and type according to weight for gestation were added in model 2 , 3 , and 4 , respectively . as a measure of goodness of fit , adjusted r was increased in each model from 0.188 in model 1 to 0.702 in model 4 , which indicates 70% of variability , now explained by model 4 ( significant r changes in each step p = 0.001 ) [ table 4 ] . substituting the variables and the unstandardized coefficients from the table 4 , the equation for model was as follows : log iri = 3.213 ( 0.173 gestational age ) ( 0.25 pi ) + ( 0.183 weight - for - length ) + ( 0.586 type ) because aga is coded zero , the final term in the equation was removed for aga . the term type indicates that after adjusting for gestational age , pi , and weight - for - length at 9 months , sga babies were 0.586 more iri than aga . in effect , this means that y - intercept was 3.799 for sga ( i.e. , 3.213 + 0.586 ) and 3.213 for aga . thus , the lines for aga and sga were parallel , but aga had a lower y - axis intercept [ figure 2 ] . scatter plot for insulin resistance index on gestational age with regression line ( appropriate for gestational age vs. small for gestational age ) standardized coefficients indicated the relative importance of each variable in comparable standardized units ( z - scores ) to predict iri . type according to weight for gestational age with a standardized coefficient of 0.505 was a more significant predictor of iri than gestational age ( = 0.481 ) , weight - for - length ( = 0.315 ) , and pi ( = 0.194 ) . as with an r - value , the negative sign was an indication of the direction of effect only [ table 4 ] . the partial correlation was the unique contribution of gestational age ( 0.596 ) , pi ( 0.258 ) , weight - for - length ( 0.504 ) , and type ( 0.579 ) to predicting iri after the effect of other three factor was removed and was an estimate of the relative importance of each predictive variable in isolation from other factors [ table 4 ] . model was validated for stability , precision , and reliability by testing for collinearity , interaction , and residuals . there was no collinearity ( significant relationship between explanatory variables ) in between variables ( correlation coefficient < 0.7 , variance inflation factor ( vif ) < 4 , and tolerance > 0.2 ) . interactions ( multiplicative rather than additive relationship between two explanatory variables ) were tested by including the interaction term ( gestational age type , pi type , and gestational age type pi ) in model , which shows no significant increase in adjusted r ( p > 0.05 ) . similarly , when iri plotted against gestational age , no significant difference between slope of regression line of aga and sga babies were present ( p > 0.05 ) [ figure 2 ] , which further favor in no interaction between gestational age and type according to weight for gestation . standard residuals ( distances between each data point and the value predicted by the regression equation ) showed normal distribution ( mean < 0.00001 , sd = 0.98 ) indicates equal spread of variance over the length of regression model , and the model was homoscedastic [ figure 3 ] . normal distribution of regression standardized residuals alternative regression model was constructed to determine role of catch - up growth in insulin resistance . in this model , gestational age , catch - up for weight , and type according to weight for gestation sequentially added ( adjusted r = 71.9% mean 71.9% variability explained by this model ) . catch - up growth ( = 0.714 ) is a significant predictor of insulin resistance independent of gestational age and sga status . weight - for - length and pi were not included in this model because of collinearity of catch - up growth with pi and weight - for - length at 9 months ( vif > 4 , tolerance < 0.2 , condition index in collinearity diagnostics > 30 ) . in collinearity , diagnostics condition index were 35.97 , 41.70 , and 57.73 for weight catch - up , weight - for - length at 9 months , and pi , respectively . substituting the variables and the unstandardized coefficients , the equation for this model was as follows : log iri = 5.621 ( 0.153 gestational age ) + ( 0.234 weight catch - up at 9 months ) + ( 0.320 type ) , the data are conflicting , with some studies demonstrating that fat mass is the major determinant of is with no effect of gestation whereas others identify a persisting effect of preterm birth . the present study demonstrates the association of prematurity with insulin resistance in infancy after adjusting for significant covariables . these findings provide additional evidence that preterm birth may be a risk factor for the future development of the insulin resistance and type-2 diabetes , and the insulin resistance may develop as early as infancy . we fill a gap in the knowledge , regarding insulin resistance in preterm born babies in 1 year of life which was unexplored till date . a particular strength of our study is that we also include term sga cohort in our study for comparison and explore relative importance of each variables to predict the insulin resistance in infants . the present study explores that sga status is the most significant predictor of insulin resistance followed by gestational age , weight - for - length , and pi in the descending order . most of the studies reveals that in preterm born babies , sga status exerts no effect on insulin sensitivity in childhood , adolescence , and adulthood . , respectively , found that sga preterm born babies have reduced insulin sensitivity in neonatal , childhood , and adolescence . gray used a milk tolerance test ( mtt ) and showed that sga preterm neonates had higher post - mtt insulin levels . reinehr study included both preterm and term sga born subjects thus insulin resistance might be due to confounding effect of term gestation . used both homa modeling and intravenous glucose tolerance test and found reduced insulin sensitivity in sga babies by using homa model but not after stimulated insulin release . however in term sga born babies ' insulin resistance in infancy , childhood , adolescent , and adulthood is well established . with concurrence to most of the studies , we also report that sga status exerts no impact on insulin sensitivity in preterm born babies , but its impact during infancy is a matter of special mention here . in multiple regression analysis , sga born infants have higher insulin resistance ( 0.586 more iri ) than aga born infants irrespective of their gestational age , pi , and current weight - for - length . this difference is insignificant in preterm babies and become significant with increasing gestational age in term babies [ figure 2 ] . furthermore , combination of both preterm cohorts does not result in significantly higher insulin resistance than term sga . these results indicate antenatal stress prior to the third trimester does not contribute in metabolic derangement , and critical window for the same is present in third trimester . similarly , hofman et al . also reported that third trimester is critical window period for metabolic imprinting , but there also remains a debate because studies in animals and humans have suggested several critical periods from the periconceptual period to later pregnancy . additional studies would require confirming the importance of this period and may provide further insight into the role of this critical period in the third trimester . our results also demonstrate that low pi ( thinness at birth ) is associated with higher insulin resistance with special concern to infancy . pi is a continuum with proportionality in sga fetuses and depends on the duration of intrauterine insult and the extent of its effects on weight and length before delivery . relatively brief duration intrauterine insult which affects more on weight than length results in lower pi ( asymmetrical intrauterine growth restriction [ iugr ] ) , and therefore higher insulin resistance . these results further favor that critical window period for metabolic derangement in lbw babies may present in third trimester in which maximum fetus weight growth occurs . insulin is an important growth factor during infancy , and insulin secretion could be relevant for fat deposition and weight gained shortly after birth . iugr followed by catch - up growth for weight in infancy and early childhood may be a sequence that led to high basal metabolic index ( bmi ) , obesity , and fat mass , and therefore insulin resistance in infancy , childhood , adolescent , and adulthood . similarly , prematurity followed by postnatal catch - up growth for weight in infancy and early childhood may be a sequence that led to high bmi , obesity , fat mass , and therefore insulin resistance in childhood , adolescent , and adulthood . in present study , we examine early changes in body composition by using weight - for - length , which provides a more accurate measure of adiposity ( bmi for > 2 years ) than weight alone . our study indicates that higher weight - for - length is associated with higher insulin resistance in infancy , and thus , it indicates an association of adiposity with insulin resistance as early as the 1 year of life . collinearity between catch - up growth for weight and weight - for - length indicate that excess adiposity is superimposed on normal growth related adiposity during catch - up growth . concomitant to this , converging evidence suggests that catch - up growth in preterm and small for gestation is intimately linked with a disproportionately faster rate to gain body fat rather than lean tissue , that is , to a preferential acceleration of fat recovery or catch - up fat . therefore , we create a hypothesis that catch - up growth for weight in infancy and early childhood and the higher bmi and obesity in letter childhood , adolescence , and adulthood are the same spectrum phenomenon in different window periods of life after exposure to environmental factors such as high energy intake or low physical activity and may be a result of metabolic imprinting after inutero stress or postnatal stress . first , it is one of the few to implement a prospective , longitudinal design to investigate the link between prematurity and insulin resistance as early as infancy . second , this study examines early changes in body composition using weight - for - length , which provides a more accurate measure of adiposity than weight alone . third , regression model is tested for stability , collinearity , and interaction between various variables and finally , this study also includes full - term sga infants , and thus can identify the relative importance of various variables to determine insulin sensitivity in infancy . our cohort did have better clinical outcomes than the total cohort of surviving children who had been born prematurely since we excluded subjects with major or moderate disability . on the basis of these neonatal characteristics and on the better developmental outcome of our cohort , we do not believe that a selection bias has occurred that influenced our findings . finally , we have drawn equation to estimate iri by using gestational age , pi , sga status , and current weight - for - length . by using weight - for - length which is age - independent variable , we can monitor insulin resistance during follow - up in both preterm and sga babies . identification of these infants may help to focus preventive measures aimed at controlling the current epidemic of obesity and its complications , but we should be cautious while making recommendations for nutrition till we have evidence available from randomized control trial

objective : this study was done to determine the role of prematurity and other variables to predict insulin sensitivity in infancy.subjects and methods : in this prospective study , 36 preterm appropriate for gestational age ( aga ) , 11 preterm small for gestational age ( sga ) , and 17 term sga included as study cohort and 36 term aga as control cohort . detailed anthropometry assessment was performed at birth , 3 , 6 , and 9 months and at 9 months , fasting plasma glucose and serum insulin was done . insulin resistance was determined by using homeostasis model assessment version 2.results:it is found that preterm aga ( mean difference 0.617 , 95% confidence interval [ ci ] ; 0.430.80 , p = 0.0001 ) , preterm sga ( mean difference 0.764 , 95% ci ; 0.441.09 , p = 0.0001 ) , and term aga ( mean difference 0.725 , 95% ci ; 0.490.96 , p = 0.0001 ) group had significantly higher insulin resistance than control . there was no significant difference in between preterm sga and preterm aga ( mean difference 0.147 95% ci ; 0.130.42 , p = 0.927 ) . in multiple regression models , sga status ( = 0.505 ) was more significant predictor of insulin resistance index than gestational age ( = 0.481 ) , weight - for - length ( = 0.315 ) , and ponderal index ( = 0.194).conclusion : preterm birth is a risk factor for the future development of insulin resistance which may develop as early as infancy .

a large number of hydrophobic compounds with potentially high pharmacological value fail to pass initial screening tests because of the perception that they will be too difficult to deliver effectively due to anticipated formulation limitations . fortunately , nanosuspensions of such drugs may be used to increase bioavailability and offer a variety of delivery options . typically these limit the dimensions obtainable since the strategies use high shear processing of preformed entities . to achieve nanoscale dimensions by these size reduction technologies ( top down processing ) , an excessive amount of energy and time needs to be expended [ 5 , 6 ] . unfortunately , they often not only proved ineffective but lead to possible product degradation . because nanosuspensions and novel targeting chaperones , for example t - cells , can deliver much larger amounts of drug in a smaller volume than the solvent diluted drug systems [ 14 , 79 ] the focus here is on how these techniques influence delivery strategies and efficacy through enhancement of the transport phenomena involved in all phases of a drug 's life cycle . for example , the ability to obtain desired drug properties , such as size , habit , and morphology , through novel manufacturing strategies permits unique formulation control for optimum delivery methodologies . the ability to transfer energy , mass , and momentum with directed purposeful outcomes is imperative in establishing higher production rates of these carefully engineered nanoparticles at elevated technoeconomic stature . the role of transport phenomena becomes critically apparent as the industry moves more aggressively toward continuous manufacturing modes , utilizing process analytical technology ( pat ) and process intensification ( pi ) concepts . although these advances rely upon more effective sensor - reporter systems , based on nanoprobe technology , they are not the focus here and therefore will only be briefly touched upon in the following discussions . the emphasis is on the clinical aspects that drive all the other phases needed to get to this stage . that is , once available , these nanoscale entities can be utilized quite effectively in both traditional and novel delivery techniques , relying heavily on in vivo transport capabilities . the topics to be addressed in the following sections all capitalize on how carefully these drugs were designed , developed , and engineered for desired properties and capabilities . specificity of uptake , clearance control , and transport to the brain via the blood brain barrier , cerebrospinal fluid , or in smart implants are a few examples this first generation of such drugs relies mainly on the small size of the particles to increase the surface area and therefore bioavailability of poorly soluble drugs , and to a lesser extent in the structure of the particle for delayed release , and so forth . examples of nanotechnology drugs in the us market include rapamune / pfizer , emend / merck , invega sustenna / janssen , all based on elan 's nanocrystal technology . abraxane / abraxis bioscience and triglide / sciele pharma are also in the us market . in emerging technologies , the particles have improved functionalities that include diagnosis , targeting , and drug delivery functions and enhance transport and uptake characteristics . the focus of this paper will be in these emerging technologies rather than the current status of the market drugs . the credibility of the techniques ( topics ) being presented here is established through either prior extensive testing , preliminary results from proof - of - concept tests , or derived from analogous successes for what are believed to be realistic projected applications . presented here therefore will be discussions relative to ( a ) crystal size and morphology control , via bottom - up processing , for direct use with traditional delivery methods , ( b ) simultaneous targeting / delivery techniques incorporating novel chaperones obtained from functionalized surfactant encapsulants and t - cells , and ( c ) controlled release using nanotechnology innovations involving single and multiple drug interventions and tissue therapies ( e.g. , angiogenesis , wound healing , and artificial organs for autoimmune diseases ) . in these cases , attempts are made to identify the underlying fundamental physicochemical principles / mechanisms associated such that projected extensions are feasible , and scaleup where necessary can be accomplished reliably . in the recent article by g. liversidge , as mentioned previously , a number of specific pharmaceutical companies and associated drugs are identified that combine control - release and nanotechnologies . this combination is identified as a key market driver for this industry . based upon documented recent advances and successful applications , powerful extensions to many of the concepts and methods mentioned there are being developed and some are currently being implemented throughout the industry . for example , the concept of minitablets has a profound impact on many release formulations , ( i ) delayed- , ( ii ) extended- , and ( iii ) pulsitile - release systems . an objective of ours via this paper is to identify the importance and effectiveness of nanotechnological innovations on the enhancement of transport processes that improve therapeutic protocols . of the techniques being discussed , the bottom - up method for nanocrystal formation will be used as an example because it provides the basis for our ability to carefully engineer the nanoparticles for the drug delivery protocols . these entities are an essential component for the clinical implementation of all the transport enhanced techniques in use and/or proposed . whenever available , the results from the various levels of experimental programs executed are presented and discussed , conclusions drawn , and recommendations for future efforts set forth . presented in table 1 below is an outline of the current and emerging methods and nanotechnology applications in drug delivery platforms . these topics will be discussed or referenced in the sections that follow . a continuous bottom - up approach to the solvent / antisolvent crystallization process allows precise control of product properties . achievement of specified quality goals associated with overall performance criteria has been demonstrated [ 1114 ] . the technique involves generating a large number of nucleation sites and limiting subsequent growth . with this method crystal size control is via molecular approaches that utilize various mechanistic pathways governed by transport phenomena , thermodynamics principles , and/or intrinsic kinetics . the design and operation of commercial scale crystallizers are optimized based on minimizing the formation of agglomerates , impurities included within crystals , liquid entrapped within crystal aggregates , and mother liquor retained by the crystal cake [ 1517 ] . the various crystallization mechanisms that contribute to the observed phenomenological events and how they affect these objectives will be addressed throughout this section . the generation of nanoscale homogeneous regions dispersed throughout the active crystallization volume is essential for the success of this bottom - up process . estimating the size of these regions is reasonably straightforward using proven turbulence calculation algorithms [ 1820 ] . the significance is that the length scale over which no further mixing takes place is established and thus molecular diffusion now dictates timing for the steps involved in the homogeneous nucleation and growth processes within these regions . since hydrodynamics has a significant impact upon mass , energy , and momentum transport rates and reaction proficiency it is imperative that the role it plays not be underestimated . it is also essential to identify the energy dissipation mechanisms present and thereby quantify the intensity of mixing ( i.e. , macro- , meso- , or micro- ) , contact efficacy , and associated level of turbulence with its resultant eddy cascade . the length scale of the kolmogorov ( i.e. , smallest ) eddies , when formed at high energy dissipation levels , can easily be at the nanoscale . the important point is that the magnitude of this energy dissipation rate per unit volume establishes both the time and length scales over which events occur . these can be key control variables manipulated by mixing intensity once the thermodynamic state of the working fluid is established through other processing variables . observed rates are highly dependent on the concentration differences beyond the solubility limit and hydrodynamic scales . hence , the local degree of supersaturation can be used as the primary metric to account for both the kinetics and thermodynamic behavior of the system [ 11 , 12 , 21 , 22 ] . crystal characteristics , such as crystal size distribution ( csd ) , surface area and topography , morphology , dissolution rate , and strength ( affected by any impurities and flaws present ) , depend heavily upon their formation processing conditions . an inclusion of mother liquor for example affects not only product quality for its desired applications but also storage stability , particularly with respect to csd and morphology . this is of considerable importance to the pharmaceutical industry since polymorphic systems exhibit different physicochemical properties due to the existence of these different crystal structures . polymorphism influences the dissolution characteristics , which along with csd affects product formulation strategies and bioavailability [ 1 , 2 , 1114 , 2326 ] . to understand how to form crystalline nanoparticles of hydrophobic active pharmaceutical ingredients ( apis ) via this bottom - up process requires knowledge of the fundamental thermodynamic and rate processes involved in the generation of solid particles from a liquid phase . this involves solubility limits of the target species ( with associated degree of supersaturation ) , nucleation and growth rates , and turbulence intensity to obtain the requisite mixing levels . it is the energy dissipation levels developed by turbulence that determine the appropriate length and time scales required to control the phenomenological events occurring . although these topics are discussed in some detail for specific applications elsewhere [ 1122 ] , a brief summary of each is included here for clarity of purpose . the various aspects and important parameters that affect the bottom - up crystallization process to be discussed are the following . thermodynamics ; describes phase characteristics , solubility limits and phase stability , establishing the driving force for crystallization . nucleation and crystal growth ; related to crystallization rates , particle sizes , and crystal structures . complications ; describes some of the issues that need to be addressed in designing a process and getting the desired product quality . flow patterns , mixing , and transport phenomena ; describes the role of mixing in crystallization processes , relevant to processes that involve mixing of multiple streams , heating or cooling . creating nanoscale entities ; describes strategies of achieving mixing in the nanometer scale and techniques used . energy dissipation ; gives an overview of the mechanisms that absorb energy during the process . generating solids from a liquid phase is initiated by changes in the thermodynamic state of the solution , thereby reducing the solubility of the target species . initiation may be through temperature adjustment(s ) , concentration changes , or by altering solution activity coefficients as in the solvent / antisolvent method . phase stability is an important factor in determining both when and how fast events progress . the temperature - composition phase behavior , see figure 1 , can be used to illustrate some important concepts . a solubility curve represents thermodynamic equilibrium between the phases . for most liquid systems with a composition and temperature above its solubility curve a stable unsaturated liquid exists . beyond this solubility limit the liquid may not be in thermodynamic equilibrium with respect to the formation of the solid phase , that is , it exists as a supersaturated ( ss ) liquid . system behavior can be determined by this degree of ss since there is a region , referred to as the metastable zone , where the system may not always be considered thermodynamically unstable . however , beyond the boundary of this metastable zone , these seed nucleation sites are no longer required . in this region a ss liquid is neither stable nor in equilibrium , and is subject to spontaneous nucleation and rapid growth of the solids . unfortunately , due to the large increase in entropy , some undesired events may occur . the crystal matrix may have flaws , such as dislocations , impurity molecules , or liquid inclusions . when a system exhibits various polymorphs , this spontaneity could be problematic or beneficial , depending on the morphology sought and its stability . since our objective is to create a large number of nucleation sites and thereby restrict the ultimate size of the individual particles , and possibly control morphology , this unstable zone is the desired initial operational region . to control the nucleation and growth rates , the strategy used must establish the desired supersaturation state , level of energy input , and energy dissipation mechanisms . the degree of supersaturation influences the rate of the individual steps involved in forming the solid as well as which crystal polymorph is formed . in general , the process proceeds as follows : ( 1 ) feed streams are mixed in a process unit selected to meet required specifications for the energy dissipation rate per unit volume . the time to achieve homogeneity is dependent on diffusivity of the target species and the distance they must travel within the smallest eddies obtained ( see the discussion on mixing for the role of turbulence and the kolmogorov scale ) ; ( 2 ) mixing to obtain the desired local degree of supersaturation , leading to a nucleation rate , which increases proportionally with ss . the features of the product formed depends significantly on this rate ; ( 3 ) growth of the nuclei is by diffusion of solute molecules from the bulk solution to the surface and then along the surface to be integrated into the matrix . this continues until a limiting particle size is reached , determined by the magnitude of the shear force present ; ( 4 ) further growth is by mechanisms whereby particles collide and adhere to each other . particle number thus decreases with time as the particle size increases . the ability to create and control it can be generated by various methods including indirect cooling , evaporation , adiabatic evaporative cooling , antisolvent addition and salting out , chemical reactions , and ph adjustment . note that temperature changes may be detrimental for some systems , for example when dealing with protein - based drugs . alternative methods most frequently used to reduce solubility are ph adjustment to the isoelectric point , increasing ionic strength , addition of nonionic polymers , and addition of a miscible nonsolvent . of particular interest for the bottom - up approach are agglomeration , liquid inclusions , and inefficient mother liquor removal . when growing crystals collide they may stick together and form new particles , that is , agglomerates form when the collisions are inelastic . the strength of the physical bonds thus formed determines their stability upon further collisions . for the bottom - up processing to be effective in limiting crystal size the probability of agglomeration needs to be low . unfortunately , a large number of small particles are produced when operating in the unstable supersaturation region and collision frequency is high . to offset this concentration effect , it is necessary to limit the time for interaction and/or relieve ss quickly . also , a surfactant may be effective in limiting the probability that the particles will stick to one another . when growing crystals collide they may stick together and form new particles , that is , agglomerates form when the collisions are inelastic . the strength of the physical bonds thus formed determines their stability upon further collisions . for the bottom - up processing to be effective in limiting crystal size the probability of agglomeration needs to be low . unfortunately , a large number of small particles are produced when operating in the unstable supersaturation region and collision frequency is high . to offset this concentration effect , it is necessary to limit the time for interaction and/or relieve ss quickly . also , a surfactant may be effective in limiting the probability that the particles will stick to one another . liquid inclusion in individual crystals and agglomeratesthis is particularly undesired when liquid impurities are present . also , liquid can get trapped between colliding particles during agglomeration and higher supersaturation levels increase the probability of that occurrence . one can mitigate any associated problems by limiting the interaction time and/or relieve the supersaturation condition rapidly . also , liquid can get trapped between colliding particles during agglomeration and higher supersaturation levels increase the probability of that occurrence . thus high supersaturation can have both beneficial and problematic outcomes . one can mitigate any associated problems by limiting the interaction time and/or relieve the supersaturation condition rapidly . mixing at the nanometer scale occurs as reactants , which may include several liquid and solid phases , are subjected to high shear stresses and turbulence . the energy dissipation rate determines whether the macro- , meso- , or micromixing level is attained . the overall mixing process occurs within a flow field continuum which covers the wide range of length and time scales indicative of each of these mixing levels , each with distinct characteristics . for example , consider two miscible fluids . next , the breakdown of large eddies into smaller ones via the eddy cascade is termed mesomixing . fluid engulfment in small eddies with subsequent laminar stretching of them , where molecular diffusion is now the final mechanism to obtain uniform composition , is referred to as micromixing [ 1820 ] . the length scale for this diffusional process is determined by the size of the smallest eddies formed and is referred to as the kolmogorov length scale . along with time and kinetic energy scales , each determined by these local flow conditions alone , ( i.e. , related to kinematic viscosity and the energy dissipation rate per unit mass ) , the so called kolmogorov scales are established . estimating the magnitude of these kolmogorov parameters can be accomplished with reasonable confidence using proven theoretical turbulence calculations . the significance is that the length scale over which no further mixing takes place is established and molecular diffusion now dictates timing for the necessary steps involved in the homogeneous nucleation and growth processes . these mixing subprocesses generally occur in series , but often to some extent , in parallel . turbulent energy dissipation rates , for example in modified impinging jet technologies [ 11 , 12 , 2729 ] , are estimated to be on the order 10 w / kg and higher when using these micromixing models . at these levels , rapid micromixing and mesomixing ( on time scales of 4 and 20 s , resp . ) are achieved , and the length scale of the smallest eddies are at the nanoscale . note that residence times in many of the microreactors systems used for pi applications , particularly those utilizing impinging jets , are of the order 1 ms and lower . incorporating these fundamental principles and using appropriately designed equipment it is possible to precisely control each step in the crystallization process . mixing at the nanometer scale provides a uniform supersaturation ratio . the onset of the nucleation process can be manipulated by controlling the timing and location of the mixing of the solvent and antisolvent streams that are used to generate the supersaturated state . this in combination with an evenly dispersed homogeneous supersaturation ratio results in uniform crystal growth and stabilization rates . the generation of nanoscale homogeneous regions dispersed throughout the system is a major requirement for the success of this bottom - up process . this concept is ideal for our purposes since both length and time scales are quite small for the processes involved in creating these monodispersed nanoparticles . consequently , it is immaterial whether or not these regions are stabilized , as for example , by use of surface active agents . it is important to reiterate that the length scale over which no further mixing takes place is established and molecular diffusion now dictates timing for the necessary steps involved in the homogeneous nucleation and growth processes . in the absence of seed crystals or other nucleation sites , a critical number of molecules must collide and remain aggregated forming stable clusters , ( i.e. , nuclei ) . subsequent growth requires diffusion to and along the surface , followed by a specific integration process that incorporates these molecules into the crystal matrix of a particular polymorph . the observed crystallization rate is , therefore , highly dependent on length scales and the local degree of supersaturation . the polymorph that is obtained is dependent on thermodynamic considerations , such as component activity coefficients ( solvent / antisolvent / solute species interactions , composition / concentrations , and temperature ) and entropy generated due to the spontaneous nature of the process , that is , rates influenced by supersaturation ratios . to generate the high energy dissipation rates used to produce nanoparticles , jet impingement , on a solid surface or with another jet , has been shown to be a highly efficient method [ 11 , 12 , 27 , 28 ] . systems that incorporate high velocity linear fluid jets that collide can rapidly reduce the scale of segregation between the streams . high - energy dissipation is observed because the kinetic energy of each stream is converted into a turbulent - like motion as the result of the collision and redirection of the flow within a very small volume . more thorough discussions on the phenomenological events , equipment design criteria , and characterization studies are given elsewhere [ 1115 , 1826 ] . surface tension and various molecular forces between the species present are key variables associated with the crystal size distribution . thus , surface active agents can play a significant role whether as a contributor to growth mechanisms or as a size stabilizer . for example , they are involved in self - assembly mechanisms , and can act as barrier components that restrict transport , as possible chaperones that target specific sites during drug delivery , as sequestering agents to facilitate contact efficacy , as promoters of interfacial phenomena , and as inhibitors to agglomeration . the fraction of the input energy available for formation of surfaces is instrumental in establishing system efficacy . performing an energy audit to determine overall requirements this entails determining the amount of input energy transformed into kinetic energy of the jets , identifying all forms of dissipation ( whether desired or not ) , and ascertaining the amount stored as internal energy . although the system energy requirements are not readily identified a priori , the total energy input and the amount dissipated and stored are measurable . estimates of the various losses occurring can be made , and the energy utilization for the desired processes can also be estimated . this permits energy considerations to be used in predicting performance from the estimated length and time scales obtained . system validation is accomplished when these length and time scales can be corroborated with observed kinetics phenomena [ 12 , 27 , 28 ] . confined impinging jet systems have been used in our laboratory to consistently produce submicron api suspensions via a continuous process that involves crystallization via the solvent / antisolvent technique to generate supersaturation conditions . microfliudics reaction technology ( mrt ) was selected for this bottom - up processing since it is based on novel multiple stream inlet capabilities coupled with the impinging jet concept [ 1114 , 26 ] . it is designed to produce jet velocities and energy dissipation orders of magnitude higher than those of conventional impinging jet reactors . the technology provides precise control of the feed rates , and the subsequent location and intensity of mixing of the reactants . it may provide significant technical and economical advantages due to its process intensification character that minimizes energy requirements , and the proven scalability of the reactor . in our first proof of concept studies performed , nanosuspensions of several apis were produced varying the key parameters of the technology . five different model apis were used for testing and were selected to belong to different chemical families that exhibit different pharmacological activities . there were two antibiotics ( azithromycin and api-2 ) , an antihistamine ( loratadine ) , an anticonvulsant ( oxycarbazepine ) and a non - steroidal anti - inflammatory ( nsais , api-1 ) . the particle size depended on the supersaturation ratio and energy dissipation expressed as process pressure . the nanosuspensions were stable with narrow particle size distributions and median particle sizes in the range of 50760 nm . top down process in which drug nanosuspensions were created as a result of particle size reduction . this first study did not attempt to identify crystalline structure and therefore no polymorph selectivity capabilities were evaluated . to accomplish this , two additional , more in depth studies were conducted on single apis : carbamazepine ( cbz ) , an anticonvulsant , and norfloxacin ( nfn ) , an antibacterial agent . the details of the experimental protocols and results are reported in separate papers , cbz and nfn . a few brief comments are given here to help validate the benefits of bottom up processing with respect to the stated objectives of creating carefully engineered particles with the nfn nanosuspensions had narrow particle size distributions and median particle sizes in the range of 170350 nm depending on the supersaturation ratio and energy dissipation expressed as process pressure . however , the particle size was found to be insensitive to the presence of the surfactant used . the crystalline structure of nfn was not affected by the processing conditions for this particular solvent / antisolvent system , but it was different than the initial crystalline structure of the drug . two miscible fluids were used as the solvent ( dmso ) and antisolvent ( water ) . the effect of process pressure ( determining the energy input ) , the nfn concentration , the supersaturation ratio , and the presence of surfactant on the particle size and the crystallized material was investigated . higher pressures resulted in smaller particle sizes , as did lowering nfn concentration and supersaturation ratios . however , the crystallite size of the material decreased threefold from no shear to high shear conditions . cbz was selected as a model system since it is known to exhibit polymorph multiplicity . several solvents and antisolvents were used to determine their effect on the crystalline structure and particle size . cbz is also known to form hydrates , therefore both aqueous and nonaqueous solvent / antisolvent systems were used for comparison . they were dichloromethane ( dcm)/hexane , poly(ethylene - glycol ) ( peg ) 300/water , and dimethyl sulfoxide ( dmso)/water . the results obtained with respect to processing conditions are consistent with those of the nfn study . particle sizes obtained with all bottom up experiments were consistently in the range of 250320 nm . unfortunately , the results obtained with respect to polymorph selectivity were not as definitive . what was observed is that the solvent / antisolvent system does matter , but it is unclear if the degrees of supersaturation or processing intensity had significant roles in that study . three different morphologies were detected via xrd patterns and a hypothesis is given to explain the detailed observations presented there . although not conclusive and thus more thorough studies must be performed , the explanations are consistent with those results . although the emphasis in the previous paragraphs was in crystallization , other processes can be used to manufacture nanosized materials with tailored properties . encapsulation of functional ingredients in polymers is another method , which will be discussed in more detail in the sections that follow . table 2 summarizes the processes used in the bottom up production of nanoparticles and the properties controlled via such methodologies . creative advances in nanotechnologies , coupled with systems biology , has led to novel chaperone systems for simultaneous targeting / delivery , and in certain instances , enhanced controlled release strategies . the systems selected for illustration here are ( 1 ) polymer nanosuspensions , ( 2 ) functionalized designer surfactant encapsulants , and ( 3 ) attachment to t - cell surfaces . the creation and use of chaperone systems in targeting , drug delivery , and diagnostic imaging has greatly broadened the applications , and thus needs , for polymer nanosuspensions . the enhanced surface to volume ratios provides unique capabilities for functionalization of the surface for these high degrees of specificity requirements . the intended use of these nanosuspensions dictates control of both the mean particle size and distribution . these parameters determine performance and toxicity through the selectivity and rate of receptor - ligand interactions and/or the ability and rate of cellular uptake . the implementation of systems that can control nanoscale phenomena is required and has been reported previously . the techniques reported there can create nanosuspensions of many different polymer types with varying particle sizes by controlling the formulation and process variables . these nanosuspensions may also contain encapsulated species via either co - precipitation or other less efficient cargo loading techniques that rely upon diffusional uptake strategies . encapsulation of active pharmaceuticals and contrast agents within these biocompatible polymers is readily accomplished using bottom - up techniques for co - precipitation processes that are reproducible and scalable . nanosuspensions in the range of 50500 nm with different polymers with high encapsulation efficiencies have been created successfully . for example , suspensions of poly(epsilon - caprolactone ) ( pcl ) ( a polymer that has been extensively used for parenteral drug delivery ) were created using mrt ( as discussed above in previous sections ) . by mixing a 20 mg / ml ( pcl / acetone ) solvent stream with water at a ratio 1 : 10 ( solvent / antisolvent ) a nanosuspension with a mean particle size of 220 nm was prepared . there has always been an active interest in targeted drug delivery to tumors to specifically kill cancer cells . ongoing research in this area has provided significant advances due to the ability to carefully engineer both the vesicle , for its specificity and imaging characteristics , and its cargo api . a collaborative team has developed a highly adaptable amphiphilic alternating copolymer system that self - assembles into micelles for therapeutic delivery applications in cancer [ 8 , 9 ] . the synthetic scheme includes the enzymatic polymerization of multifunctional linker molecules ( dimethyl 5-hydroxyisopthalate ) with poly(ethylene glycol ) . subsequent synthetic steps have been developed to attach ligands ( for targeting ) , perfluorocarbons ( 19f mr imaging ) , fluorescent dyes ( nirf imaging ) , and radioiodine ( nuclear imaging and radioimmunotherapy ) to the backbone polymer . attachment of hydrocarbon or perfluorocarbon side chains provides amphiphilicity to produce the multimodal self - assembling micelles . additionally , encapsulation procedures for chemotherapeutic agents , that is , doxorubicin and paclitaxel , have been established . these unique alternating copolymer micelle nanoparticles were designed as delivery vehicles targeted to human cancer cells expressing the underglycosylated mucin-1 antigen , which is found on almost all epithelial cell adenocarcinomas , by use of the peptide eppt , or the folate receptor ( fr ) by using folate . development of the synthetic schemes has been coupled with in vitro toxicity tests using various cell viability assays to minimize the toxic effect of these copolymer structures . similar results were obtained with paclitaxel as the cargo . cellular uptake determined by 125i or 3h radioactive analysis and fluorescence confocal microscopy was also investigated in other in vitro studies . microscopy images of the labeled polymer alone demonstrated that the polymer was most likely confined to vesicles within the cytoplasm and not found in the nucleus , whereas encapsulated doxorubicin was shown to be largely confined to the nucleus . theoretical models of polyvalent binding were employed to guide the design of the targeting polymers . unfortunately , the polymers used in this study appeared largely nonspecific for the targeted cells when studied in vitro . however , the versatility of these polymer constructs suggests that continuing to optimize for a targeting delivery system for drugs and imaging agents using this polymer platform could be extremely beneficial . before discussing the specifics of the use of t - cells in drug delivery protocols , a few general comments about the basis of this approach is attributed to the new , burgeoning field of biohybrid materials which will have a significant impact on the efficacy of drug delivery . this is in addition to their obvious use in bioimaging , cellular functionalization , immune system and tissue engineering , and cell - based therapeutics where cell - environment interactions are critical . of particular interest here are synthetic materials systems such as magnetic micromanipulators , nanoparticulate cellular patches , and functional cell backpacks [ 31 , 32 ] . these offer exciting possibilities for symbiosis between synthetic building blocks and native biological behavior . this was clearly demonstrated by the collaborative efforts of the cohen and rubner research groups with functional polyelectrolyte multilayer ( pem ) patches attached to a fraction of the surface area of living , individual lymphocytes . these cells remained viable , and with patches containing magnetic nanoparticles the cells could be spatially manipulated using a magnetic field . since the patches did not completely occlude the cellular surface from the surrounding environment a functional payload could be attached without interfering with the cells ability to perform its native functions . backpacks , nanoscale thickness , micrometer - sized , photolithographically patterned heterostructured multilayer systems capable of noncytotoxically attaching to the membrane of a living cell . it is interesting to note that these backpacks can play an integral part in tissue engineering applications , such as in cell aggregate self - assembly which will be discussed briefly in a later section . to illustrate the use of this concept in a drug delivery scenario , an extension of this technique was exploited as follows . in a recently published study , a method of attaching carefully engineered nanoparticles to the surface of t - cells although their application was for a cell therapy approach , the t - cells were used as chaperones for the stimulant drugs . they designed drug carrying nanoscale vesicles with lipid characteristics for coupling with the sulfur containing molecules on t - cell surfaces . in their study the researchers injected these cargo carrying cells , each with approximately 100 vesicles loaded with interleukins il-15 and il-21 , into mice with lung and bone marrow tumors . once reaching the tumors these packets gradually degraded releasing the drugs over a period of one week . their concept was for the drug molecules being released to reattach to these chaperone t - cells , stimulating them to replicate and thus provide the requisite tissue therapy . the techniques proved successful in that within 16 days , all tumors in the mice treated in this fashion disappeared and these mice survived for the entire 100-day experiment . mice that received no treatment died within 25 days and those that received either t - cells alone or t - cells with injections of interleukins died within 75 days . a few details of their procedure are presented here to stress the relatively straight forward nature of these protocols and instill confidence that the proposed clinical applications can be realized with a high degree of certainty . their method exploits the fact that t - cells , like many cell lines , have high levels of reduced thiol groups on their surface , and thus stable coupling of the synthetic drug carrying nanospecies to them is possible . specifically , liposomes and liposome - like synthetic entities 100300 nm in diameter , with a drug loaded core and phospholipid exterior layer , were linked to the cells via the thiol reactive maleimide head - groups . the donor cells were first incubated with nanoparticles to accomplish the thiol - maleimide coupling . this is followed by in situ conjugation to thiol - terminated poly ethylene glygol ( i.e. , pegylation ) to quench the residual reactive groups to ensure that only about 20% of the surface thiol groups were involved with the initial coupling , that is , linked with approximately 150 nanoparticles . stable , nontoxic linkages to live cells were thus accomplished with particles ranging from simple liposomes to complex multilamellar lipid nanoparticles or lipid coated polymers . this benign behavior was anticipated since only 3% of the surface of a typical 7 m diameter t - cell would be blocked by 200 nm diameter particles occupying 150 sites . these results suggest therapeutic cells are promising vectors ( chaperones ) for actively targeted imaging and drug delivery . furthermore , the attached entities can be engineered for controlled release of individual or multiple drug sequencing capabilities . what can be envisioned is the use of different vesicles with specific transport or degradation properties or a vesicle composed of , for example , multiple polymeric materials , as will be discussed in the following section devoted to release strategies . for a large number of health care / wellness interventions the release concepts employed range from ( i ) simplistic steady release rates { via dissolution , etc . } , ( ii ) intermittent timed release , ( iii ) programmed simultaneous and or sequential release of multiple species { antigenic drugs and adjuvants } , to ( iv ) smart systems responding to stimuli : including single and multiple drug interventions and tissue therapies ( e.g. , angiogenesis , wound healing , and artificial organs for autoimmune diseases ) . the applications discussed in the following sections demonstrate the breadth of nanotechnologies that impact these release strategies . these all capitalize on how carefully these drugs were designed , developed , and engineered for desired properties and capabilities . specificity of uptake , clearance control , and ability to perform extremely difficult tasks , such as drug delivery to the brain via transport across the blood brain barrier , the cerebrospinal fluid , or in smart implants , are highly desired capabilities . coupling advanced materials development and processing techniques with nanoscience and technology creates innovative opportunities not only for traditional drug delivery capabilities , but helps establish the impact platform technologies necessary for tissue engineering / therapy methodologies . they are highly dependent upon the physicochemical properties and geometric features of a drug 's formulation . in addition to solubility limits , size distribution , habit and morphology ( when applicable ) , compaction or encapsulation technique , and diffusivity / mass transfer coefficients are significant contributors to accomplishing a successful therapeutic event . for example , nanosized apis are more readily distributed uniformly with an excipient and/or adjuvant . they also exhibit greater dissolution rates than larger sized entities having the same total mass of drug retained within the product matrix . variable release rates can easily be obtained using a composite structure ; each layer having different transport properties . the design of release protocols for multiple apis , sequenced for optimum efficacy and synergism , is thus straightforward . furthermore , nontherapeutic layers can be included to ( i ) provide a delay mechanism , ( ii ) possibly be a barrier for protection until arrival to the desired local or organ system , and/or ( iii ) be a sacrificial layer containing an adjuvant or other functional component that would , for example , pre - condition the microenvironment . obvious extensions to these methods are incorporated into implant systems with hindered diffusion capabilities , in addition to facilitated delivery due to targeting features . demonstrated implementations of a few of these , along with some conceptualizations innovative drug delivery protocols have evolved capitalizing on these and recognizing the analogous processes present during successful applications in related areas . understanding the binding properties and characterization of transport mechanisms within modified hydrogels and biomembranes provides the bases for designing implants with entrapped vesicles and the controlled release of their cargo apis . included here is the concept of pulsitile release systems ; that is , the drug is released as bolus pulses in well defined time intervals ( see later section referring to future opportunities for additional comment ) . therapies that require the sequencing of multiple drugs can therefore be accomplished by logical extensions . as examples ; ( i ) amphoteric core - shell microgels , that is , contraphilic two compartment colloidal particles [ 35 , 36 ] could be used as smart systems ; either as implants or chaperones , ( ii ) the concept of chaperones within a larger vector could also prove feasible ; to minimize clearance of the smaller entities , or their catabolism , prior to their uptake at difficult to reach sites such as to the brain and subsequent transport across the blood brain barrier , and ( iii ) stimulate angiogenesis through release of multiple cytokines ( growth factors ) from nanovesicles entrapped in functionalized hydrogel beads used as immunoprotective barriers for tissue therapy applications [ 3741 ] . additional details with respect to the research studies involved in formulating these extensions and conceptualizations can be found in the following sections . transport and drug delivery through the blood - brain barrier and cerebrospinal fluidthere are multiple barriers in the central nervous system that inhibit api therapies . the blood - brain barrier ( bbb ) and blood - csf ( cerebrospinal fluid ) barriers are vascular in nature , whereas the other , the brain - csf barrier , exists between brain tissue and the csf . the wall of the cerebral microvessels in the brain parenchyma constitutes the bbb . due to its unique structure the multicell layer present in the middle of the brain parenchyma is known as the blood - csf barrier . present there are ventricular cavities ( ventricles ) filled with csf secreted by the epithelial cells of the choroid plexus , a highly vascular tissue with leaky , fenestrated capillaries covered with ependymal epithelium , which has relatively tight junctions . the third barrier , the interface between the csf and brain tissue , is unlike the other two tight blood barriers since it is relatively leaky . since it does not prove to be a significant resistance to mass transport it is a probable route for drug delivery once the transport issues with the other barriers are resolved . given that the area of the bbb is about 1000 times that of the blood - csf barrier , it is more important to circumvent its impermeability , and therefore that is the focus for continued discussion . furthermore , since it is not considered as limiting as compared to the bbb , further discussions related to csf transport are not given here but can be found elsewhere .the transport of substances from capillary blood into the brain tissue is dependent upon molecular size , lipid solubility , binding to specific transporters , and electrical charge . compared to the peripheral microvessel wall , the additional structure of the bbb and tighter endothelial junctions greatly restricts transport of hydrophilic molecules through the gaps between the cells , that is , the paracellular pathway of the bbb . in contrast , small hydrophobic molecules such as o2 and co2 diffuse freely across plasma membranes following their concentration gradients , that is , the transcellular lipophilic diffusion pathway . the bbb permeability to most molecules can be estimated on the basis of their octanol / water partition coefficients . for example , diphenhydramine ( benadryl ) , which has a high partition coefficient , can cross the bbb with relative ease , whereas water - soluble loratadine ( claritin ) is blocked . some solutes with low partition coefficients easily cross the bbb by active or facilitated transport mechanisms , which rely on ion channels , specific transporters , energy - dependent pumps , and a limited amount of receptor - mediated transcytosis . small drug molecules analogous to glucose , amino acids , and small intermediate metabolites , for example , reach brain tissue via facilitated transport mediated by specific transport proteins , whereas larger molecules , such as insulin and other protein type therapeutic agents , are carried across the bbb via receptor - mediated or adsorptive transcytosis . furthermore , some small molecules with high octanol / water partition coefficients are seemingly blocked . thorough data analysis suggests that they are actively pumped back into the blood by efflux systems . for instance , members of the adenosine triphosphate - binding cassette family of exporters are potent energy - dependent transporters . they contribute greatly to the efflux of xenobiotics and due to this protective role impede the delivery of therapeutic agents . consequently , to develop effective and efficient methods for drug delivery to the brain through the bbb , it is imperative to control its permeability . this requires understanding the mechanism by which these structural components , as well as transporters , receptors , efflux pumps and other components at the endothelium and astrocyte foot processes determine it.various methods such as intracerebral implantation , microdialysis , convection - enhanced distribution ( ced ) , osmotic shock , and chemical modification of the bbb have been developed for delivering drugs into the brain . however , the applications of these methods are limited and they can only partially keep with the demands of modern therapies . for instance , the efficiency of intracerebral implantation , microdialysis and ced methods are low since their major transport mechanisms are diffusion and convection of interstitial fluid . for effective treatment of cns diseases , an adequate amount of therapeutic agents must reach the specific regions of the brain . they can directly deliver therapeutic agents via these transporters by closely mimicking their substrates , or conjugating the drugs to ligands of the specific surface receptors expressed for transcytosis ( receptor - mediated transcytosis , rmt - trojan horse approach ) . furthermore , these functionalized target chaperones are used in delivering cationized proteins , peptides , and as nanoparticle carriers for adsorptive mediated transcytosis ( amt).although the exact mechanisms of rmt are not fully understood , the development of drug delivery protocols using receptor targeting has been successful [ 4650 ] . this physiological approach is often referred to as the molecular trojan horse approach since the therapeutic compounds are delivered to specific sites for transcytosis by various forms of vector carriers . the technique is very promising , but unfortunately there remain a number of hurdles to overcome [ 4850 ] . in particular , even if the total amount of drug transported to the brain is large , most of it may not be efficacious since it might remain associated with brain microvessel endothelial cells and not reach the brain parenchyma . if drug translocation is accomplished by conjugation with an antibody , there exists the challenge of dissociation due to the high affinity of antibodies . furthermore , specificity for uptake in the brain may be compromised since the bbb receptors utilized there could also have a widespread distribution on peripheral organs ; in effect , resulting in a seemingly nonspecific uptake . there are multiple barriers in the central nervous system that inhibit api therapies . the blood - brain barrier ( bbb ) and blood - csf ( cerebrospinal fluid ) barriers are vascular in nature , whereas the other , the brain - csf barrier , exists between brain tissue and the csf . the wall of the cerebral microvessels in the brain parenchyma constitutes the bbb . due to its unique structure the multicell layer present in the middle of the brain parenchyma is known as the blood - csf barrier . present there are ventricular cavities ( ventricles ) filled with csf secreted by the epithelial cells of the choroid plexus , a highly vascular tissue with leaky , fenestrated capillaries covered with ependymal epithelium , which has relatively tight junctions . the third barrier , the interface between the csf and brain tissue , is unlike the other two tight blood barriers since it is relatively leaky . since it does not prove to be a significant resistance to mass transport it is a probable route for drug delivery once the transport issues with the other barriers are resolved . given that the area of the bbb is about 1000 times that of the blood - csf barrier , it is more important to circumvent its impermeability , and therefore that is the focus for continued discussion . furthermore , since it is not considered as limiting as compared to the bbb , further discussions related to csf transport are not given here but can be found elsewhere . the transport of substances from capillary blood into the brain tissue is dependent upon molecular size , lipid solubility , binding to specific transporters , and electrical charge . compared to the peripheral microvessel wall , the additional structure of the bbb and tighter endothelial junctions greatly restricts transport of hydrophilic molecules through the gaps between the cells , that is , the paracellular pathway of the bbb . in contrast , small hydrophobic molecules such as o2 and co2 diffuse freely across plasma membranes following their concentration gradients , that is , the transcellular lipophilic diffusion pathway . the bbb permeability to most molecules can be estimated on the basis of their octanol / water partition coefficients . for example , diphenhydramine ( benadryl ) , which has a high partition coefficient , can cross the bbb with relative ease , whereas water - soluble loratadine ( claritin ) is blocked . some solutes with low partition coefficients easily cross the bbb by active or facilitated transport mechanisms , which rely on ion channels , specific transporters , energy - dependent pumps , and a limited amount of receptor - mediated transcytosis . small drug molecules analogous to glucose , amino acids , and small intermediate metabolites , for example , reach brain tissue via facilitated transport mediated by specific transport proteins , whereas larger molecules , such as insulin and other protein type therapeutic agents , are carried across the bbb via receptor - mediated or adsorptive transcytosis . furthermore , some small molecules with high octanol / water partition coefficients are seemingly blocked . thorough data analysis suggests that they are actively pumped back into the blood by efflux systems . for instance , members of the adenosine triphosphate - binding cassette family of exporters are potent energy - dependent transporters . they contribute greatly to the efflux of xenobiotics and due to this protective role impede the delivery of therapeutic agents . consequently , to develop effective and efficient methods for drug delivery to the brain through the bbb , it is imperative to control its permeability . this requires understanding the mechanism by which these structural components , as well as transporters , receptors , efflux pumps and other components at the endothelium and astrocyte foot processes determine it . various methods such as intracerebral implantation , microdialysis , convection - enhanced distribution ( ced ) , osmotic shock , and chemical modification of the bbb have been developed for delivering drugs into the brain . however , the applications of these methods are limited and they can only partially keep with the demands of modern therapies . for instance , the efficiency of intracerebral implantation , microdialysis and ced methods are low since their major transport mechanisms are diffusion and convection of interstitial fluid . for effective treatment of cns diseases , an adequate amount of therapeutic agents must reach the specific regions of the brain . as discussed earlier , they can directly deliver therapeutic agents via these transporters by closely mimicking their substrates , or conjugating the drugs to ligands of the specific surface receptors expressed for transcytosis ( receptor - mediated transcytosis , rmt - trojan horse approach ) . furthermore , these functionalized target chaperones are used in delivering cationized proteins , peptides , and as nanoparticle carriers for adsorptive mediated transcytosis ( amt ) . although the exact mechanisms of rmt are not fully understood , the development of drug delivery protocols using receptor targeting has been successful [ 4650 ] . this physiological approach is often referred to as the molecular trojan horse approach since the therapeutic compounds are delivered to specific sites for transcytosis by various forms of vector carriers . the technique is very promising , but unfortunately there remain a number of hurdles to overcome [ 4850 ] . in particular , even if the total amount of drug transported to the brain is large , most of it may not be efficacious since it might remain associated with brain microvessel endothelial cells and not reach the brain parenchyma . if drug translocation is accomplished by conjugation with an antibody , there exists the challenge of dissociation due to the high affinity of antibodies . furthermore , specificity for uptake in the brain may be compromised since the bbb receptors utilized there could also have a widespread distribution on peripheral organs ; in effect , resulting in a seemingly nonspecific uptake . improvements in encapsulation technologies for tissue therapiesthe success of an implant protocol utilizing entrapped tissue for a therapeutic intervention is highly dependent upon controllability of transport characteristics and the microenvironment . the basic concepts are to improve the permeability of the encapsulating hydrogel and maintain a high oxygen partial pressure in the surrounding microenvironment . those that utilize nanotechnology , with their inherent improvement qualities , are the focus in this section . the results of two independent studies that address the individual concepts mentioned above will be discussed briefly . oxygen supply to the capsule surfaces was enhanced through greater vascularization in the microenvironment by stimulation of angiogenesis by cytokines released from the implant [ 3741 ] . use of cargo - loaded functionalized nanovesicles that control individual cytokine release rates is an obvious extension to that work . one important goal of these angiogenesis studies was to quantitatively evaluate the rates at which different individual growth factors ( gfs ) are released from their hyaluronic acid hydrogel implants . the ability of added amounts of heparin to specifically regulate basic fibroblast growth factor ( bfgf ) or vascular endothelial growth factor ( vegf ) , release from their gels without loss of ability to stimulate a neovascularization response was investigated both in vitro and in vivo . for both of these growth factors , as little as 0.03% w / w hp significantly moderated the time course of release , while inclusion of 0.3% hp resulted in sustained release over several weeks .the results of that study suggest the possibility of delivery of growth factors in specified sequences at regulated rates , simply by controlling the composition of the gels . inclusion of as little as 0.3% hp in the gels led to significant differences in the rates of release of individual gfs . by taking advantage of those differences , it may be possible to design implants that are capable of both storing and providing sustained , localized in vivo release of the growth factors , without loss of their biologic effectiveness.co-delivery of a combination of more rapidly released gfs together with more slowly released factors may then permit engineered control of desired physiologic processes such as angiogenesis through use of this selective release sequence concept.the johnson et al . study is an example that illustrates the usefulness of permeability enhancement , through nanotechnology techniques , for delivery of tissue based therapeutic agents . their efforts were to enhance the performance of a bioartificial pancreas to treat diabetes that uses microencapsulation as an immune barrier for transplanted islets of langerhans . unfortunately , the barrier also imposes oxygen diffusional limitations that can result in loss of viability and function . it is critical that the necessary amount of oxygen be delivered to encapsulated tissue after transplantation in order to maintain normal levels of insulin secretion . without a solution that allows for effective oxygen delivery , transplantation of encapsulated tissue may never be successful.their investigation included methods to reduce oxygen transport limitations by enhancing encapsulant oxygen permeability , for example , by combination of a highly concentrated perfluorocarbon ( pfc ) nanoemulsion with alginate ( pfc alginate ) . a theoretical reaction diffusion model was used to predict the three - dimensional distribution of oxygen partial pressure in a spherical microcapsule and a planar slab containing islet tissue , from which the loss of cell viability and the reduction in insulin secretion rate are estimated . numerical simulations were carried out for normal alginate and pfc alginate to examine the effect of surface oxygen partial pressure , capsule diameter , slab thickness , and the size and density of dispersed islet tissue . results show that hypoxic conditions can be reduced , thereby enhancing islet viability and substantially maintaining insulin secretion rate when the pfc nanoemulsion is incorporated in the encapsulation material for both geometries.the approach was also evaluated experimentally , and the ability to enhance encapsulated tissue survival and function was successfully demonstrated , both in vitro and in vivo . intact islets encapsulated in normal alginate and in pfc alginates having the composition described in the numerical predictions were used as model systems . recovery of viable tissue after culture under various o2 partial pressure conditions was expressed as the oxygen consumption rate ( ocr)/unit volume of capsule divided by the same parameter measured immediately after encapsulation and before culture . when cultured at very low po2 , fractional ocr recovery was substantially greater with pfc alginate than with normal alginate . furthermore , examination of histological sections revealed necrosis in some islets in normal alginate capsules cultured at 3.5 and 142 mm hg , whereas no necrosis was observed in islets within pfc alginate capsules . the findings and insights gained from both the theoretical and experimental studies will increase the probability of a successful cell therapy for the treatment of diseases such as diabetes.the concept of backpacks discussed earlier with respect to drug chaperones can also be applied to encapsulation techniques and tissue therapies . the commonality rests with the use of nanofabrication approaches to create these entities , for example , the photolithographic method reported previously [ 31 , 32 ] . the product of this manufacturing step can be either the cell - backpack complexes or freely suspended backpacks . since these backpacks can carry a myriad of compounds with differing functionalities , their applications seem boundless . of particular interest here with respect to tissue engineering is the ability of these freely suspended backpacks to promote cell aggregate self - assembly . the size of these aggregates , as influenced by backpack diameter and ratio of cells to backpacks in the culture medium , has been shown to be reproducible . furthermore , the binding strength is quite strong ; which was demonstrated by forcing the complexes through small pores and noting that the backpacks were not removed from the surface of the cells . the importance lies in the ability to use injection techniques ( as in a needle tip of a syringe assemble ) , or for the movement from blood to tissue ( extravasation ) via narrow gaps . based on these successes , one can envision applications that would create organoids of various types , such as lymphoid and beta cell clusters ( analogous to islet of langerhans ) . in these cases , the cargo could consist of drugs , adjuvants , and/or growth factors ( for angiogenesis stimulation , reproduction , etc . ) . there also appears the potential for wound healing protocols.to support our conjectures , some specific results should be elucidated . in their paper , the cohen group presents fundamental studies on forming cellular aggregates using injectable cellular backpacks , how to control aggregate size , and observations on association strength . using confocal microscopy , flow cytometry , and laser diffraction , they observed that , while very large ( > 1 mm ) aggregates can form , they may also dissociate and reform . aggregates were forced through a nylon mesh filter and observed afterward : as the filter size decreased , resultant aggregates were smaller . when the pore size was reduced to less than the diameter of an individual cell , this implied to them that the attachment is sufficiently strong such that the backpacks would remain attached to a lymphocyte undergoing extravasation in vivo . in conclusion , they feel that an injectable backpack system could have applications in lymphoid tissue engineering as described by others [ 52 , 53 ] , as well as more general cellular engineering applications requiring close cell association . the success of an implant protocol utilizing entrapped tissue for a therapeutic intervention is highly dependent upon controllability of transport characteristics and the microenvironment . the basic concepts are to improve the permeability of the encapsulating hydrogel and maintain a high oxygen partial pressure in the surrounding microenvironment . those that utilize nanotechnology , with their inherent improvement qualities , are the focus in this section . the results of two independent studies that address the individual concepts mentioned above will be discussed briefly . oxygen supply to the capsule surfaces was enhanced through greater vascularization in the microenvironment by stimulation of angiogenesis by cytokines released from the implant [ 3741 ] . use of cargo - loaded functionalized nanovesicles that control individual cytokine release rates is an obvious extension to that work . one important goal of these angiogenesis studies was to quantitatively evaluate the rates at which different individual growth factors ( gfs ) are released from their hyaluronic acid hydrogel implants . the ability of added amounts of heparin to specifically regulate basic fibroblast growth factor ( bfgf ) or vascular endothelial growth factor ( vegf ) , release from their gels without loss of ability to stimulate a neovascularization response was investigated both in vitro and in vivo . for both of these growth factors , as little as 0.03% w / w hp significantly moderated the time course of release , while inclusion of 0.3% hp resulted in sustained release over several weeks . the results of that study suggest the possibility of delivery of growth factors in specified sequences at regulated rates , simply by controlling the composition of the gels . inclusion of as little as 0.3% hp in the gels led to significant differences in the rates of release of individual gfs . by taking advantage of those differences , it may be possible to design implants that are capable of both storing and providing sustained , localized in vivo release of the growth factors , without loss of their biologic effectiveness . co - delivery of a combination of more rapidly released gfs together with more slowly released factors may then permit engineered control of desired physiologic processes such as angiogenesis through use of this selective release sequence concept . study is an example that illustrates the usefulness of permeability enhancement , through nanotechnology techniques , for delivery of tissue based therapeutic agents . their efforts were to enhance the performance of a bioartificial pancreas to treat diabetes that uses microencapsulation as an immune barrier for transplanted islets of langerhans . unfortunately , the barrier also imposes oxygen diffusional limitations that can result in loss of viability and function . it is critical that the necessary amount of oxygen be delivered to encapsulated tissue after transplantation in order to maintain normal levels of insulin secretion . without a solution that allows for effective oxygen delivery , transplantation of encapsulated tissue their investigation included methods to reduce oxygen transport limitations by enhancing encapsulant oxygen permeability , for example , by combination of a highly concentrated perfluorocarbon ( pfc ) nanoemulsion with alginate ( pfc alginate ) . a theoretical reaction diffusion model was used to predict the three - dimensional distribution of oxygen partial pressure in a spherical microcapsule and a planar slab containing islet tissue , from which the loss of cell viability and the reduction in insulin secretion rate are estimated . numerical simulations were carried out for normal alginate and pfc alginate to examine the effect of surface oxygen partial pressure , capsule diameter , slab thickness , and the size and density of dispersed islet tissue . results show that hypoxic conditions can be reduced , thereby enhancing islet viability and substantially maintaining insulin secretion rate when the pfc nanoemulsion is incorporated in the encapsulation material for both geometries . the approach was also evaluated experimentally , and the ability to enhance encapsulated tissue survival and function was successfully demonstrated , both in vitro and in vivo . intact islets encapsulated in normal alginate and in pfc alginates having the composition described in the numerical predictions were used as model systems . recovery of viable tissue after culture under various o2 partial pressure conditions was expressed as the oxygen consumption rate ( ocr)/unit volume of capsule divided by the same parameter measured immediately after encapsulation and before culture . when cultured at very low po2 , fractional ocr recovery was substantially greater with pfc alginate than with normal alginate . furthermore , examination of histological sections revealed necrosis in some islets in normal alginate capsules cultured at 3.5 and 142 mm hg , whereas no necrosis was observed in islets within pfc alginate capsules . the findings and insights gained from both the theoretical and experimental studies will increase the probability of a successful cell therapy for the treatment of diseases such as diabetes . the concept of backpacks discussed earlier with respect to drug chaperones can also be applied to encapsulation techniques and tissue therapies . the commonality rests with the use of nanofabrication approaches to create these entities , for example , the photolithographic method reported previously [ 31 , 32 ] . the product of this manufacturing step can be either the cell - backpack complexes or freely suspended backpacks . since these backpacks can carry a myriad of compounds with differing functionalities , their applications seem boundless . of particular interest here with respect to tissue engineering is the ability of these freely suspended backpacks to promote cell aggregate self - assembly . the size of these aggregates , as influenced by backpack diameter and ratio of cells to backpacks in the culture medium , has been shown to be reproducible . furthermore , the binding strength is quite strong ; which was demonstrated by forcing the complexes through small pores and noting that the backpacks were not removed from the surface of the cells . the importance lies in the ability to use injection techniques ( as in a needle tip of a syringe assemble ) , or for the movement from blood to tissue ( extravasation ) via narrow gaps . based on these successes , one can envision applications that would create organoids of various types , such as lymphoid and beta cell clusters ( analogous to islet of langerhans ) . in these cases , the cargo could consist of drugs , adjuvants , and/or growth factors ( for angiogenesis stimulation , reproduction , etc . ) . the cohen group presents fundamental studies on forming cellular aggregates using injectable cellular backpacks , how to control aggregate size , and observations on association strength . using confocal microscopy , flow cytometry , and laser diffraction , they observed that , while very large ( > 1 mm ) aggregates can form , they may also dissociate and reform . aggregates were forced through a nylon mesh filter and observed afterward : as the filter size decreased , resultant aggregates were smaller . when the pore size was reduced to less than the diameter of an individual cell , this implied to them that the attachment is sufficiently strong such that the backpacks would remain attached to a lymphocyte undergoing extravasation in vivo . in conclusion , they feel that an injectable backpack system could have applications in lymphoid tissue engineering as described by others [ 52 , 53 ] , as well as more general cellular engineering applications requiring close cell association . in this section , challenges such as safety considerations and reformulation strategies to overcome loading limitations , overdosing , and clearance issues are addressed . the opportunities lie in the enhanced capabilities with respect to improves therapeutic intervention strategies and additional applications for nanomedicine in the healthcare sector . the perception that nanomaterials have inherent incompatibility issues with respect to the uptake into the human systemic environment has been addressed by many nanobiotechnology researchers ( see zook et al . for a representative paper from the biochemical science division of the national institute of standards and technology ) . concerns such as toxicity , leaching , clearance , reproducibility / nonuniformity , chaperone characteristics / use of surface active agents and stability are major factors affecting the revolutionization of nanomedicine . the presence of multiple nanotechnology based drugs in the market place attests to the resolution of many of these issues . however , many more related to bioefficacy , loading capacity , and other features associated with performance optimization present ongoing challenges and opportunities for advances in nanomedicine thereby ensuring that it represents the future of medical care . general discussions , with key literature references , can be found in sources such as the biomedical engineering handbook . of particular interest would be the section devoted to bionanotechnology with specific articles related to nanomaterials : perspectives and possibilities in nanomedicine . the following comments are excerpts from their work and that of many other previously mentioned researchers [ 110 , 31 , 32 , 35 , 45 , 52 ] , along with summary statements from previous sections of this paper . specific illness treatments via nanomedicine protocols each have unique detriments that can be remedied by providing a range of delivery systems . the concept is to develop methods of controlled therapeutic delivery and release to specific tissues and tumors over a desired timeline . these systems are designed specifically to deliver soluble drugs , proteins , vaccine adjuvants , and plasmid dna for gene therapy by exposing target cells to their cargo . the chaperone is thus required to enter the cells via endocytic or phagocytic pathways and release its payload through degradation and diffusion mechanisms . the major challenge here is to accomplish these tasks while addressing the issues of biocompatibility , biodegradation , and the capture and clearance by the reticuloendothelial system ( res ) . although excelling at some aspects , the current systems often fail to incorporate all required characteristics for high in vivo performance . the chaperones for therapeutic nanoentities include viral carriers , organic and inorganic nanoparticles , and peptides . although the efficient targeted delivery of therapeutic drugs continues to present challenges ( with tremendous potential benefits ) , the emerging research into proteomics , for gene therapy as the future of nanomedicine treatments is attracting more attention . fortunately , the necessary gene transfection considerations are directly applicable to drug delivery systems also . although highly efficient they pose immunogenic and mutagenic hazards which led researches to seek nonviral vectors . these include liposomes and nanoparticles of peptides and polymers , both synthetic and natural . selection of vector type is dictated by the therapeutic agent , required pharmacokinetics , and the target cellular system , in addition to physical properties such as zeta potential ( positive surface charge ) . the binding to blood proteins , clearance by the res , and circulation times in the range of hours , rather than minutes , can be key performance targets / specifications . hydrophilic polyethylene glycol ( peg ) or longer chain polyethylene oxide ( peo ) are commonly used synthetic polymers . specific details can be found in douglas et al . and their accompanying literature references . the practical considerations enumerated there stress the need for the control of zeta potential , surface functionality via physical and chemical modifications , and the attainment of desired sizing . the method used to determine size is also important since dynamic light scattering ( dls ) frequently gives larger measurement values than electron microscopy . furthermore , dls is particularly dependent on the presence of aggregate - inducing ions and proteins . vehicle surface characteristics are essential to control the contact time these vectors remain in the vasculature of a target region with respect to endocytosis and/or cargo release kinetics . thus , in addition to chemical functionalization there exists numerous opportunities for magnetic , heat , and light affected systems influenced by external stimulus / fields . for example , nanosizing of current marketed products is a means of providing these old drugs a new delivery platform offering new benefits and improved performance . fda records indicate that the majority of approvals are reformulations or combinations of previously approved products . as a new candidate proceeds through its clinical testing program , it can be refined and/or postprocessed from its discovery formulation to meet the requirements of the emerging target product profile ; that is , its delivery route , dosage , and pharmacokinetic behavior . considering its vast potential it becomes evident that nanotechnology will have a significant impact upon the drug delivery sector and its ability to provide sound technological solutions for drug development programs . consequently , market expectations for the nanotechnology drug delivery platform are high , and it is estimated that it will increase to about $ 16 billion ( usd ) by 2014 . novel nanomaterial manufacturing methods and emerging nanotechnology applications for the pharmaceutical industry have been discussed in this paper . these manufacturing methods combine features such as bottom up nanoparticle formation for control of size and crystal structure with continuous manufacturing and process analytical technology ( pat ) for quality control and compatibility with the strict requirements imposed upon the pharmaceutical industry . the production of carefully engineered nanoparticles produced at high throughput rates and elevated technoeconomic stature demonstrates the role that transport phenomena has in path forward approaches for advanced drug delivery .

emerging nanotechnologies have , and will continue to have , a major impact on the pharmaceutical industry . their influence on a drug 's life cycle , inception to delivery , is rapidly expanding . as the industry moves more aggressively toward continuous manufacturing modes , utilizing process analytical technology ( pat ) and process intensification ( pi ) concepts , the critical role of transport phenomena becomes elucidated . the ability to transfer energy , mass , and momentum with directed purposeful outcomes is a worthwhile endeavor in establishing higher production rates more economically . furthermore , the ability to obtain desired drug properties , such as size , habit , and morphology , through novel manufacturing strategies permits unique formulation control for optimum delivery methodologies . bottom - up processing to obtain nano - sized crystals is an excellent example . formulation and delivery are intimately coupled in improving bio - efficacy at reduced loading and/or better controlled release capabilities , minimizing side affects and providing improved therapeutic interventions . innovative nanotechnology applications , such as simultaneous targeting , imaging and delivery to tumors , are now possible through use of novel chaperones . other examples include nanoparticles attachment to t - cells , release from novel hydrogel implants , and functionalized encapsulants . difficult tasks such as drug delivery to the brain via the blood brain barrier and/or the cerebrospinal fluid are now easier to accomplish .

caudal regression syndrome ( crs ) is a rare disorder of distal spinal segments affecting the development of the spinal cord , with attendant sequelae . the exact etiology is elusive , though maternal diabetes , genetic factors , and hypoperfusion might play roles . we report late presentation of crs in a 9 year old with scoliotic deformity with an asymptomatic cervical syrinx , in absence of any systemic abnormality . a 9-year - old girl was referred in view of a lateral deformity of dorso lumbar spine to explore the possibility of an underlying skeletal dysplasia . there was no history of trauma , associated weakness of any part of body , bowel and bladder involvement or protruding mass over the spine . child was born of third degree consanguious mating at full term by normal vaginal delivery at home . according to mother she was of normal intelligence with intelligence quotient ( iq ) of 105 on developmental assessment scales for indian infants ( dasii ) score . clinical examination revealed short stature with short trunk , the height being less than 3 centile with upper to lower segment ratio 0.9 . examination revealed multiple hypo pigmented patches over the left ear lobule , left angle of mouth , and mid dorsum of right leg . additional finding was evidence of dysgenesis at lumbo sacral region with abnormal orientation of sacrum [ figure 1 ] . magnetic resonance imaging ( mri ) spine revealed butterfly vertebrae at d10 level , the first three sacral segments were hypoplastic , distal sacrum and coccyx were absent . a syrinx was present in cervico - thoracic region opposite c5-t1 vertebrae [ figure 2 ] . x - ray showing lumbosacral dysgenesis with abnormal orientation of sacrum saggital neuroimaging shows rudimentary disc at l1-l2 , l3-l4 , visualization of only s1 and s2 segments and non - visualization of distal sacral segments and coccyx crs is a rare congenital malformation characterized by varying degrees of developmental failure first described by duhmel in 1964 to explain the spectrum of sacrococcygeal malformations . the developmental defects include the lower extremities , the lumbar spine , the coccygeal and thoracic vertebrae , and corresponding segments of spinal cord . a male to female ratio of 2.7:1 has been reported . however , upto 22% cases of crs are associated with diabetes mellitus in the mother , diabetic women being 200 times to 400 times more likely to have a child with crs . group 1 has blunt spinal cord termination above l1 , and is the most severely affected . group 2 has less severe dysgenesis with low - lying tapered spinal cord and tethered cord , which may be caused by tight filum , lipoma , etc . , thus , the best diagnostic clue for crs is dysgenetic lumbosacral vertebrae and abnormal distal spinal cord . the latter may explain or at least aggravate the scoliosis that was the only clinical sign in this child . the sacral dysgenesis below s2 suggests this is a group 2 case , therefore an associated tethering mechanism should be suspected . alternatively , probably the scoliosis may be explained by vertebral anomalies at d10 , l1 - 2 and l3 - 4 instead of a tethering mechanism , it is unlikely that a child with spinal cord tethering associated with a crs would merely present with a scoliosis . on the other hand , it is even more unlikely to presume there would be a scoliosis without an associated tethering mechanism , unless the scoliosis is related to the malaligned dysgenetic vertebra as seen in our case . the exact etiology of crs at embryonic level is thought to result from defect in induction of caudal elements in the embryo before the 4 week of gestation . the insult occurs at midposterior axis mesoderm , causing the absence of the development of the mesoblastic caudal bud . the proximity and interdependence of developing caudal neurons , spinal , hindgut and mesonephric elements involved in closure of the neural tube result , in the constellation of neural , distal vertebral , anorectal , renal , and genital abnormalities . the attenuation of bone morphogenetic protein signaling at the posterior primitive streak of embryos leads to the caudal dysmorphogenesis including the cloaca and fusion of both hind limbs . hedgehog - responding cells derived from peri - cloacal mesenchyme contribute to the urogenital / reproductive organs . these findings indicate the existence of developmental programs for the coordinated organogenesis of urogenital / reproductive tissues based on growth factor function and crosstalk . interestingly , structures that are developmentally separate from these caudal elements such as the brain , proximal spine and spinal cord , are generally spared by crs . the consequences of the disruption after the maturation of spinal cord 's caudal portion ensue after the 4 week of gestation are different . this results in motor deficits and neurologic impairment , varying from incontinence of urine and feces to complete neurologic loss . understandably , most children affected by crs- except the very mild cases - would have some problem with genito - urinary and/or anorectal anatomy and function while our case was surprisingly asymptomatic till 7 years of age . short trunk , late onset scoliosis and absence of anomaly of any other system are the unusual manifestations of our case . the presence of an asymptomatic cervical syrinx in association with crs in our case is also intriguing . the overall radiological incidence of syringomyelia in patients with distal spinal abnormalities has varied from 19% to 22.5% in asymptomatic group to 48.5% in symptomatic group . if one were to consider the most accepted theories concerning the pathogenesis of syringomyelia in children with crs , chiari ii malformation , hydrocephalus and tethered cord favour the formation of a cavitating lesion of the spinal cord . in fact , a sizeable spina bifida population , screened with cranial and spinal ultrasound in infancy , showed the absence of syringomyelia . the syrinx may start after the 1 year of life , and remain asymptomatic for a variable amount of time . in our child it was interesting to note the presence of segmental vitiligo in our patient with crs , which might be a chance assoction . neurosurgical consultation taken advocated stringent follow - up , but decided against prophylactic untethering in absence of neurological symptoms . given the fact that genetic and environmental causes may produce a very similar phenotype , a sharp division between syndrome and association is nearly impossible and makes counselling for recurrence risk challenging . the term caudal regression is probably incorrect since more than caudal structures are involved and nothing regressed that was previously present .

we report late presentation of caudal regression syndrome in a 9 year old presenting with a scoliotic deformity . she in addition had an asymptomatic cervical syrinx and vitiligo . we discuss the reasons for this unusual constellation of symptomatology present in our case .

tumour necrosis factor alpha ( tnf - alpha ) is one of the most frequently studied pro - inflammatory cytokines . it drives the activation and recruitment of inflammatory cells , amplifies the production of other pro - inflammatory cytokines , and activates nuclear transcription factors , thereby promoting and maintaining the inflammatory response . tnf - alpha is likely to be a key cytokine in several autoimmune diseases such as rheumatoid arthritis ( ra ) , inflammatory bowel disease , systemic sclerosis , and systemic lupus erythematosus [ 13 ] . a very large number of studies have been performed investigating the importance of tnf - alpha in arthritis , especially ra . tnf - alpha has also attracted interest in recent years for its possible role in skeletal muscle damage . increased protein degradation , as well as decreased body weight and food consumption , was demonstrated when tnf - alpha was administered to rats via a catheter into the external jugular vein . crush injury in mice leads to elevated tnf - alpha levels in skeletal muscle tissue and there is an increase in tnf - alpha serum levels in response to repetitive strain injuries . for example , an experiment in which tnf - alpha was administered to mice via an osmotic pump led to accumulation of inflammatory cells in skeletal muscles but no signs of atrophy or injury . furthermore , the results of studies on tnf receptor knockout and tnf - alpha antibody - neutralized mice indicate that tnf - alpha can actually be involved in the recovery of muscle function after traumatic muscle injury . therefore , it might be that the role of tnf - alpha in muscle injury varies with the type , severity , and stage of the injury . in humans , tnf - alpha is known to be intimately involved in cachexia , a complex condition characterised by progressive muscle loss that affects up to 13% of patients with ra . nevertheless , a recent study showed acute elevation of tnf - alpha not to affect markers of systemic or skeletal muscle turnover in healthy humans . remarkably little data is available on the role of tnf - alpha in situations where there is a pronounced infiltration of inflammatory cells in the muscle tissue , that is , myositis . from studies in tissue other than muscle , it is known that macrophages and other immunoactive cells such as monocytes , mast cells , and neutrophils are responsible for tnf - alpha production [ 1316 ] . data addressing a possible tnf - alpha production by inflammatory cells in myositis comes almost entirely from studies of patients affected by a group of diseases known as idiopathic inflammatory myopathies these autoimmune diseases include mainly the subgroups inflammatory myopathic polymyositis , dermatomyositis , and inclusion body myositis . in these conditions , tnf - alpha is also expressed in the inflammatory cells in crush - injured and transplanted muscle autografts in mice . finally , blockade of tnf - alpha in the dystrophic ( mdx ) mouse , which is the most frequently used model of duchenne 's muscular dystrophy , reduces tnf - mediated adverse responses to exercise - induced muscle damage [ 20 , 21 ] . however , without further information , it is difficult to reach conclusions on the importance of tnf - alpha and the possible usefulness of tnf - blocking in muscle disorders , including in myositis . furthermore , it should be stressed that the majority of information on the tnf system for skeletal muscle tissue has come from studies of cultured myoblasts ( e.g. , ) . animal models are needed to advance our understanding of the disease mechanisms of tnf - alpha that are involved in myositis . our laboratory has developed a rabbit model of marked muscle ( m. triceps surae ) and tendon overuse that , when combined with injections of substances eliciting pro - inflammatory effects , results in significant myositis . this model causes myositis that morphologically resembles that seen in inflammatory myopathies [ 17 , 18 ] but without having an apparent autoimmune origin . the model leads to a muscle pathology that to some extent resembles the morphology seen in overuse musculoskeletal disorders ( see , for a review , see ) . in studies using this model , we noted evidence of local glutamate signaling in the cells of the inflammatory infiltrates within the muscle tissue . we have taken advantage of this model in order to examine the tnf - system during myositis development . thus , the aim of this study was to examine the pattern of tnf - alpha expression in one segment of the triceps surae muscle ( the soleus muscle ) affected by myositis using immunohistochemistry and in situ hybridization in order to get an insight into the possible usefulness of this model for further studies on the importance of tnf - alpha in myositis . the animals had an average weight of 4 kg and ranged in age from 6 to 9 months . six of the animals corresponded to control nonexercised animals ( subgroup 1 ) and 22 were assigned to an exercise protocol leading to marked overuse of the triceps surae muscle ( subgroups 25 ) . in order to increase the muscle affection , including the degree of inflammation , the muscle overuse was combined with paratendinous injection treatment ( cf . the following ) . as a control for this , five of the exercised animals ( subgroup 2 ) were given control substance ( nacl ) just outside the tendon of the triceps surae muscle ( i.e. , the achilles tendon ) . 17 of the exercised animals ( subgroups 35 ) were in parallel to being subjected to marked overuse , given local injections of pro - inflammatory substance ( substance p and/or endopeptidase inhibitors ; captopril , dl - thiorphan ) outside the achilles tendon . substance p was given as this neuropeptide has well - known pro - inflammatory effects and the injections of the endopeptidase inhibitors were given in order to diminish endopeptidase activities and thereby lead to more pronounced effects of substance p. for clarification of all the various animal groups , see table 1 . the animals were exposed to an exercise procedure designed to cause marked overuse of the triceps surae muscle and the associated tendon ( the achilles tendon ) . the procedure is performed according to previously described procedures , with some modifications . throughout the experiment , injections of diazepam ( 5 mg / ml ; 0.2 ml / kg ) and fentanylfluanison ( 0.2 - 0.3 ml / kg ) . fentanylfluanison ( 0.1 ml / kg ) was injected each 3045 min during the experiment in order to maintain the anaesthesia . mg / kg ) was given subcutaneously ( s.c . ) after each experiment session . the experiment was repeated every second day for one week ( 4 exercise sessions in total ) . an apparatus ( kicking machine ) was used to achieve passive repetitive flexions and extensions of the right ankle joint ; a pneumatic piston attached to the right foot produced the movements . during the plantar flexion , an active contraction was furthermore induced by electrical stimulation via surface electrodes ( pediatric electrode 40 426 a , hewlett packard , andover , ma , usa ) placed 2 cm apart over the triceps surae muscle of the right leg . the stimulation was synchronized with the plantar flexion movement of the piston by a microswitch , which trigged the stimulator unit ( disa stimulator type 14e10 , disa elektronik a / s , herlev , denmark ) . an impulse of 0.2 ms duration was delivered 85 ms after the initiation of the plantar flexion at an amplitude of 3550 v. the movement frequency was 150 repetitions per minute . the pelvis was strapped down and there were no ankle movements on the left side . one day after the final exercise session , the animals were sacrificed by an overdose of pentobarbital . for further details about injections were given into the loose connective tissue around the achilles tendon , that is , in the paratenon region . the substances injected were ( a ) nacl ( 0.91% w / v , volume : 1 ml ) ( subgroup 2 ; n = 5 ) , ( b ) substance p ( 10mol / ml ) and captopril ( sigma ) ( c4042 , 30 mol / kg ) both in distilled water ( volume : 1 ml ) and dl - thiorphan ( n-[(rs)-2-benzyl-3-mercaptopropanoyl]-glycine ) ( sigma ) ( 500 g / ml ; 0.02 ml ) ( subgroup 3 ; n = 5 ) , ( c ) captopril ( sigma ) ( c4042 , 30 mol / kg , dissolved in distilled water , volume 1 ml ) + dl - thiorphan ( sigma ) ( 500 g / ml , 0.02 ml ) ( subgroup 4 ; n = 6 ) , and ( d ) captopril ( sigma ) alone ( c4042 , 30 mol / kg , dissolved in distilled water , volume 1 ml ) ( subgroup 5 ; n = 6 ) . , unpublished observations ) and as recently reported , it has become obvious that subgroups 35 develop myositis . therefore , these subgroups were grouped together and further on referred to as the myositis group . as there are minimal or no signs of myositis in the nacl - treated subgroup ( subgroup 2 ) , the animals in this group were grouped together with the nonexercised animals ( subgroup 1 ) , comprising the non - myositis group . muscle samplesafter the animals were sacrificed , the right triceps surae muscle was dissected out and immediately transported on ice to the laboratory . samples conforming to the soleus muscle part ( 58 10 mm ) were dissected out and fixed by immersion overnight at 4c in an ice - cold solution of 4% formaldehyde in 0.1 m phosphate buffer ( ph 7.0 ) . the samples were thereafter thoroughly washed in tyrode 's solution containing 10% sucrose at 4c overnight , mounted on thin cardboard in oct embedding medium ( miles laboratories , naperville , ill , usa ) , frozen in propane chilled with liquid nitrogen , and stored at 80c . the sections were mounted on slides precoated with chrome - alum gelatine and were then processed for immunohistochemistry . after the animals were sacrificed , the right triceps surae muscle was dissected out and immediately transported on ice to the laboratory . samples conforming to the soleus muscle part ( 58 10 mm ) were dissected out and fixed by immersion overnight at 4c in an ice - cold solution of 4% formaldehyde in 0.1 m phosphate buffer ( ph 7.0 ) . the samples were thereafter thoroughly washed in tyrode 's solution containing 10% sucrose at 4c overnight , mounted on thin cardboard in oct embedding medium ( miles laboratories , naperville , ill , usa ) , frozen in propane chilled with liquid nitrogen , and stored at 80c . the sections were mounted on slides precoated with chrome - alum gelatine and were then processed for immunohistochemistry . other sections were processed for morphology or in situ hybridization . as a reference , human ra synovial tissue was analyzed . the tissue was fixed and further processed in the same way as were the rabbit specimens ( cf . one section from all specimens was stained in harris haematoxylin solution for 2 min . these sections were then rinsed in distilled water , dipped in 0.1% acetic acid for a few seconds , and then washed in running water . the sections were pretreated with acid potassium for 2 min , a procedure found to enhance specific immunofluorescence reactions . thereafter followed incubation for 20 min in a 1% solution of triton x-100 ( kebo lab , stockholm ) in 0.01 m phosphate buffer saline ( pbs ) , ph 7.2 , containing 0.1% sodium azide as preservative , and three 5 min washes in pbs . the sections were then incubated for 15 min in 5% normal donkey serum ( code no : 017 - 000 - 121 , jackson immune research lab . inc . ) in pbs . next , incubation with the primary antibody , diluted in pbs ( ph 7.4 ) , occurred in a humid environment for 60 min at 37c . after incubation with specific antiserum , and three 5 min washes in pbs , another 15 min incubation in normal donkey serum followed . next , the sections were incubated with either of these donkey antigoat iggs for 30 min at 37 : fitc-(fluorescein isothiocyanate- ) conjugated affinipure donkey antigoat igg ( jackson immunoresearch lab inc , dilution 1 : 100 ) or alexa fluoro 488 donkey antigoat ( invitrogen , dilution 1 : 300 ) . the sections were thereafter washed in pbs and then mounted in vectashield mounting medium ( h-1000 ) ( vector laboratories , burlingame , ca , usa ) . examination was carried out in a zeiss axioscope 2 plus microscope equipped with epifluorescence optics and an olympus dp70 digital camera . to clarify the tnf - alpha immunoreaction pattern in relation to that of white blood cells , double stainings were made . as it is frequently emphasized that macrophages [ 13 , 16 ] show tnf - alpha expression , double stainings for tnf - alpha / macrophage marker ( cd68 ) were performed . double stainings for tnf - alpha / t - cell - neutrophil marker were also performed . alexa fluoro donkey antigoat was used as secondary antibody for tnf - alpha immunolabelling , and tritc ( tetramethylrhodamine isothiocyanate- ) conjugated rabbit antimouse antibody was used for stainings for cd68 and t - cell / neutrophil marker . for detailed information about the staining procedures for tnf - alpha , when doing double stainings for cd68 and t - cell / neutrophil marker , 5% normal rabbit serum ( code no : x0902 , dako cytomation , glostrup , denmark ) , diluted in 0.1% bsa ( bovine serum albumin ) in pbs , was used as normal serum , and tritc - conjugated rabbit antimouse antibody ( r0276 , dako cytomation ) , diluted 1 : 40 in 0.1% bsa in pbs , as the secondary antibody . an antibody against tnf - alpha produced in goats was used ( af-210-na ; r&d systems ) . various dilutions were trialled to achieve the optimal fluorescence to background ratio , with a dilution of 1 : 50 found to be optimal . the supplier reports that this antibody is directed against e. coli - derived recombinant human tnf - alpha . the tnf - alpha - specific igg was purified by human tnf - alpha affinity chromatography . it is described to be specific via having the ability to neutralize the biological activity of recombinant human tnf - alpha . of note , the tnf - alpha amino acid sequence homology between species is reported to be highly conserved and tnf - alpha dna sequence comparison shows an overall high sequence homology between various species ( including rabbit ) . in control stainings , preabsorption of the primary antibody with tnf - alpha antigen ( t6674 ; sigma ; 20 g / ml antiserum ) was performed overnight at 4c . a macrophage ( cd68 ) antibody ( m0814 ) from dako cytomation ( glostrup , denmark ) was used . it is an affinity purified mouse monoclonal antibody and was used at a dilution of 1 : 100 in 0.1% bsa in pbs . the antigen for this antibody is glycosylated transmembrane glycoprotein , which is mainly located in lysosomes . a mouse antirabbit t - cell and neutrophil antibody ( mca805 g ) from abd serotec ( oxford , uk ) was furthermore used . it is an affinity purified mouse monoclonal antibody against a cell surface antigen , which is expressed by a subset of t - cells , thymocytes , neutrophils , and platelets in rabbits . stainings performed in a parallel project on rabbit soleus muscle were used as a reference ( control ) for the current double stainings . in that project , double stainings were performed using the same cd68 and t - cell / neutrophil marker antibodies as in the current double stainings . in this previous study , double stainings were made against an antibody produced in goats ( against vglut2 ; santa cruz ) , that is , being of the same type as the tnf - alpha antibody used in the current study . in these reference stainings , the same types of secondary antibodies as described previously were utilized . in situ hybridization was used as a complementary method to detect the expression of tnf - alpha , namely , at the mrna level . a digoxigenin-(dig ) hyperlabeled oligonucleotide probe ( ssdna ) for detection of rabbit tnf - alpha mrna was used on sections from myositis ( 4 specimens ) and nonmyositis ( 1 specimen ) groups ( gd1001-ds custom designed ; genedetect , new zealand ) . the procedures were performed according to an established protocol , using an alkaline phosphatase - labeled anti - dig antibody for detection . the probe for tnf - alpha mrna the tissue specimens were cut into 10 m thick fresh cryosections using a cryostat ( with a knife washed in 70% etoh in depc [ diethylpyrocarbonate]-h2o ) and mounted onto super frost plus slides ( nr.041200 , menzel glser ) . the protocol that thereafter followed was that previously used in our laboratory for detection of mrna for other substances ( e.g. , [ 3436 ] ) . an alkaline phosphatase-(ap ) labelled anti - dig antibody ( roche , germany , 11 093 274 910 ) was used for detection . the corresponding sense dig - hyperlabeled ssdna probe was used as a negative control . as a positive control probe , a -actin antisense probe ( gd5000-op ) was used , comparisons being made with sense -actin probe ( genedetect , new zealand ) . myositis was observed in subgroups 35 , and these are now collectively being referred to as the myositis group ( cf . above ) . the most noteworthy feature was the presence of an inflammatory infiltrate ( figure 1 ) , although muscle fiber changes were also observed , including muscle fiber necrosis ( cf . ) . variations in the levels of myositis were observed between the different subgroups , as well as between different animals within the subgroups . the inflammatory infiltrates were seen in some parts of the specimens . in the other groups ( subgroups 1 and 2 ) , there were no or very marginal changes seen and these are now collectively referred to as the nonmyositis group . it was considered relevant to examine a reference tissue regarding the demonstration of tnf - alpha . human ra synovial tissue was therefore analysed to provide reference information for the particular tnf - alpha antibody used , as it a well - known fact that there is a marked tnf - alpha expression in the inflammatory infiltrates in the synovial tissue of patients with ra . we observed that mononuclear - like cells of the human synovial tissue exhibited immunoreactions when incubated with the tnf - alpha antiserum ( figures 2 and 3 ) . the reactions were in high magnification seen in the form of intracellular granular reactions ( cf . figures 2 and 3 ) . the specificity of the reactions was confirmed via preabsorption with synthetic antigen ( figure 2 ) . the cells occurred as parts of immune cell aggregates or as isolated cells in the synovial tissue . pronounced tnf - alpha immunoreactions were observed in cells of the inflammatory infiltrates ( figures 4 and 5 ) in all subgroups in the myositis group . it was noteworthy that the immunoreaction patterns seen in the cells resembled those observed for the mononuclear - like cells of the human ra synovial tissue . thus , the reactions showed a granular pattern in high magnification ( figures 4 and 5 ) . in lower magnification , fibroblasts in the connective tissue did also to some extent display tnf - alpha immunoreactions , however the reactions were very faint . no specific tnf - alpha reactions were noted for blood vessel walls , the nerve fascicles , muscle spindles , and the muscle fibers ( not shown ) . in order to clarify the patterns of cellular reactions for tnf - alpha in the inflammatory infiltrates , double stainings for tnf - alpha / cd-68 and tnf - alpha / t cells and neutrophil marker were performed . it was found that cd68 coexisted with tnfalpha in cells in the inflammatory infiltrates ( figure 6 ) . on the other hand , colocalization between tnf - alpha and t cells and neutrophil marker was not observed ( not shown ) . in the reference studies ( cf . section 2 ) using the same secondary antisera and the same white blood cell markers but a primary goat antibody not directed against tnf - alpha , completely different colocalization patterns were noted . reactions for tnf - alpha mrna were revealed for white blood cells of the inflammatory infiltrates in the myositis specimens ( figure 7 ) . reactions were also seen for fibroblasts ( figure 8) and sometimes for blood vessel walls ( figure 9 ) and muscle fibers ( figure 10 ) . the muscle fibers and blood vessels for which reactions were seen were located in the regions with inflammatory infiltrates . it was noted that the muscle fibers with reactions for tnf - alpha mrna were often infiltrated by inflammatory cells . the majority of the muscle fibers and blood vessels in the tissue of the myositis samples had no demonstrable reaction . there were no reactions at all in the musculature and the blood vessel walls in the nonmyositis samples . no reactions were noted for nerve fascicles and muscle spindles in any of the specimens from the myositis or non - myositis groups . it is well known that tnf - alpha is highly involved in arthritis , notably in ra . accordingly , in our reference studies in the present investigation we found that tnf - alpha was expressed in mononuclear - like cells in the ra synovial tissue . detection of tnf - alpha reactions was thus clarified from the methodological point of view , and verifications were obtained via preabsorption stainings . with this as a basis , studies on tnf - alpha in myositis were performed . a unique model for the production of myositis in rabbit musculature ( the soleus muscle ) was utilized . the main finding was that cells in the inflammatory infiltrates in the myositis muscles were found to express tnf - alpha at both at the mrna and protein levels . expression of tnf - alpha in macrophages has previously been noted in other situations ( e.g. , [ 13 , 16 ] ) , including inflammatory myopathies . in contrast , in our recent studies using the current myositis model , expression of the vesicular glutamate transporter vglut2 was noted in white blood cells in the inflammatory infiltrates other than macrophages . a further main finding was that the muscle fibers and blood vessel walls in areas showing inflammatory infiltration exhibited tnf - alpha mrna and that fibroblasts also were seen to exhibit tnf - alpha mrna . from a methodological point of view , it was clear that muscle fibers , blood vessel walls , and fibroblasts exhibited tnf - alpha mrna but that no reactivity ( muscle fibers , blood vessel walls ) or very weak reactivity ( fibroblasts ) was noted at the protein level . the production level in these locations is therefore likely to be low , which precluded clear detection with our immunohistochemical methods . it is also possible that our in situ hybridization method detects very small quantities of tnf - alpha mrna . nevertheless , it has previously been shown that tnf - alpha can be expressed not only in inflammatory cells but also in injured muscle fibers and fibroblasts in response to muscle injury ( crush - injury ) as well as in muscle fibers and cells in the connective tissue in inflammatory myopathies [ 17 , 18 ] . the patterns of morphologic appearances of the inflammatory infiltrates and other morphologic changes seen resembled the appearances that can be seen in the muscle tissue in inflammatory myopathies . nevertheless , preliminary analysis using elisa detecting anti - jo-1 antibodies , which are known to correlate with disease activity for patients with inflammatory myopathy , does not lend proof to the theory that the myositis in our model is autoimmune in origin ( unpublished observations ) . however , further studies on this aspect are warranted . as noted previously , tnf - alpha immunoreactions in inflammatory cells invading muscles affected by myositis have previously only been documented in biopsies from patients with inflammatory myopathies [ 17 , 18 ] and in muscle of mice in response to crush - injury . thus , in combination with this previous work , our results imply that tnf - alpha is intimately involved in the inflammatory process in myositis . indeed , it has been suggested that tnf - alpha may have a role in the pathogenesis of the myositis in the inflammatory myopathies [ 40 , 41 ] and that a marked inflammatory response involving tnf - alpha may be directly responsible for damaging muscle fibres in myopathic conditions . whether or not the tnf - alpha produced by the cells of the inflammatory infiltrates is entirely responsible for pro - inflammatory and damaging effects remains open to speculation . it is well known that tnf - alpha administration can have pro - inflammatory and detrimental effects , for example , leading to various catabolic changes as seen in studies on cultured skeletal muscle cells [ 4 , 43 , 44 ] . however , there is a marked discrepancy in the literature regarding the effect of tnf - alpha on the musculature . some studies on myoblast cell culture show that tnf - alpha administration does not have catabolic effects ( e.g. , ) , and other studies documenting accumulations of inflammatory cells in skeletal muscle in response to tnf - alpha administration show no decrease in skeletal muscle proteins and no signs of muscle atrophy or injury . perhaps these discrepancies reflect a dual role of tnf - alpha where in some circumstances inflammatory cell derived tnf - alpha can play a protective role and also be involved in the recovery of muscle function after traumatic injury and in muscle regeneration . the discrepancies may also reflect the fact that different methods have been used in the studies that have been performed . the results in preliminary studies on inflammatory myopathies suggest that tnf blocking might be useful , but it is also emphasized that further studies are needed in order to clarify if this type of treatment is indeed useful . results of in vitro studies suggest that targeting tnf - alpha might be worthwhile in myositis [ 49 , 50 ] and studies on dystrophic mdx mice subjected to wheel exercise indicate that tnf blockade can reduce myofiber necrosis [ 20 , 21 ] . the use of anti - tnf treatment in studies on a rat model of repetitive reaching and grasping leads to an improvement in grip strength and attenuated task - induced increases in inflammatory cytokines , including tnf - alpha . although the use of other animal models have shown inflammation in muscle tissue in response to various forms of exercise [ 50 , 52 ] , the myositis model used in the current experiment is clearly distinguishable from these models . thus , in contrast to these models , it leads to a marked presence of inflammatory infiltrates in the muscle tissue , that is , a morphology resembling that seen in inflammatory myopathies . in fact , no experimental myositis model exists which resembles the one used here and in which a marked presence of inflammatory infiltrates becomes present in the muscle tissue . those for which such an infiltration has been demonstrated are the model of crush - injury described above and models designed to help understand the mechanisms of inflammatory myopathies that occur in man . in these latter cases , myositis is induced by various infectious agents [ 53 , 54 ] , immunization with muscle components , for example , myosin [ 55 , 56 ] , and intraperitoneal injections with lipopolysaccharide . the tnf system has not been examined in any of these myositis models replicating the inflammatory myopathies seen in man . interestingly , there is evidence indicating a relationship between the inflammatory myopathies and another condition , muscular dystrophy , in the form of complex interactions between immunological and nonimmunological features of the diseases . a noteworthy aspect with the currently used model is that marked overuse is applied in the procedures . nevertheless , a limitation of the present study is that the relative contributions of the exercise protocol and the injections of the proinflammatory substances to the observed myositis are unclear . forthcoming studies will clarify this issue . in any case , the model will , as it is currently used , provide the opportunity to evaluate the effects of interference with tnf - alpha actions in myositis development . an animal model in which the importance of tnf - alpha for myositis development can be followed has previously been lacking . using a newly established rabbit model of myositis development , a marked tnf - alpha expression has here been shown for the cells of the inflammatory infiltrates within damaged muscle . there was thus a clear evidence of local tnf - alpha production via infiltrated inflammatory cells , presumably leading to secondary inflammation - modifying effects . using in situ hybridization , it was also seen that tnf - alpha mrna was detected for muscle fibers and blood vessel walls in regions of inflammatory infiltrates . the current model can be used for further studies on the importance of tnf - alpha in the development of myositis and to document the expression patterns of other cytokines and signal substances in this condition .

the importance of tnf - alpha in arthritis is well documented . it may be that tnf - alpha is also markedly involved in muscle inflammation ( myositis ) . an animal model where this can be investigated is needed . a newly developed rabbit myositis model involving pronounced muscle overuse and local injections of substances having proinflammatory effects was therefore used in the present study . the aim was to investigate the patterns of tnf - alpha expression in the developing myositis and to evaluate the usefulness of this myositis model for further tnf - alpha research . human rheumatoid arthritis ( ra ) synovial tissue was examined as a reference . tnf - alpha immunoexpression and tnf - alpha mrna , visualized via in situ hybridization , were detected in cells in the inflammatory infiltrates of the affected muscle ( soleus muscle ) . coexistence of tnf - alpha and cd68 immunoreactions was noted , suggesting that the tnf - alpha reactive cells are macrophages . expression of tnf - alpha mrna was also noted in muscle fibers and blood vessel walls in areas with inflammation . these findings demonstrate that tnf - alpha is highly involved in the myositis process . the model can be used in further studies evaluating the importance of tnf - alpha in developing myositis .

pre - pubertal testicular and paratesticular tumors represent 1 - 2% of all solid pediatric lesions with an incidence of 0.5 - 2 per 100,000 children . malignant paratesticular tumors do arise , the most common being rhabdomyosarcoma , a non - germ cell tumor ( gct ) . gcts arising from paratesticular tissues are rare , because of which , treatment options are not standardized . an infant with yolk sac paratesticular tumor who underwent high inguinal orchiectomy is reported because of its rarity . a 12-month - old boy presented with painless progressive swelling of the right scrotum of 1 month duration . there was no history of trauma , fever , weight loss or any constitutional symptoms . the general physical examination revealed an active playful child with normal developmental milestones . on local examination , a right scrotal mass of size 3.5 cm 4.5 cm , non - tender , firm to hard in consistency with smooth surface and regular margin was felt just above and separately from the testes . an ultrasonography ( usg ) revealed a large heterogeneous mass lesion in the right hemiscrotum , separately seen from the right testis . the chest radiograph was normal and computed tomography ( ct ) scan of the abdomen and pelvis did not reveal any retroperitoneal lymphadenopathy . the serum alpha fetoprotein ( afp ) level was found to be markedly elevated for age ( 555.1 ng / ml ) with normal beta human chorionic gonadotrophin ( hcg ) . through an inguinal incision , the tumor mass ( which appeared to be slightly adherent to the testes ) along with the testes was delivered with an intact tunica vaginalis . the gross sectioned specimen showed the paratesticular location of the tumor with normal tunica albuginea [ figure 2 ] . the histopathological examination revealed a tumor arranged predominantly in glandular - alveolar pattern with formation of schiller - duval bodies with occasional solid and papillary pattern with hyperchromatic nucleus and moderate amount of clear cytoplasm [ figure 3 ] suggestive of yolk sac ( endodermal sinus ) tumor without any other germ cell component . photograph of the excised tumor following high inguinal orchiectomy with intact tunica vaginalis gross sectioned specimen showing paratesticular location of the tumor with normal testis and intact tunica albuginea photomicrograph of the same tumor showing glandular - alveolar pattern of tumors ( straight arrows ) with formation of characteristic schiller - duval bodies ( black curved arrow ) at 18 months follow - up , the patient was doing well with afp level of 5 ng / ml and no radiological evidence of metastasis . paratesticular tissues consist of the epididymis , spermatic cord , vestigial remnants and fascia derived from abdominal wall during testicular descent . unlike testicular masses , most paratesticular masses are benign . malignant paratesticular tumors are rare and they usually occur from 2 decades to 5 decades . yolk sac tumors are the most common pediatric malignant extragonadal gct . in neonates and young children , these neoplasms are found primarily in extragonadal sites , particularly the sacrococcygeal area . they are also seen in the testes of infants and young boys but rarely at paratesticular sites . to our knowledge , friedman , et al . suggested that extragonadal gct arises when there is aberrant migration from its normal pathway or remains outside the coalescence of gonadal tissue . it is imperative to ensure that testicular tissue is not involved in order to label the tumor as primary paratesticular . however , pain may be associated when torsion of the testes or intra tumoral hemorrhage occurs which may lead to erroneous diagnosis of a benign scrotal lesion leading to scrotal approach and potential violation of onco surgical principles . however , it is often non - specific with malignant tumors mimicking the more common benign lesions and vice versa . chest radiography should be performed as 20% of yolk sac tumors occur with lung metastasis . ct scan of the chest , abdomen and pelvis is mandatory if there is high suspicion of malignant gct . however , the correct diagnosis is made only after histopathological examination , which demonstrates eosinophilic periodic acid - schiff ( pas ) positive inclusions in the cytoplasm of clear cells that consist of afp and schiller - duval bodies . studies have also shown deletions in chromosomes 1p36 in 80 - 100% cases of infantile gct arising from testicular and paratesticular sites . they are generally diploid or tetraploid unlike those in adults which tend to have aneuploid dna . more than 85% cases present in stage i and are predominantly of pure histology , unlike mixed gct in adults . afp levels are often elevated in yolk sac tumors but not beta hcg and serve as a tumor marker which after complete excision should return to normal ( < 20 ng / ml ) within 1 month . persistently elevated afp levels after surgery suggest tumor metastasis or recurrence . the age of the patient should be considered while interpreting levels of tumor markers . in our patient , afp levels were 555.1 ng / ml , which came down to 40.4 ng / ml and 12.1 ng / ml at 1 month and 3 months respectively after surgery . the treatment for paratesticular yolk sac tumor is not standardized . no chemotherapy was given to our patient as it was a stage i disease as per staging system of the children 's oncology group and the national cancer institute . high inguinal orchiectomy was justified as serum afp was highly elevated , the testis appeared to be adherent to the tumor clinically and micrometastasis to the testis could not be ruled out pre - operatively . spread to the retroperitoneal lymph nodes is uncommon in children so routine prophylactic dissection of the nodes is not recommended . however , it may be considered when us shows salvageable testicular tissue in benign tumors with normal serum afp levels . summary of reported cases of paratesticular yolk sac tumor the risk of recurrence in imaging and pathologically proven stage i disease with normal serum afp levels after inguinal orchiectomy is 15 - 22% and is greatest in the first 2 years . survival with modern chemotherapeutic regimen approaches 100% even in metastatic yolk sac tumor in children . chemotherapy is indicated in patients with radiographic evidence of metastatic disease or persistently elevated serum afp levels . our patient has been kept under close surveillance with serum afp estimation and chest radiograph every 3 months for the 1 year and will be monitored 6 monthly in the 2 year . clinical findings along with us and tumor markers help in differentiating this tumor from non - neoplastic mimics . high inguinal orchiectomy is warranted as tumor microinvasion of the testes can not be ruled out pre - operatively , even if the testis is salvageable .

paratesticular germ cell tumors are extremely rare . a 12-month - old boy with yolk sac tumor involving only the paratesticular tissue is reported . pre - operatively raised alpha fetoprotein levels fell to normal levels after high inguinal orchiectomy . this appears to be the youngest and only the 3rd case reported in the english literature .

although hepatolithiasis is a common disease , the combination of hepatolithiasis and bile duct carcinoma only accounts for 1~3% of total cases . but once being complicated with bile duct cancer , patients often present typical symptoms similar to those of hepatolithiasis with cholangitis . imaging examination , on the other hand , may not clearly visualize the tumor due to the coverage by the lithiasis . due to such diagnostic difficulty and its insidious onset , hepatolithiasis with bile duct carcinoma patients often receive confirmed diagnosis only at a late stage , thus severely compromising patient prognosis . it is well known that angiogenesis is closely related with the invasion and metastasis of malignant tumors . there are 2 isozymes of cox : cox-1 potentiates the formation of new vessels in tumors and cox-2 has high expression under the stimuli of inflammatory mediator or growth factors . ki-67 , on the other hand , is a proliferative antigen closely related with cell cycle regulation , as supported by its pleiotropic functions in cytokinetics and oncogenesis . studies have shown that the co - assay of cox-2 and ki-67 expression had significantly higher sensitivity or specificity in breast cancer invasive ductal carcinoma when compared to either single test . the present study therefore investigated the co - expression of cox-2 and ki-67 in hepatolithiasis with bile duct carcinoma patients , in an attempt to find the significance of gene expression in disease occurrence and progression . a total of 44 patients diagnosed with hepatolithiasis with bile duct carcinoma in our hospital between april 2012 and january 2014 were recruited in this study , along with 47 hepatolithiasis patients . inclusion criteria : ( 1 ) had not received surgical resection or radio-/chemo-/hormonal treatment before tissue collection ; ( 2 ) with full information of clinical / tnm staging ; ( 3 ) with confirmed diagnosis of hepatolithiasis with bile duct carcinoma by post - operative pathological examination . exclusion criteria : ( 1 ) complicated with other malignant tumors ; ( 2 ) incomplete medical history ; ( 3 ) accompanied with acute or chronic infection . there were 27 males and 20 females in the cancer group , ages 37~73 years old ( average age=62.53.9 years ) . a further tnm staging following the american joint committee on cancer ( ajcc ) standard identified 6 stage i patients , 11 stage ii patients , 12 stage iii patients , and 18 stage v patients . lymph node metastasis were discovered in 13 patients . a further differentiation score based on gleason system showed 7 low , 21 moderate , and 19 highly differentiated tumors . the invasive condition of tumors was : 11 cases of deep muscular layer invasion ; 26 cases of shallow muscular layer invasion , and 10 cases without any invasion . among 47 hepatolithiasis patients , 26 were males and 21 were females , ages 36~77 years old ( average age=61.84.5 years ) . thirty samples of normal bile duct tissues from hemangioma resection were recruited in the control group . there were 17 males and 13 females , ages 35~79 years old ( average age=62.14.5 years ) . the study protocol was approved by the research ethics committee of our hospital , and all patients gave their informed consent before study commencement . all tissue samples were frozen and sectioned for further hematoxylin - eosin staining and immunohistochemical ( ihc ) staining following standard protocols as stipulated in the ihc kit ( gibco , us ) . mouse anti - human cox-2 and rabbit anti - human ki-67 antibody were purchased from baijing biotech ( china ) . the staining score was calculated as the product of : ( 1 ) percentage of positive cells and ( 2 ) staining intensity . in brief , the positive cell% was deduced as 0 , 1 , 2 , and 3 scores when positive cells accounts for less than 5% , between 5% and 25% , between 26% and 50% , and more than 50% of total cells , respectively . the staining intensity score was 0 for no color , 1 for moderate staining , and 2 for intensive staining . the total scores ( cell percentage score x staining intensity score ) thus range between 0 and 6 . negative ( ) , weak positive ( + ) , moderate positive ( + + ) , and strong positive ( + + + ) were defined for scores of 0~1 , 2~3 , 3~4 , and 5~6 , respectively . the spss 20.0 software package was used to process all collected data , among which analysis of variance ( anova ) was used for multiple group comparisons . enumeration data , on the other hand , was presented as percentage ( % ) and was tested by chi - square method . a total of 44 patients diagnosed with hepatolithiasis with bile duct carcinoma in our hospital between april 2012 and january 2014 were recruited in this study , along with 47 hepatolithiasis patients . inclusion criteria : ( 1 ) had not received surgical resection or radio-/chemo-/hormonal treatment before tissue collection ; ( 2 ) with full information of clinical / tnm staging ; ( 3 ) with confirmed diagnosis of hepatolithiasis with bile duct carcinoma by post - operative pathological examination . exclusion criteria : ( 1 ) complicated with other malignant tumors ; ( 2 ) incomplete medical history ; ( 3 ) accompanied with acute or chronic infection . there were 27 males and 20 females in the cancer group , ages 37~73 years old ( average age=62.53.9 years ) . a further tnm staging following the american joint committee on cancer ( ajcc ) standard identified 6 stage i patients , 11 stage ii patients , 12 stage iii patients , and 18 stage v patients . lymph node metastasis were discovered in 13 patients . a further differentiation score based on gleason system showed 7 low , 21 moderate , and 19 highly differentiated tumors . the invasive condition of tumors was : 11 cases of deep muscular layer invasion ; 26 cases of shallow muscular layer invasion , and 10 cases without any invasion . among 47 hepatolithiasis patients , 26 were males and 21 were females , ages 36~77 years old ( average age=61.84.5 years ) . thirty samples of normal bile duct tissues from hemangioma resection were recruited in the control group . there were 17 males and 13 females , ages 35~79 years old ( average age=62.14.5 years ) . the study protocol was approved by the research ethics committee of our hospital , and all patients gave their informed consent before study commencement . all tissue samples were frozen and sectioned for further hematoxylin - eosin staining and immunohistochemical ( ihc ) staining following standard protocols as stipulated in the ihc kit ( gibco , us ) . mouse anti - human cox-2 and rabbit anti - human ki-67 antibody were purchased from baijing biotech ( china ) . the staining score was calculated as the product of : ( 1 ) percentage of positive cells and ( 2 ) staining intensity . in brief , the positive cell% was deduced as 0 , 1 , 2 , and 3 scores when positive cells accounts for less than 5% , between 5% and 25% , between 26% and 50% , and more than 50% of total cells , respectively . the staining intensity score was 0 for no color , 1 for moderate staining , and 2 for intensive staining . the total scores ( cell percentage score x staining intensity score ) thus range between 0 and 6 . negative ( ) , weak positive ( + ) , moderate positive ( + + ) , and strong positive ( + + + ) were defined for scores of 0~1 , 2~3 , 3~4 , and 5~6 , respectively . the spss 20.0 software package was used to process all collected data , among which analysis of variance ( anova ) was used for multiple group comparisons . enumeration data , on the other hand , was presented as percentage ( % ) and was tested by chi - square method . as shown in figure 1 , cox-2 mainly existed in the cytoplasm in tumor cells , while ki-67 protein was concentrated in the nucleus of tumor cells . further analysis showed that the positive rate of cox-2 and ki-67 in bile duct carcinoma group was 76.60% and 80.85% , respectively , with significant elevation compared to those in control or hepatolithiasis group ( p<0.05 ) . those rates in the hepatolithiasis tissues were also significantly higher than control group ( p<0.05 ) , as shown in table 1 and figure 2 . as shown in tables 2 and 3 , positive expression rate of both cox-2 and ki-67 were significantly correlated with various clinical parameters including tnm staging , lymph node metastasis and differentiation grade ( p<0.05 ) . other general parameters such as patient age , sex , pathological type and size of tumors were not related to protein expression ( p>0.05 ) . within 12-month follow - up , 43 patients survived within a total of 47 bile duct cancer patients , making the 1-year survival rate 91.59% . we further performed a cox regression survival model , considering patient age , sex , disease period , lymph node metastasis , tnm staging , differentiation grade , tumor size , tumor type , and expression of cox-2 and ki-67 . results ( table 4 ) identified 4 factors significantly related to the survival rate : ki-67 positive rate , cox-2 positive rate , tumor differentiation grade , and tnm staging . as shown in figure 1 , cox-2 mainly existed in the cytoplasm in tumor cells , while ki-67 protein was concentrated in the nucleus of tumor cells . further analysis showed that the positive rate of cox-2 and ki-67 in bile duct carcinoma group was 76.60% and 80.85% , respectively , with significant elevation compared to those in control or hepatolithiasis group ( p<0.05 ) . those rates in the hepatolithiasis tissues were also significantly higher than control group ( p<0.05 ) , as shown in table 1 and figure 2 . as shown in tables 2 and 3 , positive expression rate of both cox-2 and ki-67 were significantly correlated with various clinical parameters including tnm staging , lymph node metastasis and differentiation grade ( p<0.05 ) . other general parameters such as patient age , sex , pathological type and size of tumors were not related to protein expression ( p>0.05 ) . within 12-month follow - up , 43 patients survived within a total of 47 bile duct cancer patients , making the 1-year survival rate 91.59% . we further performed a cox regression survival model , considering patient age , sex , disease period , lymph node metastasis , tnm staging , differentiation grade , tumor size , tumor type , and expression of cox-2 and ki-67 . results ( table 4 ) identified 4 factors significantly related to the survival rate : ki-67 positive rate , cox-2 positive rate , tumor differentiation grade , and tnm staging . the combined treatment plan involving surgical resection and chemo-/radio - therapy has obtained satisfactory results in bile duct carcinoma , especially for those early - stage tumors . postoperative follow - ups , however , showed that about 17~51% of patients develop distal metastasis , in addition to the 6~21% rate of local recurrence within 3~5 years after surgery . such recurrent tumors are more difficult to treat and usually lead to death within 1 year . past studies have identified the history of hepatolithiasis as a risk factor of bile duct carcinoma , mainly due to the persistent mechanical stimuli on the bile duct wall . currently , there are many biomarkers for evaluating the prognosis of bile duct carcinoma patients , such as growth factor , vesicular endothelial growth factor ( vegf ) , and transforming growth factor ( tgf)-1 . as shown in this study ( figure 1 ) , ki-67 mainly locates within the nucleus , consistent with its major functions in mitosis . previous studies have found an important role of ki-67 in cell proliferation , as it is both a structural protein and scaffold of chromosomes . because an important feature of tumor cells is the loss of cell cycle control and hyper - proliferation , ki-67 has been widely used as an index reflecting the proliferative activity and recurrence possibility of malignant tumors , including urinary cancer , breast cancer , gastrointestinal cancer , and lung cancer . for example , ki-67 level was significantly elevated in primary stomach cancer patients , especially those patients with lymph node metastasis , distal recurrence , and lower - differentiation tumors . cox-2 belongs to the immediate early gene family and has trace amounts in placenta , kidney , and brain tissues . the expression of cox-2 is only elevated when cells receive inflammatory stimuli and growth factor - like molecules , with its start codon playing an important role in transcriptional modulation . recently , cox-2 has been suggested to be involved in the pathogenesis and progression of malignant tumors , as it has higher expression in most malignant tumor tissues , and may participate in the differentiation , proliferation , metastasis , neo - angiogenesis , and anti - apoptosis , thereby lowering the cell - to - cell adhesion and facilitating disease progression . within the occurrence and progression of malignant tumors , angiogenesis plays an important role . high - level expression of cox-2 may inhibit the immune response and escape from immune surveillance , thus facilitating formation of tumor vessels . for example , cox-2 has been shown to be highly expressed in upper urinary epithelioma by cdna microarray analysis . other studies also reported the correlation between cox-2 expression level and the invasion , metastasis , and prognosis of bladder papillary tumors . in this study , we analyzed the expression pattern of cox-2 and ki-67 in hepatolithiasis with bile duct carcinoma tissues and its clinical significance . table 1 shows that only 1 out of 30 normal bile duct tissue had cox-2 expression , significantly lower than that in the hepatolithiasis ( 29.79% ) or bile duct carcinoma group ( 76.60% ) . these results suggest the possible involvement of cox-2 in the pathogenesis of hepatolithiasis with bile duct carcinoma , consistent with results of clinical studies . other studies found that the inflammation of gland and bile duct epithelial and secondary hyperplasia are indicators of bile duct cancer , which is preceded by hepatolithiasis , cholangitis , and bile duct epithelial hyperplasia . in this study , we also discovered higher expression level of cox-2 in hepatolithiasis compared to normal bile duct , suggesting that the abnormal cox-2 expression may be an early indicator of bile duct cancer . further correlation analysis found elevated cox-2 expression in bile duct cancer patients with advanced tnm stage , higher malignancy , and lymph node metastasis ( table 2 ) . therefore , cox-2 expression may be closely related to the occurrence and metastasis of bile duct cancer . the assay of cox-2 expression in patients who are susceptible to hepatolithiasis with bile duct cancer may help to make early diagnosis . no ki-67 protein has been found in normal bile duct tissues ( table 1 , figure 1 ) , suggesting its lower expression under normal circumstances . however , hepatolithiasis and bile duct cancer tissues had 25.35% and 80.85% ki-67 expression , respectively , significantly higher than in normal tissues . our results are consistent with previous reports , suggesting the elevated ki-67 expression in hepatolithiasis and bile duct carcinoma . correlation analysis showed a strong relationship between ki-67 and tnm stage , lymph node metastasis , and differentiation degree ( table 3 ) . further analysis showed that bile duct cancer with positive ki-67 or cox-2 expression had 1.54- and 1.59-fold mortality rates , respectively , compared to those with negative expression . those 2 proteins , although they had minimal expression in normal cells , can be activated by tumor - related pathways such as growth factor , oncogene , and cytokines . ki-67 and cox-2 are closely related with short - term prognosis of bile duct patients complicated with hepatolithiasis . our results collectively showed that cox-2 and ki-67 were highly expressed in hepatolithiasis with bile duct carcinoma tissues , and might promote the pathogenesis , progression , and metastasis of bile duct tumors .

backgroundas an induced enzyme , cox-2 expression is elevated under stimuli from inflammatory mediator or growth factor product . ki-67 , a cell cycle - related proliferative antigen , reflects the tissue proliferative activity . this study analyzed the expressional profile of cyclooxygenase-2 ( cox-2 ) and ki-67 in hepatolithiasis and bile duct carcinoma tissues , in an attempt to provide evidence for diagnosis and prognosis prediction of disease.material/methodsa cohort of tissue samples from hepatolithiasis with bile duct carcinoma ( n=47 ) patients were analyzed using immunohistochemical ( ihc ) staining method for the expression of cox-2 and ki-67 , in parallel with hepatolithiasis ( n=44 ) and normal bile duct tissues ( n=30 ) . the relationship between expression pattern of cox-2 and ki-67 and pathological conditions was also analyzed , in addition to the correlation with positive expression in hepatolithiasis samples.resultsthe positive expression rate of cox-2 and ki-67 in bile duct carcinoma was 76.6% and 80.9% , respectively , and was significantly higher than those in the hepatolithiasis group , which was also higher than the control group . expression of both cox-2 and ki-67 is closely related to tnm staging , lymph node metastasis , and differentiation stage . they were also correlated with the mortality rate of patients.conclusionsboth cox-2 and ki-67 are abundantly expressed in hepatolithiasis and bile duct carcinoma tissues and may play an important role in the disease occurrence , progression , and metastasis .

one problem may be that it is difficult to present a comprehensive picture of the scope of the problem assessable to other than clinical experts . resistance is a global phenomenon involving many types of infections , bacteria and substances and data are scarce , particularly so in developing countries . nevertheless , using data from the european antimicrobial resistance surveillance system ( earss ) , the european centre for disease prevention and control ( ecdc ) and the european medicines agency ( emea ) issued a report in 2007 on the situation in the eu , norway and iceland for six commonly isolated , often multi - resistant , bacteria ( 4 ) . for the us , information on resistance in , more or less , the same bacteria are available from intensive care units ( 11 , 13 ) . though the figures for the eu and the us are not quite comparable , table 1 gives an indication of the prevalence of resistance in these two regions . shares of resistant isolates in selected bacteria in the eu , norway and iceland and in the us sources : data for the eu , norway and iceland are from earss and refer to 2007 ( cf . data for the us are from cdc national nosocomial infections surveillance system and refer to 2002/2003 ( cf . references 11 and 13 ) . it should be noted that , for the european countries , there are large differences in the shares of resistant isolates in table 1 . in particular , there seems to be a north - south gradient , suggesting that they are less frequent in the scandinavian countries and iceland , and more frequent in the mediterranean countries . it has been suggested that these differences can be partly explained by differences in antibiotic use between countries ( 9 ) . as to trends in resistance , the findings in the ecdc / emea joint technical report indicate that , for europe as a whole , shares of resistant isolates , despite some variation from one year to another , have been fairly constant over time for most of the bacteria in table 1 . exceptions are mrsa , which declined from 2004 to 2007 , and 3rd generation cephalosporin - resistant escherichia coli isolates , which have been rising steadily since 2002 . again , there are differences between countries . for instance , despite the decline in the overall european share of mrsa isolates , two of the 28 countries reported increasing shares . the ecdc / ema - report also presents estimates of the consequences of resistance in terms of annual number of infections , additional deaths , extra hospital days and societal costs ( direct health care costs and the value of productivity lost ) caused by the selected bacteria ( cf . number of infections , deaths and hospital days caused by resistant bacteria in the eu , norway and iceland 2007 source : earss ( cf . reference 4 ) . societal costs of infections caused by antibiotic resistant bacteria in the eu , norway and iceland 2007 ( million ) source : earss ( cf . reference 4 ) . data for the us are more scattered and often concern effects of all infections caused by resistant bacteria in singular hospitals ( 14 ) or , alternatively , the consequences of all hospital acquired infections ( which may not necessarily have been caused by resistant bacteria ) ( 15 ) . however , there are studies reporting that mrsa caused 250,000300,000 hospital acquired infections , about 2.7 million hospital days , and 12,000 deaths annually , resulting in annual costs of about us $ 9.5 billion ( 10.1 billion ) in the early 2000s and that vre caused about 26,000 infections in us hospitals in 2004 ( 16 , 17 ) . regarding the development of new antibiotic substances , the ecdc / emea joint technical report ( 4 ) , as well as successive reports from the infectious diseases society of america ( idsa ) ( 1 , 11 , 13 ) and institute of medicine of the national academies ( iom ) ( 12 ) , moreover , most of them do not represent a novel mode of action associated with a new chemical structure ( 18 ) . iii ) , ecdc / emea and idsa concluded that expectations for a rapid change are low . in particular there seems to be a lack of new antibiotics expected to be effective in resistant gram - negative bacteria ( 3rd generation cephalosporin resistant e. coli and klebsiella pneumonia , and carbapenem resistant pseudomonas aeruginosa ) . a further cause for concern is the fact that pharmaceutical firms seem to have reduced investment in r&d for antibiotics in favour of other drugs with more promising financial incentives ( 1 , 1825 ) . thus , although the data above do not present an encompassing picture of the resistance problem , they do suggest that it is significant and causes substantial societal costs . on a general level , there seems to be consensus that there is a need for doing two things ( 1 ) preserve the efficiency of present substances by restricting use and , ( 2 ) increase the supply of new substances brought to the market ( 1 , 4 , 12 ) . unfortunately , as preserving the efficiency of present substances entails restricting use , the two objectives are partly opposing and not easy to achieve simultaneously . the discussion below uses examples from human medicine and there are concerns that the use of antibiotics in veterinary medicine may be an equally large problem . moreover , resistance to drugs used in veterinary medicine may potentially affect resistance to drugs used in human medicine , and vice versa . however , as the problem is similar , the measures to address it will also be similar . to preserve the efficiency of existing drugs , the need for guidelines for good antibiotic stewardship has been emphasised . such guidelines have also been developed in several countries , although their implementation is not straight forward ( 2630 ) . they include , inter alia , recommendations to increase hygienic measures to control resistance in hospitals and community homes , to condition all use of antibiotics on prescription , to restrict use to bacterial infections only , and emphasise the importance of choosing the optimal therapy with regard to substance , administration , dosage and duration ( 2735 ) . however , hygienic measures to control resistance in the institutional setting are not without costs to hospitals and nursing homes . restricting use to bacterial infections and choosing the optimal therapy requires access to quick and reliable diagnostic facilities which may be a problem outside the hospital setting . thus , faced with concerned patients presenting symptoms not easily diagnosed , physicians may have incentives to prescribe broad spectrum antibiotics with potential adverse effects on resistance . informing the public on the issues involved might make a decision not to prescribe ( until test - results are available ) more acceptable , but this is by no means certain as the individual patient , while gaining the full benefit if treatment is effective , will not bear the full ( societal ) costs of increased resistance if it is not ( 3638 ) . note that the opposite problem applies to hospitals and other institutions when determining if the implementation of stricter hygienic procedures is worthwhile ( i.e. the institution will bear the full costs of the procedures in question while sharing the benefits with society at large ) . to provide patients and institutions with incentives to consider the full societal costs of antibiotic use , it has been suggested that a fee corresponding to the expected costs of resistance should be levied on the use of antibiotics ( 1 , 38 ) . one problem with this solution is that the fee 's demand - constraining effect may be limited if expenses for antibiotics are covered by insurance- or other third party payer systems . . however , that could raise concern regarding the access to antibiotics for less affluent patients ( and countries ) and , therefore , not be acceptable to policy makers . the optimal fee should equal the expected marginal costs of resistance caused by antibiotic use ( 39 ) . if it is set higher , demand will be reduced by too much ( i.e. the health benefits from a marginal increase in use will exceed the expected costs of resistance caused by that marginal increase in use ) and , if it its set lower , demand will be reduced by too little ( health benefits from a marginal increase in use will be smaller than the expected costs of resistance caused by that marginal increase in use ) . as the effects of use on resistance are not fully understood , and may differ between substances , optimal fees can not be expected . one might consider determining the fee on the basis that it , for a given antibiotic , should generate enough returns to finance the development of a successor during the period it takes until resistance becomes a problem for that substance . this requires information on development costs and time until resistance becomes a problem which is likely to depend on the antibiotic in question . however , some knowledge may be gained from past experience . while development costs are the property of pharmaceutical firms and , therefore , not easily accessible , some estimates do exist ( 4044 ) . similarly , there are estimates of the time from introduction to detection of resistance for several substances ( 45 ) . to determine the fee per unit of substance required to raise the necessary funds , information on the annual amounts consumed of different substances would also be needed . a fee determined in this way is of course unlikely to be optimal in the sense defined above . however , it is also unlikely to be too high since one result from studies estimating development costs is that these have been rising continuously since the 1970s . accordingly , while it may not be large enough to achieve an optimal reduction in antibiotic consumption from the perspective of preserving efficiency , it may at least go part of the way . on the other hand , if the consumption of antibiotics is reduced , this could increase the problem of achieving the other objective increasing the supply of new antibiotics . the supply of new antibiotics is dependent on research and development ( r&d ) to discover and market them . the outcome of this process depends on how difficult it is to identify substances with the desired characteristics . this , in turn , is a function of our understanding of the biological mechanisms involved , and of the requirements of the approval procedure . the less that is known about the biological mechanisms , the more resources are needed to uncover the issues involved . similarly , the stricter the requirements for approval , the more resources are needed to satisfy them . our understanding of the biological mechanisms is imperfect ( not least regarding the mechanisms causing resistance ) , suggesting that r&d costs may be substantial . however , the literature does not indicate that they are significantly higher than for other types of pharmaceuticals ( 46 ) . the bulk of the r&d work is carried out by pharmaceutical firms that depend on sales revenues for financing their operation . of course , the larger the costs of r&d are in relation to the ( expected ) revenues , the smaller the incentives for pharmaceutical firms to engage in the process . the revenues are a function of the amounts sold and the price of the drug in question . here , there are indications that revenues from antibiotics may be lower than those from other classes of pharmaceuticals that are prescribed for longer periods ( 18 , 20 , 25 ) . accordingly , it appears that insufficient financial incentives to engage in r&d is an important reason for the paucity of new antibiotic substances . several measures to increase the supply of antibiotics by changing incentives , ranging from changing the requirements for approval of new drugs , over tax - credits for research and public - private partnerships , to prolongation of patent duration , have therefore been discussed and suggested ( 1 , 13 , 18 , 19 , 46 ) . there certainly appears to be good reason to make adjustments in the requirement for approval . for instance , it is not clear why clinical trials for each infection pneumonia , urinary tract infection , skin and soft tissue infection , etc . should be required for approval of the use of a certain drug in their treatment if these infections are caused by the same bacteria . thus , one suggestion is to condition approval on demonstrated efficacy towards specific organisms rather than specific infections ( 13 , 19 ) . this might also increase incentives for pharmaceutical firms to invest in developing drugs with a narrow spectrum relative to broad spectrum drugs , which would reduce resistance caused by the use of antibiotics . the evidence is mixed in that , while they do increase incentives , effects appear to be limited since taxes only constitute a small part of the development costs . public - private partnerships in r&d for antibiotics may be a solution for specific drugs with very limited markets ( xdr / pdr acineobacter and kleibsella ) but could not , and should not , replace the efforts of private firms in r&d to replace antibiotics not facing these market limitations . a patent excludes the holder from competition from generic substances which provides a monopoly position with the potential of gaining larger revenues from higher prices . however , patents are often sought early in the development process and , due to the longevity of this process , they may only last for a short period after the drugs are approved and marketed . increasing the duration of patents for antibiotics might , therefore , provide better incentives for their development . however , the higher prices charged by the monopolist will also affect demand , and it has been shown that this may restrict the use of antibiotics by too much from the societal perspective ( 4750 ) . in addition , the revenues obtained may not necessarily be invested in the development of new antibiotics as they compete for resources with other pharmaceuticals that may be perceived to have better net present earnings ( 18 , 20 , 25 ) . finally , strengthening the monopoly position of patent holders may have detrimental effects on competition which could reduce incentives to develop new substances . an alternative strategy may be subsidies targeted directly at the development of new antibiotics as this would compensate for lower expected sales revenues in comparison to other pharmaceuticals , particularly if measures to constrain the use of antibiotics are successfully applied . these subsidies of course needs finance which may be difficult to secure given tight government budgets . however , as noted above , if a fee is levied on the consumption of antibiotics as a measure to preserve the efficiency of existing drugs by containing their use , the proceeds from this fee could be used to finance such targeted subsidies . in this context , it may be noted that the demand - constraining effects of the fee would be less of a problem ( i.e. if the fee does not reduce the use of antibiotics very much , the revenues raised and available for the financing of subsidies to develop new substances would be larger ) . that is , the responsible body must have the competence to evaluate the potential of different research ideas , which is a very demanding task . to preserve the efficiency of existing drugs , the need for guidelines for good antibiotic stewardship has been emphasised . such guidelines have also been developed in several countries , although their implementation is not straight forward ( 2630 ) . they include , inter alia , recommendations to increase hygienic measures to control resistance in hospitals and community homes , to condition all use of antibiotics on prescription , to restrict use to bacterial infections only , and emphasise the importance of choosing the optimal therapy with regard to substance , administration , dosage and duration ( 2735 ) . however , hygienic measures to control resistance in the institutional setting are not without costs to hospitals and nursing homes . restricting use to bacterial infections and choosing the optimal therapy requires access to quick and reliable diagnostic facilities which may be a problem outside the hospital setting . thus , faced with concerned patients presenting symptoms not easily diagnosed , physicians may have incentives to prescribe broad spectrum antibiotics with potential adverse effects on resistance . informing the public on the issues involved might make a decision not to prescribe ( until test - results are available ) more acceptable , but this is by no means certain as the individual patient , while gaining the full benefit if treatment is effective , will not bear the full ( societal ) costs of increased resistance if it is not ( 3638 ) . note that the opposite problem applies to hospitals and other institutions when determining if the implementation of stricter hygienic procedures is worthwhile ( i.e. the institution will bear the full costs of the procedures in question while sharing the benefits with society at large ) . to provide patients and institutions with incentives to consider the full societal costs of antibiotic use , it has been suggested that a fee corresponding to the expected costs of resistance should be levied on the use of antibiotics ( 1 , 38 ) . one problem with this solution is that the fee 's demand - constraining effect may be limited if expenses for antibiotics are covered by insurance- or other third party payer systems . however , that could raise concern regarding the access to antibiotics for less affluent patients ( and countries ) and , therefore , not be acceptable to policy makers . the optimal fee should equal the expected marginal costs of resistance caused by antibiotic use ( 39 ) . if it is set higher , demand will be reduced by too much ( i.e. the health benefits from a marginal increase in use will exceed the expected costs of resistance caused by that marginal increase in use ) and , if it its set lower , demand will be reduced by too little ( health benefits from a marginal increase in use will be smaller than the expected costs of resistance caused by that marginal increase in use ) . as the effects of use on resistance are not fully understood , and may differ between substances one might consider determining the fee on the basis that it , for a given antibiotic , should generate enough returns to finance the development of a successor during the period it takes until resistance becomes a problem for that substance . this requires information on development costs and time until resistance becomes a problem which is likely to depend on the antibiotic in question . while development costs are the property of pharmaceutical firms and , therefore , not easily accessible , some estimates do exist ( 4044 ) . similarly , there are estimates of the time from introduction to detection of resistance for several substances ( 45 ) . to determine the fee per unit of substance required to raise the necessary funds , information on the annual amounts consumed of different substances would also be needed . a fee determined in this way is of course unlikely to be optimal in the sense defined above . however , it is also unlikely to be too high since one result from studies estimating development costs is that these have been rising continuously since the 1970s . accordingly , while it may not be large enough to achieve an optimal reduction in antibiotic consumption from the perspective of preserving efficiency , it may at least go part of the way . on the other hand , if the consumption of antibiotics is reduced , this could increase the problem of achieving the other objective increasing the supply of new antibiotics . the supply of new antibiotics is dependent on research and development ( r&d ) to discover and market them . the outcome of this process depends on how difficult it is to identify substances with the desired characteristics . this , in turn , is a function of our understanding of the biological mechanisms involved , and of the requirements of the approval procedure . the less that is known about the biological mechanisms , the more resources are needed to uncover the issues involved . similarly , the stricter the requirements for approval , the more resources are needed to satisfy them . , our understanding of the biological mechanisms is imperfect ( not least regarding the mechanisms causing resistance ) , suggesting that r&d costs may be substantial . however , the literature does not indicate that they are significantly higher than for other types of pharmaceuticals ( 46 ) . the bulk of the r&d work is carried out by pharmaceutical firms that depend on sales revenues for financing their operation . of course , the larger the costs of r&d are in relation to the ( expected ) revenues , the smaller the incentives for pharmaceutical firms to engage in the process . the revenues are a function of the amounts sold and the price of the drug in question . here , there are indications that revenues from antibiotics may be lower than those from other classes of pharmaceuticals that are prescribed for longer periods ( 18 , 20 , 25 ) . accordingly , it appears that insufficient financial incentives to engage in r&d is an important reason for the paucity of new antibiotic substances . several measures to increase the supply of antibiotics by changing incentives , ranging from changing the requirements for approval of new drugs , over tax - credits for research and public - private partnerships , to prolongation of patent duration , have therefore been discussed and suggested ( 1 , 13 , 18 , 19 , 46 ) . there certainly appears to be good reason to make adjustments in the requirement for approval . for instance , it is not clear why clinical trials for each infection pneumonia , urinary tract infection , skin and soft tissue infection , etc . should be required for approval of the use of a certain drug in their treatment if these infections are caused by the same bacteria . thus , one suggestion is to condition approval on demonstrated efficacy towards specific organisms rather than specific infections ( 13 , 19 ) . this might also increase incentives for pharmaceutical firms to invest in developing drugs with a narrow spectrum relative to broad spectrum drugs , which would reduce resistance caused by the use of antibiotics . the evidence is mixed in that , while they do increase incentives , effects appear to be limited since taxes only constitute a small part of the development costs . public - private partnerships in r&d for antibiotics may be a solution for specific drugs with very limited markets ( xdr / pdr acineobacter and kleibsella ) but could not , and should not , replace the efforts of private firms in r&d to replace antibiotics not facing these market limitations . a patent excludes the holder from competition from generic substances which provides a monopoly position with the potential of gaining larger revenues from higher prices . however , patents are often sought early in the development process and , due to the longevity of this process , they may only last for a short period after the drugs are approved and marketed . increasing the duration of patents for antibiotics might , therefore , provide better incentives for their development . however , the higher prices charged by the monopolist will also affect demand , and it has been shown that this may restrict the use of antibiotics by too much from the societal perspective ( 4750 ) . in addition , the revenues obtained may not necessarily be invested in the development of new antibiotics as they compete for resources with other pharmaceuticals that may be perceived to have better net present earnings ( 18 , 20 , 25 ) . finally , strengthening the monopoly position of patent holders may have detrimental effects on competition which could reduce incentives to develop new substances . an alternative strategy may be subsidies targeted directly at the development of new antibiotics as this would compensate for lower expected sales revenues in comparison to other pharmaceuticals , particularly if measures to constrain the use of antibiotics are successfully applied . these subsidies of course needs finance which may be difficult to secure given tight government budgets . however , as noted above , if a fee is levied on the consumption of antibiotics as a measure to preserve the efficiency of existing drugs by containing their use , the proceeds from this fee could be used to finance such targeted subsidies . in this context , it may be noted that the demand - constraining effects of the fee would be less of a problem ( i.e. if the fee does not reduce the use of antibiotics very much , the revenues raised and available for the financing of subsidies to develop new substances would be larger ) . that is , the responsible body must have the competence to evaluate the potential of different research ideas , which is a very demanding task . the previous review has , while by no means being exhaustive , pointed at some of the problems in addressing the challenge from antibiotic resistance . basically , they arise from the fact that the two objectives preserving the efficiency of existing substances and increasing the supply of new antibiotics are partially opposing . thus , while several measures to achieve either one of them have been proposed in the literature , each with their own pros and cons that need to be considered , the problem remains that the more successful measures are in achieving one of the objectives , the more difficult will it be to achieve the other . notwithstanding , the idea of levying a fee on antibiotics and use the proceeds to finance targeted subsidies may , at least partly , reconcile them . also , measures aimed at facilitating the approval process do not necessarily limit the possibilities of containing the use of antibiotics . this suggests that more resources should be directed to the further development of these two strategies . a further problem is that , ideally , measures to address antibiotic resistance should be applied on a global basis . this , of course , requires international cooperation which has proved to be complicated in other circumstances . given the global nature of the resistance problem , as well as the fact that pharmaceutical firms operate on a global basis , the who seems to be a natural candidate for shouldering the responsibility of developing solutions to the implementation of the measures suggested in the literature . however , national governments may be reluctant to give up legislative power ( which might be called for if principles of good antibiotic stewardship should be equally enforced in all countries ) or the control of funds ( which may be the implication of using the proceeds from a fee on antibiotics strictly for the development of new substances ) . nevertheless , actions taken by national governments may go a long way to reduce the resistance problem , as visualised by the differences between countries in the eu in the magnitude of the problem . lack of incentives and awareness are important reasons for why the problem of antibiotic resistance is difficult to overcome . increasing incentives for supplying new antibiotics requires funding to subsidise r&d costs . to increase awareness , this funding could , at least partially , be provided by a fee levied on the use of antibiotics . as the fee would raise the price of antibiotics , it would also serve as an incentive to reduce antibiotic consumption , thereby contributing to preserving the efficiency of present substances . the author has not received any funding or benefits from industry or elsewhere to conduct this study .

antibiotic resistance has been increasing along with antibiotic use . at the same time , the supply of new drugs to replace those rendered inefficient by the development has been dwindling , leading to concerns that we may soon lack efficient means to treat bacterial infections . though the problem has received considerable interest , there are no indications that the situation is about to change . the present review maintains that this is because the two objectives - preserving the efficiency of existing drugs and increasing the supply of new ones - are partly opposing . hence , creating an incentive structure compatible with both of them is not easy . nevertheless , it is suggested that levying a fee on the use of antibiotics , and earmarking the proceeds from this fee for subsidizing development of new antibiotics , would be an important step towards increasing incentives for a better antibiotic stewardship while preserving incentives to develop new substances .

rheumatoid arthritis ( ra ) is a widespread chronic systemic inflammatory disorder that affects approximately 1% of the worldwide population . the female - to - male ratio of the disease is 3 : 1 , and although it can occur at any age , it is more common between ages 40 and 70 years . in this context , pharmaceutical companies are interested in developing new anti - inflammatory treatments for the disease , including the use of p2x7 receptor antagonists or bisphosphonates [ 2 , 3 ] . this paper will focus on the role of p2x7 receptors in the pathophysiology of ra and the possible therapeutic connection of bisphosphonates with p2x7 receptor signaling . bone is a specialized connective tissue composed of mineralized extracellular matrix and distinct cell populations including osteoblasts , osteocytes , and osteoclasts . under physiological conditions , however , disturbances of this balance can lead to various diseases such as osteoporosis , ra , or periodontitis . the balance is regulated in bone by a complex network of factors , including hormones and mechanical stimulation . the latter , in turn , induces nucleotide release to the extracellular space and purinergic p2-receptor signaling . p2 receptors are expressed in a variety of cell types in the bone and cartilage , including osteoblasts , osteoclasts , chondrocytes , and synoviocytes and are subdivided into two classes : the p2y family of g - protein - coupled receptors and the p2x family of ligand - gated cation channels . provides specific insight into the role of the p2x7 receptor subtype in osteoblasts and osteoclasts . additionally , p2x7 receptor knockout mice exhibit decreased periosteal bone formation , increased trabecular bone resorption , and impaired response to mechanical stimulation , leading to a reduction in total bone content [ 8 , 9 ] . p2x7 receptor activation in osteoblasts enhances differentiation and bone formation , whereas its activation in osteoclasts results in apoptosis . these differences in the function of p2x7 receptor reflect a sophisticated mechanism whereby the skeleton responds to mechanical stimulation by simultaneously increasing bone formation and suppressing its resorption . furthermore , genetic loss - of - function polymorphisms of the human p2x7 receptor are related with increased skeletal fragility , which is consistent with decreased susceptibility of osteoclasts to apoptosis , as well as impaired osteoblast differentiation and bone formation [ 12 , 13 ] . p2x7 receptor modulation could also play an important role in regulating bone - cell response , and atp appears to mediate internalization of p2x7 receptors in osteoclast - like cells . mechanical stimulation of different cell types , including osteoblasts and chondrocytes , induces atp release through hemichannel opening [ 1517 ] . atp released into the extracellular compartment of the bone could activate p2x7 receptors on osteoblasts and osteoclasts and the function of p2x7 receptor in bone is consistent with the altered skeleton phenotype of p2x7 receptor knockout mice described by ke et al . . in this regard , pores formed in response to p2x7 receptor activation induces additional atp release , initiating a positive purinergic feedback loop . although the physiological importance of this phenomenon remains unknown , in vitro mechanical stimulation of osteoblasts leads to cell permeabilization via a mechanism dependent on p2-receptor signaling . purinergic signaling has been implicated in the pathophysiology of various bone and cartilage diseases , including bone loss , ra , osteoarthritis , and bone cancer pain [ 2024 ] . ra is a widespread and complex chronic inflammatory disorder with no current successful treatment . because it is a complex multifactorial disease , its pathophysiology is not fully understood ; however , there is evidence to suggest that t lymphocytes and macrophages play a critical role in the initiation and perpetuation of synovial inflammation . interleukin ( il)-1 and tumor necrosis factor ( tnf)- are macrophage - derived cytokines that play a primary role in the pathogenesis of ra . one effect of these cytokines is to regulate the production of matrix metalloproteinases ( mmps ) , which are directly involved in extracellular matrix degradation during ra . in fact , the serum and synovial concentrations of tnf- and il-1 are high in patients with active ra , and drugs targeting tnf- were the first biologics to be approved and widely used to treat ra . at present , food and drug administration ; all of these agents have been shown to be effective in reducing the clinical signs of inflammation in ra patients . experimental injection of il-1 into the knee joints of rabbits resulted , within hours , in leukocyte accumulation in the synovial fluid as well as substantial proteoglycan loss from joint cartilage [ 28 , 29 ] . unlike tnf- , which is predominantly detected in the early stages of disease , il-1 is a potent stimulator of leukocytic infiltration , synovial hyperplasia , cell activation , cartilage breakdown , and inhibition of cartilage matrix synthesis . the first step regulates the synthesis of il-1 precursor ( pro - il-1 ) by engagement of innate pattern recognition receptors such as toll - like receptors ( tlr ) , activating nuclear factor-b ( nf-b ) , and mitogen - activated protein kinase ( mapk ) transcription pathways . the second stimulation signal leads to the formation of a multiprotein complex called the inflammasome , which culminates in caspase-1 activation and pro - il-1 maturation . the best - characterized inflammasome is formed by the nlrp3 receptor ( nucleotide - binding domain and leucine - rich repeat receptor containing pyrin domain 3 ) , the adaptor protein asc ( apoptotic speck - like protein with a caspase - activating recruiting domain ) , and caspase-1 . numerous stimuli trigger the nlrp3 inflammasome , including pathogen- or danger - associated molecular patterns ( pamps and damps , resp . ) . extracellular atp activating p2x7 receptor is one of the most widely investigated damps that activate the nlrp3 inflammasome and is probably one of the most important pathways for il-1 release in ra . extracellular atp is found at high concentrations in the synovial fluid of patients with ra , and p2x7 receptor deficiency leads to a lower incidence and lower severity of joint inflammation in animal models of arthritis induced by anticollagen antibodies . ra - associated synoviocytes express functional p2x7 receptors , and their activation upregulates the production of proinflammatory cytokine il-6 from these cells . interest in developing new drugs that target the synthesis , processing , and/or release of il-1 has risen in recent years . blocking antibodies for soluble il-1 have been shown to reduce joint destruction in several animal models of ra . in a phase ii clinical trial with ra patients , 24 weeks of treatment with the recombinant il-1-receptor antagonist ( il-1ra or anakinra ) provided significantly greater clinical improvement of ra symptoms than placebo . anakinra treatment is safe and highly effective for patients with systemic - onset juvenile idiopathic arthritis , adult - onset still 's disease , hereditary autoinflammatory syndromes , schnitzler 's syndrome , and gouty attacks . however , this treatment has been associated with liver toxicity , and long - term follow - up analyses are essential to guide appropriate management strategies . selective drug - like p2x7 receptor antagonists have already been tested in clinical trials for ra . nonetheless , after initial positive outcomes in phase ii trials , astrazeneca and pfizer discontinued the development of their compounds following negative phase iib / iii results in subjects receiving methotrexate [ 2 , 41 ] . the poor pharmacokinetics and pharmacodynamics of astrazeneca 's and pfizer 's p2x7 receptor antagonists could partially explain the lack of effectiveness . however , as described above , the fact that ra pathophysiology could be strongly mediated by tnf- might also explain such poor outcomes . recently , a new generation of p2x7 receptor antagonists with better drug - like properties is entering early - phase clinical studies . new evidence suggests that p2x7 receptor activation also triggers inflammasome- and cytokine - independent pathways that play an active role in the pathogenesis of ra . in fact , an effective anti - p2x7 receptor therapy would be more beneficial than anticytokine directed therapy . p2x7 receptor activation has been associated with the release of the tissue - destroying proteases mmps and cathepsins [ 22 , 42 , 43 ] . cathepsin is a family of lysosomal proteases implicated in cartilage joint destruction and found in the synovial fluid of patients with active ra [ 44 , 45 ] . treatment with p2x7 antagonist , but not with other active ra drugs such as methotrexate , abolished cathepsin release from atp - activated macrophages . p2x7 receptor activation has also been associated with the release of prostaglandin e2 ( pge2 ) and other autacoids . in particular , pge2 induces the release of mmps in the joint during ra and is a key mediator of pain sensation . because of their anti - inflammatory properties , bisphosphonates may represent a promising new drug for the treatment of ra . bisphosphonates are metabolically stable analogues of pyrophosphate , where the central oxygen atom is replaced by a carbon atom ( figure 1 ) . the r1 and r2 side chains bonded to the central carbon confer different properties to the bisphosphonate molecule , whereas r1 is usually short and is involved in binding to bone mineral and r2 is responsible for its biological effects [ 47 , 48 ] . depending on the side - chain structure of r2 , bisphosphonates are classified into nitrogen - containing bisphosphonates ( n - bisphosphonates , such as pamidronate , risedronate , ibandronate or zolendronate ) and nonnitrogen - containing bisphosphonates ( non - n - bisphosphonates , such as etidronate or clodronate ) ( figure 1 ) . bisphosphonates are able to bind divalent ions , such as ca , and therefore target exposed bone mineral surfaces in vivo . after bone binding , bisphosphonates are incorporated in bone - resorbing osteoclasts , resulting in osteoclast function inhibition and apoptosis . bisphosphonates that selectively affect osteoclasts have become a major class of antiresorptive drug for the treatment of osteoporosis and paget 's disease [ 50 , 51 ] . bisphosphonates can also be encapsulated in liposomes and then selectively used to target phagocytic cells in vivo . liposome - encapsulated clodronate is widely used to eliminate macrophages in vivo and has been shown to reduce ra - associated inflammation in animal models and in patients . non - n - bisphosphonates can be metabolized to nonhydrolyzable and , -methylene - containing ( appcp - type ) analogues of atp . the in vivo accumulation of appccl2p in osteoclasts induces apoptosis and inhibits bone resorption [ 54 , 55 ] . n - bisphosphonates are also known to affect normal cellular function by inhibiting farnesyl diphosphate synthase and thereby preventing the prenylation of small gtpases [ 47 , 56 ] . reactive oxygen species ( ros ) are known to contribute to the inflammatory process in ra by degrading cartilage and bone . in an inflamed joint , . showed that bisphosphonates inhibit ros production in human neutrophils by acting before or on nadph oxidase . bisphosphonates also have antioxidant properties as iron chelators and block chondrocyte lipid peroxidation , suggesting a protective role for bisphosphonates in ra . our group recently identified clodronate and its physiological analogue pyrophosphate as a new molecule structure able to block il-1 release . this inhibition was also found by using high atp concentrations during macrophage polarization towards anti - inflammatory or alternatively activated m2 states , where p2x7 receptor signaling was uncoupled from inflammasome activation . in proinflammatory m1 macrophages , we previously proposed a model for inflammasome regulation by pyrophosphates and extracellular atp during macrophage polarization gradient towards m2 where p2x7 receptor uncouples from both ros production and the nlrp3-inflammasome / caspase-1 pathway , while p2x7 receptor remains functional in terms of its ion channel activity . under these conditions , pyrophosphates act to inhibit ros and cluster actin dynamics induced by other inflammasome activators ( e.g. , maitotoxin ) , resulting in blockage of caspase-1 and il-1 release our evidence suggested that the diphosphate group resident in nucleotides was blocking inflammasome activation , because pyrophosphates and triphosphates but not monophosphates ( inorganic phosphate ) inhibited caspase-1 activation and il-1 release , with a potency order of clodronate > triphosphate = diphosphate > atp adp . it is still unknown if pyrophosphates act by phosphate chains remaining attached to the nucleotide molecule or if they result from atp metabolism by ectonucleotidases . ectonucleotidases might play a play in this process , because the expression of ectonucleotidases and other genes involved in the extracellular generation of pyrophosphate is highly expressed by m2 compared to m1 macrophages . m2 macrophages accumulate the transcript for ectonucleotide pyrophosphatase / phosphodiesterase 1 ( enpp1 ) , which primarily degrades atp to produce amp and pyrophosphate . m2 macrophages also accumulate the transcript for ank , a plasma - membrane protein involved in progressive ankylosis that mediates cytosolic pyrophosphate release into the extracellular space . pyrophosphates were also able to alter the lps - induced proinflammatory gene expression profile in a similar manner to the action of il-4 ( cytokine responsible for polarizing macrophages to m2 ) . interestingly , pyrophosphates were also able to upregulate enpp1 expression after lps stimulation , suggesting that over the course of the inflammatory process , extracellular pyrophosphate production could promote the initiation of resolution by shifting macrophage polarization to m2 . in m1 macrophages , pyrophosphate and clodronate significantly inhibited gene expression dependent on nf-b activation , such as the lps - induced production of proinflammatory cytokines ( il-1 , il-6 , and tnf- ) and the recovery of ib protein . redox balance has been extensively implicated in nf-b activation , and it has been found that pyrophosphates , so as clodronate , chelating the actions of ros , could specifically affect the nuclear actions of translocated nf-b to dampen proinflammatory gene expression and enhance enpp1 gene expression . additionally , pyrophosphates are able to enhance the resolution of peritonitis in vivo by reducing proinflammatory cytokine production . taken together , these results suggest that extracellular atp and its metabolism to pyrophosphate are key triggers in the switch from a proinflammatory macrophage towards its alternative functions in the resolution of inflammation . pharmacologically , this effect could be mimicked and enhanced by the use of bisphosphonates as anti - inflammatory compounds . several approaches can be taken to reduce synovial inflammation in ra , and many of them use the p2x7 receptor as a central signaling molecule . these approaches include direct p2x7 receptor antagonism and extracellular il-1 blocking ( figure 2 ) . in fact , there is increasing evidence that bisphosphonates may be useful as novel anti - inflammatory drugs in ra . these compounds present various modes of action ; for example , they deplete macrophages when administered encapsulated in liposomes . in contrast , free bisphosphonates present pyrophosphate - like activities that chelate ros and block inflammasome activation during the resolving phase of inflammation , when p2x7 receptor stimulation is uncoupled from proinflammatory signaling or when the p2x7 receptor is pharmacologically inhibited . in the latter case , bisphosphonates produce a switch in p2x7 receptor signaling and extracellular nucleotide metabolism to pyrophosphates during the resolution of inflammation [ 61 , 62 ] . therefore , a combination of p2x7 receptor antagonists with bisphosphonates could be more successful in treating chronic inflammation in ra .

p2x7 receptor - mediated purinergic signaling is a well - known mechanism involved in bone remodeling . the p2x7 receptor has been implicated in the pathophysiology of various bone and cartilage diseases , including rheumatoid arthritis ( ra ) , a widespread and complex chronic inflammatory disorder . the p2x7 receptor induces the release into the synovial fluid of the proinflammatory factors ( e.g. , interleukin-1 , prostaglandins , and proteases ) responsible for the clinical symptoms of ra . thus , the p2x7 receptor is emerging as a novel anti - inflammatory therapeutic target , and various selective p2x7 receptor antagonists are under clinical trials . extracellular atp signaling acting through the p2x7 receptor is a complex and dynamic scenario , which varies over the course of inflammation . this signaling is partially modulated by the activity of ectonucleotidases , which degrade extracellular atp to generate other active molecules such as adenosine or pyrophosphates . recent evidence suggests differential extracellular metabolism of atp during the resolution of inflammation to generate pyrophosphates . extracellular pyrophosphate dampens proinflammatory signaling by promoting alternative macrophage activation . our paper shows that bisphosphonates are metabolically stable pyrophosphate analogues that are able to mimic the anti - inflammatory function of pyrophosphates . bisphosphonates are arising per se as promising anti - inflammatory drugs to treat ra , and this therapy could be improved when administrated in combination with p2x7 receptor antagonists .

in the united states , foodborne pathogens cause 76 million illnesses , 325,000 hospitalizations , and 5,000 deaths annually . some of the main pathogens behind these sicknesses are gram - negative bacteria such as salmonella , escherichia , and campylobacter . infections with gram - negative bacteria may lead to endotoxemia caused by lipopolysaccharides ( lps ) which is a complex of glycolipids made up of two distinct regions . these are hydrophilic polysaccharide region ( composed of o antigen and core oligosaccharide ) and hydrophobic regions known as lipid a . lipid a is responsible for most of the lps induced biological effects . since lipid a is the innermost component of lps , it is closely adherent to the inner cell wall of bacteria and is generally not released ( and therefore not toxic ) until the death of the bacterial cell . lps is heat stable and not strongly immunogenic , so it can not be converted to a toxoid . histone modification is the central point for the control of cardiac growth and gene expression in response to acute and chronic stress stimuli . in the prevention and treatment of cardiac disease , histone modification and the signaling pathways manipulation is a major therapeutic step . histone acetyltransferases ( hats ) mediate acetylation of histone tails , loosening the interaction between dna and histones . acetylation of histones reduces their overall positive charge , thus decreasing their tight interactions with negatively charged dna . histone deacetylases ( hdacs ) can remove the acetyl groups on amino - terminus of histones , restore tight interactions between dna and histones , and deactivate the transcriptional pathway . therefore , hats ( histone acetyltransferases ) and hdacs ( histone deacetylases ) activity determines the transcriptional activation or repression . hat activity in cardiac muscle is determined by p300 that causes modification of chromatin and associated transcription factors and gene expression . studies done by yanazume et al . , 2003 , showed that agonist - induced cardiac hypertrophy accentuates p300 transcriptional activity and that this agonist - mediated cardiac hypertrophy is reversed by the blocking of p300-hat activity . curcumin is an inhibitor of p300-hat but very little is known about whether this regulatory effect is related to a protective role in cardiac dysfunction . curcumin which is derived from turmeric ( curcuma longa plant ) is a tropical plant that is native to southern and southeastern tropical asia . it is a perennial herb within the ginger family and is known for its yellow - orange color and for numerous therapeutic applications . curcumin is a polyphenolic compound and has been used in the treatment of many conditions , including cardiovascular diseases . this study was designed to determine the potential for curcumin to attenuate lps induced cardiac hypertrophy in rodents . curcumin c3 complex ( r ) was a kind gift from sabinsa corporation , hyderabad , india . the patented c3 complex ( r ) contains curcumin and its derivates demethoxycurcumin and bisdemethoxycurcumin , also known as curcuminoids . lps sc-3535 was obtained from santa cruz biotechnology as a white to yellow lyophilized powder . for the live and dead experiment , lps was used as 1.5 mg / mice ( dissolved in distilled water ) , and in curcumin attenuation of lps induced cardiac hypertrophy , lps was used as 60 mg / kg body weight dose ( dissolved in distilled water ) . histone h3 ( n-20 ) , was produced against a peptide mapping at the n - terminus of histone h3 of human origin . histone h4 ( n-18 ) , purified goat polyclonal antibody , was produced against a peptide mapping at the n - terminus of histone h4 of human origin . p300 ( c-20 ) was obtained from santa cruz biotechnology , santa cruz , ca . p300 ( c-20 ) , affinity purified rabbit polyclonal antibody , was raised against a peptide mapping at the c - terminus of p300 of human origin . acetyl - histone h3 ( lys9 ) antibody was obtained from cell signaling technology , danvers , ma . endogenous levels of histone h3 were detected by acetyl - histone h3 ( lys9 ) antibody only when acetylated at lysine 9 . acetyl - histone h4 ( lys12 ) antibody was obtained from cell signaling technology , danvers , ma . endogenous levels of histone h4 were detected by acetyl - histone h4 ( lys12 ) antibody only when acetylated at lys12 . acetyl - cbp ( lys1535)/p300 ( lys1499 ) antibody was obtained from cell signaling technology , danvers , ma . acetyl - cbp ( lys1535)/p300 ( lys1499 ) antibody detects endogenous levels of cbp or p300 only when acetylated at lysine 1535 or lysine 1499 , respectively . the animal care and use committee of tuskegee university approved the experimental protocol ( protocol number r0804 - 12 - 1 ) . eight breeding pairs of mice ( mus musculus ) strain 129s6/sveev , il-10 , were received from the gnotobiotic unit of the mutant mouse resource center , university of north carolina , chapel hill , nc ( iacuc approval number r076 - 10 - 2 ) . the mice were housed under specific pathogen - free ( spf ) conditions in the comparative medicine resource center ( cmrc ) , tuskegee university , and bred via brother : sister mating to produce the mice used in this study . subsequently , 48 of the resulting mice were randomly assigned to one of four treatment groups . the adult 129svev mice ( 68 weeks old ) used in the current study were assigned to 4 groups ( 3 males and 3 females in each group ) : ( a ) control , ( b ) curcumin : 100 g / kg of body weight , ( c ) lps : 60 mg / kg , and ( d ) lps ( 60 mg / kg ) + curcumin ( 100 g / kg of body weight ) . then , following a 24 acclimation period , curcumin and curcumin + lps treated groups received intraperitoneal ( i.p . ) injections of curcumin ( 100 g / kg of body weight ) . one day after the pretreatment with 100 g / kg curcumin , the lps and curcumin + lps groups received lps : 60 mg / kg i.p . all mice in the 12 hrs subgroups were sacrificed with co2 12 hrs after lps injection . all mice in the 24 hrs subgroups were sacrificed with co2 , 24 hrs after lps injection . hearts and lungs of the sacrificed mice were dissected and weighed to compare heart weight / brain weight ( mg / g ) and lung weight / brain weight ( mg / g ) ratios in lps and curcumin treated mice . the collected organs were then preserved in 10% regular formalin in 50 ml tubes . immunohistochemistry is the process of detecting antigens in cells of a tissue section by exploiting the principle of antigen - antibody binding specificity . the sections of murine tissue were placed on the heated water bath and picked up , placed on the slides , and placed in a rack . after all slides had been cut and placed in a rack they were put into a 60 degrees celsius oven over night . the slides were deparaffinized by going through 3 changes of xylene 2 minutes each , followed by 3 changes of 100% alcohol at 1 minute and one change of 95% alcohol at 1 minute . slides were washed in running tap water for 1 minute and stored in distilled water slides . each group was normally distributed , and the variances were equal in the two groups . the test statistic , ts , was calculated using a formula that has the difference between the means in the numerator . the denominator was the standard error of the difference in the means , which got smaller as the sample variances decrease or the sample sizes increase . thus ts got larger as the means got farther apart , the variances got smaller , or the sample sizes increased . mice in the lps treated group showed significant cardiac hypertrophy as measured by heart weight / brain weight ( hw / bw ) ratio . figure 1 illustrates that the lps treated group showed significant cardiomegaly . in 12 hrs subgroup , the p = .04 denotes a significant difference between the control and lps , and in the 24 hrs subgroup , the p = .0026 denotes a significant difference between the control and lps treated groups . mice , in the lps + curcumin treated group , showed attenuation of cardiac hypertrophy as measured by heart weight / brain weight ( hw / bw ) ratio ( figure 1 ) . in 12 hrs subgroup , the p = .005 denotes a significant difference between the lps and lps + curcumin , and in 24 hrs subgroup , the p = .04 denotes a significant difference between the lps and lps + curcumin treated groups . no significant changes were observed in the mice treated with either curcumin or control group ( figure 1 ) . in figure 2 , the heart collected from lps treated mice group showed significant cardiomegaly . dna wound around eight core histone proteins ( two each of h2a , h2b , h3 , and h4 ) form the nucleosome which is the primary building block of chromatin . the amino - terminal tails of core histones pass through various posttranslational modifications , including acetylation , phosphorylation , methylation , and ubiquitination . these modifications happen in response to various stimuli and serve to open the transcriptional machinery , increase the accessibility of chromatin to transcription factors , and ultimately result gene expression . acetylation of h3 has a central role in histone deposition and chromatin assembly in some organisms . hats acetylate histone proteins , relax chromatin , and expose prohypertrophic genes for activation by cardiogenic transcription factors . inhibition of histone acetylation has a central role for the antihypertrophic activity of curcumin . in order to evaluate the mechanism of action of curcumin against lps induced cardiac hypertrophy , we determined the condition of histone h3 acetylation by using acetylation specific antibodies . in this study , curcumin repressed the lps induced acetylation of histone-3 in mice ( figure 4 ) . as a control , the paraffin blocks were exposed to histone h3 antibody , and endogenous levels of histone h3 were detected by h3 antibody ( figure 3 ) . as expected , lps induced a significant increase in histone acetylation that was blocked and sustained by curcumin ( figure 4 ) . histone proteins were detected by histone h3 antibody in control ( figure 3(a ) ) , curcumin ( figure 3(b ) ) , lps ( figure 3(c ) ) , and curcumin + lps ( figure 3(d ) ) groups . but the cardiac cells in the lps treated group showed more staining in the cytoplasm indicating hypertrophy . figure 4 illustrates the degree of histone acetylation in cardiac tissue occurring in each treatment group . as can be seen in figure 4(c ) , lps induced a significant increase in histone acetylation as indicated by the intense staining . the histone proteins were mainly acetylated in the n - terminal end of the histone protein on lysine residues . in the curcumin + lps treatment group ( figure 4(d ) ) , there was a much lesser degree of nuclear staining with staining being similar to the control group , indicating that curcumin inhibited the histone acetylation activity of lps . these findings indicate that acetylation of histone tails by histone specific antibodies , mediated by histone acetyltransferases , is involved in activation of gene expression in the myocardium . conversely , repression of gene expression can be induced by histone deacetylation , which is mediated by histone deacetylases ( hdacs ) . modulation of chromatin structure has a central role in the regulation of transcription in eukaryotes . in eukaryotes , histone h4 acetylation at the position of lysine 16 ( h4-k16ac ) is a reversible posttranslational chromatin modification . the incorporation of this modified histone into the nucleosome inhibits the formation of compact 30 fibers and inhibits the ability of chromatin to form cross - fiber interactions . acetylation of histone tails , mediated by histone acetyltransferases , is involved in activation of gene expression in the myocardium . conversely , repression of gene expression can be induced by histone deacetylation , which is mediated by histone deacetylases ( hdacs ) . histone h4 acetylation at the position of lysine 16 ( h4-k16ac ) is a posttranslational chromatin modification . in addition to histone h3 we also measured histone h4 acetylation by measuring the [ h ] acetate incorporation into histones using h4 acetylation specific antibody . figure 5 illustrates the level of histone proteins as detected by histone h4 antibody in cardiac tissue from control ( figure 5(a ) ) , curcumin ( figure 5(b ) ) , lps ( figure 5(c ) ) , and curcumin + lps ( figure 5(d ) ) treatment groups . as can be seen in the figure , there was little variation in histone h4 basal expression levels among any of the treatment groups . as can be seen in figure 6(c ) , the lps treated group showed a significant increase in histone [ h ] acetylation . control and curcumin alone treatment groups , figures 6(a ) and 6(b ) , respectively , showed little difference in the staining of the acetylated histone h4 . similar to histone h3 acetylation , lps induced h4 acetylation was also blocked by curcumin treatment ( figure 6(d ) ) . this data strongly indicates that curcumin inhibits the acetylation of histones induced by lps . hats ( histone acetyl - transferase ) acetylate histone proteins , relax chromatin , and expose genes for activation by cardiogenic transcription factors . two highly related and widely expressed molecules , camp regulated enhancer binding protein ( cbp ) and p300 , have emerged as important cofactors for a broad number of transcription factors . cbp was originally discovered based on its ability and to interact with the camp response element binding protein , whereas p300 was isolated as a cellular target of the adenoviral oncoprotein e1a . sequence analysis of cbp and p300 has shown substantial homology that these proteins are transcriptional coactivators . p300 , high molecular weight transcriptional factor , has three cysteine and histidine - rich regions . p300 knockout mice showed the defects of cardiac muscle differentiation and trabeculation , indicating the importance of p300 for early cardiac morphogenesis and heart development . curcumin ( diferuloylmethane ) , a major curcumanoid in the spice turmeric , is a specific inhibitor of the p300/creb - binding protein ( cbp ) . to further check the mechanism of action of curcumin , we analyzed p300 protein acetylation levels . curcumin inhibited the lipopolysaccharide - induced acetylation resulting in an increase in the binding of p300 to the gene promoter ( figure 7(c ) ) . as was seen with histone acetylation , p300 acetylation was also inhibited by curcumin ( figure 7(d ) ) . the results of the present study indicate that inhibition of histone acetylation is a key mechanism for the anti - cardiac hypertrophy activity of curcumin and that p300-hat serves as its molecular target . p300 may maintain basal function in the normal heart but it also promotes cardiac hypertrophy under lps infusion . we can conclude from our study that curcumin attenuated lps induced cardiac hypertrophy in vivo . curcumin plays its anti - cardiac hypertrophy role by the involvement of chromatin remodeling , especially histone acetylation . acetylation of lysine residues upon a single histone tail ( hyperacetylation ) would produce a stronger effect . histone acetyltransferases have a critical role to acetylate histone proteins , relax chromatin , and expose cardiac hypertrophy genes for activation by cardiogenic transcription factors . hat activity in muscle is determined by p300 that causes modification of chromatin and associated transcription factors and gene expression . lps induced cardiac hypertrophy accentuates p300 transcriptional activity , and lps mediated cardiac hypertrophy is reversed by blocking of p300-hat activity . therefore , blocking p300-hat activity may be a viable method for the treatment or prevention of myocardial hypertrophy . the results showed that curcumin attenuated lps induced cardiac hypertrophy in rodents and that the most probable mechanism of action involves inhibiting the p300-hat activity .

to study the ameliorating effects of curcumin in lipopolysaccharide ( lps ) induced cardiac hypertrophy , mice were assigned to 4 groups ( 3 males and 3 females in each group ) : ( a ) control , ( b ) curcumin : 100 g / kg of body weight by intraperitoneal route ( ip ) , ( c ) lps : 60 mg / kg ( ip ) , and ( d ) lps + curcumin : both at previously stated concentrations by ip route . all mice were sacrificed as 12 hr and 24 hrs groups accordingly after lps injection . the hearts were collected , photographed for cardiomegaly , and weighed to compare heart weight / brain weight ( hw / bw ) in mg / mg . for immunohistochemistry , the tissue sections were exposed to histone h3 , h4 and acetylated histone h3 , h4 antibody . lps induced a significant increase in histone acetylation as shown by intense staining . in curcumin + lps treated mice nuclear staining was similar to the control group indicating that curcumin traversed the histone acetylation activity of the lps . to further check the mechanism of action of curcumin , p300 protein acetylation levels were analyzed . this study suggests that the probable mechanism of action of curcumin is via the reduction of p300 hat activity .

first performed in 1989 , laparoscopic cholecystectomy quickly became the gold standard for surgical extirpation of the gallbladder . soon after , reports began to emerge of an increased incidence of significant bile duct injuries . reported injury rates varied from 0.1% to 2.2% compared with 0.1% for open cholecystectomy . the increased rate of bile duct injury has been attributed to a steep learning curve for laparoscopic cholecystectomy . subsequent referral to tertiary centers for repair of transected common bile ducts resulted in published series that outlined the mechanisms of injury . risk factors for iatrogenic injury to the common duct appear to be the surgeon 's inexperience , patient obesity , scarring and acute inflammation . a common factor in the reported cases of bile duct injury is the misidentification of common duct for cystic duct , resulting in resection of a portion of the common bile and hepatic ducts and a right hepatic artery injury . precautions suggested to decrease the possibility of bile duct injury have included : routine cystic duct cholangiography , cholecystocholangiography , confirmed identification of the junction of the cystic duct with the common bile and hepatic ducts and identification of the junction of the cystic artery with the right hepatic artery . our experience with laparoscopic cholecystectomy supports the importance of anatomic identification in the safe performance of the procedure and also suggests the presence of a useful and reproducible anatomic landmark which can help define a zone of safe dissection within the triangle of calot . this study is derived from a single surgeon 's experience with more than 300 laparoscopic cholecystectomies performed since 1991 . all patients with acute or chronic gallbladder disease were considered for the laparoscopic approach with the exception of those with suspected gallbladder masses or neoplasm . the anatomy of the biliary tree was defined prior to division of the cystic duct and artery . cholangiograms were abandoned only if the lumen of the cystic duct would not accommodate the epidural catheter used for this purpose . during 1996 , patients were chosen in one of three representative categories as they randomly presented in the course of our practice . these were chronic disease , acutely inflamed gallbladder and intra - operatively identified aberrant anatomy . i.m . , a 67 year - old - female with biliary colic systems and ultrasound proven cholecystolithiasis , underwent outpatient laparoscopic cholecystectomy . two 1 mm veins were demonstrated within the triangle of calot between the cystic duct and cystic artery ( figure 1 ) . case i : acute chdeaystititis and multiple cystic veins m.b . , a 56-year - old female with biliary colic symptoms and cholecystolithiasis on ultrasound , underwent outpatient laparoscopic cholecystectomy . a small venous structure was found to be crossing it and extending to the gallbladder ( figure 2 ) . further dissection showed the artery to be an aberrant right hepatic artery with a short cystic artery identified later . , a 49-year - old female , was admitted with acute abdominal pain , fever , leukocytosis and thickened gallbladder containing stones on ultrasound . dissection within the triangle of calot demonstrated venous structures between the later confirmed cystic duct and artery ( figure 3 ) . early in our experience with laparoscopic cholecystectomy , the presence of small tubular structures within the triangle of calot became evident as being necessarily divided to proceed with the dissection . among these was a structure that presented as a bridge between the cystic artery and duct ; the division of which , unless controlled , resulted in insignificant but annoying bleeding . it quickly became a standard part of the procedure to clip or cauterize this ves sel during the dissection . its color and non - pulsatile bleeding when divided , indicated that this was a vein , ostensibly unnamed . it was subsequently determined that these venous channels were named as the cystic veins and their courses documented in early textbooks of anatomy . i.m . , a 67 year - old - female with biliary colic systems and ultrasound proven cholecystolithiasis , underwent outpatient laparoscopic cholecystectomy . two 1 mm veins were demonstrated within the triangle of calot between the cystic duct and cystic artery ( figure 1 ) . m.b . , a 56-year - old female with biliary colic symptoms and cholecystolithiasis on ultrasound , underwent outpatient laparoscopic cholecystectomy . a small venous structure was found to be crossing it and extending to the gallbladder ( figure 2 ) . further dissection showed the artery to be an aberrant right hepatic artery with a short cystic artery identified later . n.m . , a 49-year - old female , was admitted with acute abdominal pain , fever , leukocytosis and thickened gallbladder containing stones on ultrasound . dissection within the triangle of calot demonstrated venous structures between the later confirmed cystic duct and artery ( figure 3 ) . early in our experience with laparoscopic cholecystectomy , the presence of small tubular structures within the triangle of calot became evident as being necessarily divided to proceed with the dissection . among these was a structure that presented as a bridge between the cystic artery and duct ; the division of which , unless controlled , resulted in insignificant but annoying bleeding . it quickly became a standard part of the procedure to clip or cauterize this ves sel during the dissection . its color and non - pulsatile bleeding when divided , indicated that this was a vein , ostensibly unnamed . it was subsequently determined that these venous channels were named as the cystic veins and their courses documented in early textbooks of anatomy . a consistent anatomic feature during laparoscopic cholecystectomy has been the presence of venous twigs that run parallel to the cystic duct and perpendicular to the common bile duct , bridging the gap between cystic artery and cystic duct . as the veins do not cross the common hepatic duct , their presence can serve as an anatomic feature of a safe dissection space between the important structures of the triangle of calot . during surgical residency a common question intended to " trip up " junior residents during division of the cystic artery , has been to ask them to define the cystic vein . it is usually pointed out to the less assured that there is no cystic vein per se , but that the venous drainage is through the bed of the liver . in contradistinction to this , however , grant 's anatomy , in illustrating the veins of the extrahepatic bile passages and gallbladder ( figure 4 ) , clearly demonstrates venous drainage directly into the liver , and describes venous twigs which clinging to the passages .... join branches of the portal vein . gray 's anatomy of the human body likewise describes cystic veins which join at the neck of the gallbladder to form either single or double cystic veins which flow along the cystic duct and upward along the hepatic ducts . textbooks of surgery , however , do not assign much importance to the venous drainage of the gallbladder . the textbook of surgery edited by sabiston reports only that the venous drainage of the gallbladder and extrahepatic ducts is into the portal vein . schwartz 's principles of surgery describes the venous drainage as variable and generally does not run parallel with the arteries . a prominent surgical atlas similarly omits all veins from it 's anatomic diagrams and does not describe operative management of these structures during the procedure . one of the differences between laparoscopic and open surgery is that magnification of the anatomy gives a new perspective to the dissection process . the magnified field of view demands meticulous attention to operative detail lest bleeding from a source not significant during open cholecystectomy , completely obscures the field during laparoscopic cholecystectomy . this fractal * anatomic view uncovered the presence of venous structures within the triangle of calot , which , if not clipped or cauterized prior to division , would obscure the operative field . only with experience did we find the presence of the cystic veins to be constant . during difficult cases , the cystic veins can serve as a landmark for that space between cystic duct , cystic artery and common bile duct , that allows for safe handling and isolation of these vital structures . a review of anatomy texts has shown that the cystic veins were known to classical anatomists . it was only later that , without apparent clinical relevance , a description of the cystic veins was omitted from surgical textbooks and their very existence devolved into a trick question for unsuspecting house staff . i do not doubt that experienced laparoscopic surgeons have encountered these venous structures and are dealing with them on a regular basis , but that they exist as a reliable anatomic feature of the region is not well appreciated . its importance , however , may lie in the fact that the cystic vein can serve as a landmark during a difficult laparoscopic cholecystectomy to decrease the risk of inadvertent bile duct injury .

the cystic vein , a portion of biliary anatomy whose insignificance in open gallbladder surgery led to its being relegated to mythology , has been rediscovered by the magnified view of laparoscopic surgery . its presence is an important anatomic feature that helps distinguish between cystic duct and common hepatic duct , thus diminishing the risk of inadvertent bile duct injury during laparoscopic cholecystectomy .

the rate of admission to the icu and intensive care mortality in patients with hiv infection have shifted repeatedly during the aids epidemic , influenced by attitudes of patients and providers toward utility of care . respiratory failure remains the most common diagnosis for icu admission in patients with hiv infection , and , despite advances in medical therapy and intensive care medicine , the mortality of these patients remains substantial [ 1 , 2 ] . however , the incidence of aids - associated illnesses has significantly decreased , and the overall life expectancy of hiv - infected patients markedly increased after the introduction of haart [ 3 , 4 ] . nevertheless , infectious and noninfectious complications that may require critical care support continue to occur in this patient population . in the past decade , the number of patients with aids - defining conditions admitted to the icu decreased dramatically . this decreasing pattern has been offset by a parallel increase in the number of patients with hiv infection admitted for medical problems nonrelated to hiv infection . several studies have suggested that the introduction of haart not only improved the survival of hiv - infected patients admitted to icu but also changed the etiology of admissions of these patients to the critical care units [ 57 ] . unfortunately , only 25%50% of hiv - infected patients receive haart at the time of admission to the icu [ 5 , 6 ] . retrospective cohort studies have shown that prognostic factors of mortality in hiv - infected patients admitted to icu are diverse , including severity of acute illness , poor functional status , and respiratory failure requiring mechanical ventilation [ 8 , 9 ] . in this study , the authors evaluated the etiology of respiratory failure among hiv - infected patients requiring icu admission , its relationship with their cd4 lymphocyte count as well as the use of haart , and its impact on outcome . it was conducted in a general , community innercity hospital located in brooklyn , new york ( woodhull medical and mental health center ) , which serves a multiethnic hiv population . the hospital has a total of 346 beds distributed among general internal medicine , general surgery , psychiatry , obstetrics / gynecology , and pediatrics wards . the icu at our institution works under a closed system with the supervision of 5 board - certified intensivists and is composed of 12 beds , with an annual admission average of 700 patients . all patients , 18 years or older , with known hiv infection and respiratory failure admitted to the icu , either from the emergency department or transfered from the medical or surgical wards , were enrolled in the study . we excluded patients with hiv infection admitted to the icu for reasons other than respiratory failure . for the purpose of this study , only data from the first admission was analyzed in patients readmitted to icu within 30 days . variables such as age , gender , comorbid conditions , acute physiology and chronic health evaluation- ( apache- ) ii score at the time of icu admission , t - lymphocytes count ( cd4 ) , hiv viral load by pcr ( test was performed using the cobas ampliprep / cobas taqman , hiv-1 test kit version 2.0 , roche molecular systems , inc . ) , use of haart at the time of admission , needs for mechanical ventilation or noninvasive ventilation and/or vasopressors , and outcome at hospital and icu discharge were measured . computerized medical records were reviewed and clinical information was abstracted for each patient . after waiving the need for informed consent , the institutional review board approved the study . a total of 42 patients with hiv infection and respiratory failure were admitted to icu during the 15-month study period . on admission to icu , nineteen patients ( 45.2% ) were not on antiretroviral therapy , either because of poor adherence or lack of access to medical care . their median cd4 cell count was 123.0 cells/l ( mean 205.7 , range 2.0694.0 ) , with a median hiv viral load of 203.5 copies / ml ( mean 58,676 , range < 20367,649 ) . among patients receiving haart , the median cd4 cell count was 187.9 cells/l ( mean 218.5 , range 2.0694.0 ) , with a correspondent median hiv viral load of 51 copies / ml ( mean 27,356 , and range < 20235,769 ) . patients not receiving haart had lower cd4 cell count ( median 51.0 cells/l , mean 190.2 , and range 3.0689.6 ) and higher viral load ( median 55,851 copies / ml , mean 96,594 , range < 20367,649 ) . the median apache - ii score was 15 ( mean 15.9 , range 527 ) . patients with noninfectious etiology of respiratory failure had higher apache - ii score ( median 17 , range 827 versus median 13 , range 523 ) . infectious etiology of respiratory failure was identified in 17 patients ( 40.5% ) , while a noninfectious reason was accountable in 25 patients ( 59.5% ) . both patients on haart and those not receiving antiretroviral therapy were most likely to have a noninfectious etiology for the respiratory failure ( 14 , 60.9% and 11 , 57.9% , resp . ) . infectious etiology of respiratory failure in patients with hiv infection is as follows : bacterial pneumonia ( 6)pneumocystis jirovecii pneumonia ( 4)intra - abdominal sepsis ( 3 ) : abscess ( 1 ) , biliary tract sepsis ( 1 ) , vancomycin - resistant enterococcus ( vre ) bacteremia ( 1)lung abscess ( 1)cns infection ( 2 ) : bacterial meningitis ( 1 ) , cryptococcus neoformans meningitis ( 1)nasopharyngeal abscess ( 1 ) bacterial pneumonia ( 6 ) pneumocystis jirovecii pneumonia ( 4 ) intra - abdominal sepsis ( 3 ) : abscess ( 1 ) , biliary tract sepsis ( 1 ) , vancomycin - resistant enterococcus ( vre ) bacteremia ( 1 ) cns infection ( 2 ) : bacterial meningitis ( 1 ) , cryptococcus neoformans meningitis ( 1 ) nasopharyngeal abscess ( 1 ) noninfectious etiology of respiratory failure in patients with hiv infection is as follows : decompensated congestive heart failure ( chf ) ( 8),drug overdose ( 4),cardiac arrest ( 4),electrolyte imbalance ( 2),hemorrhagic shock ( 2),chronic obstructive pulmonary disease ( copd ) exacerbation ( 1),status epilepticus ( 1),upper airways obstruction ( angioedema , 2),thrombotic thrombocytopenic purpura ( ttp ) ( 1).thirty - six patients ( 85.1% ) required mechanical ventilation , while 2 patients ( 4.8% ) needed noninvasive ventilation . four patients ( 9.5% ) achieved acceptable oxygen saturation by receiving oxygen via nasal cannula . the total number of ventilator days required was 297 ( median 5.5 , mean 8.3 , and range 138 ) . the group of patients with infectious etiology of respiratory failure required mechanical ventilation for longer period ( median 7 , range 138 days versus median 4 , range 117 days ) . both patients on haart and those not on antiretroviral therapy had comparable length of stay in icu ( median 5 , mean 7.8 , and range 131 days versus median 4 , mean 6.8 , and range 140 days , resp . ) . a total of 18 patients ( 42.9% ) required vasoactive therapy , either in the form of vasopressors or inotropes . there was no significant difference in the apache - ii scores of survivors and nonsurvivors ( median 15 , range 827 versus median 15 , range 526 , resp . ) . the median cd4 cell count of the patients who did not survived was 27.0 cells/l ( mean 110.3 , range 3.7622.0 ) , and their median viral load was 476 copies / ml ( mean 79,015 , range < 20367,649 ) . majority were males ( 11 , 84.6% ) , and their median age was 50 years ( range 2069 ) . mortality rate was higher in patients with infectious etiology of respiratory failure ( 8 , 61.5% ) . the total number of icu days utilized by all patients was 299 ( median 4.5 , mean 6.8 , and range 140 ) . decompensated congestive heart failure ( chf ) ( 8) , electrolyte imbalance ( 2 ) , hemorrhagic shock ( 2 ) , chronic obstructive pulmonary disease ( copd ) exacerbation ( 1 ) , status epilepticus ( 1 ) , upper airways obstruction ( angioedema , 2 ) , thrombotic thrombocytopenic purpura ( ttp ) ( 1 ) . this study examined the characteristics of hiv - infected patients with respiratory failure admitted to icu . it provides important insights into the critically ill patient with hiv infection , through identification of variables influencing the outcome of these patients . to our knowledge , this is the first study to analyze the etiology of respiratory failure in hiv - infected patients requiring icu admission and their outcome in the new york city public hospitals system ( new york city health and hospitals corporation ) . since the onset of the hiv epidemic , there has been a significant shift away from aids - defining diagnoses as indications for icu admission . in this study , non - hiv - related illnesses accounted for majority of admissions , which goes in accordance to results from other studies [ 6 , 9 , 10 ] . the overall hospital survival rate in this report is comparable to data previously published [ 1012 ] . although the reported overall hospital mortality rate in this study was lower than in other series [ 6 , 13 , 14 ] , our icu survival rate was remarkably lower when compared with similar studies [ 5 , 9 , 11 , 1315 ] . majority of our patients were receiving antiretroviral therapy at the time of death . on the other hand , none of patients that were not receiving haart were initiated on antiretroviral therapy in icu . while the long - term benefits of haart on survival among hiv - infected outpatients are indisputable , there may be no short - term benefits in icu patients with critical illnesses . data on the impact of haart on the incidence of critical illness among hiv - infected patients are limited . haart leads to decreased opportunistic infections that collectively could contribute to lower risk of icu admission . because hiv - infected patients are living longer , however , they are at increasing risk of developing comorbid illnesses not previously thought to be hiv related , and these non - aids - related conditions account proportionally for the majority of deaths [ 15 , 16 ] . our results failed to demonstrate the potential protective and beneficial effect of haart in both icu admission and mortality , contradicting results from previous reports [ 5 , 1720 ] . it is well known that the use of haart is associated with a dramatic reduction in hiv viral load and with a concomitant increase in cd4 cell counts . management of antiretroviral therapy is a challenge in icu , and introducing haart in a critically ill patient is a difficult decision . concerns about drug - to - drug interactions and toxicities , medication absorption , and variability of drug levels are common challenges among intensivists . since the authors of this study did not collect information on adherence to haart prior to icu admission and our observational study did not measure haart resistance , the impact of nonadherence or drug resistance as explanations can not be excluded . therefore , our study probably does not reflect the effect that haart has on the survival of patients with hiv infection admitted to the icu . in addition , this study does not address the real value of intensive care for critically ill patients with hiv infection , because it is possible that a significant selection bias existed during the triaging process . although haart use may not be associated with short - term icu survival , long - term survival may be improved in the group of icu survivors who received antiretroviral therapy [ 5 , 2022 ] . in one study , morquin et al . showed that introducing haart on admission to icu seems to be protective in both short- and long - term outcomes . nevertheless , the overall mortality rate in this study falls into the range observed in other series [ 2 , 6 , 11 , 23 ] . as previously described [ 9 , 11 , 20 , 2426 ] , the authors of this study identified independent predictors of hospital mortality : low cd4 cell count , infectious etiology of respiratory failure or sepsis , and needs for mechanical ventilation and/or vasopressors . although comparable to results from other similar studies [ 12 , 14 , 27 ] , the median cd4 cell count in our patients was higher than those reported in other series [ 9 , 10 , 13 , 28 ] . a large proportion of our hiv - infected population is known for poor adherence to antiretrovirals and ongoing substance abuse . the substandard treatment responses to haart in our patients may be due to the presence of transmitted drug resistance of the hiv strains , poor adherence to haart that may result in emergence of antiretroviral drug resistance mutations , and suboptimal or nonimmunological response . the median apache - ii score in our patients was lower than reported in previous studies [ 10 , 14 , 18 , 21 ] , and we found no significant difference in the apache - ii score of survivors and nonsurvivors . this result contradicted data from other studies , where elevated apache - ii scores were prognostic of adverse outcomes in these patients [ 5 , 9 , 10 , 12 ] . interesting is the high number of patients who received mechanical ventilation in this study , outweighing results from other reports [ 911 , 13 , 14 , 20 , 23 , 28 ] . however , our patients ' length of stay in icu is comparable to figures shown previously [ 11 , 14 , 18 , 27 ] . we hypothesized that our lengthened icu stay was the result of some patients who developed complications such as acute respiratory distress syndrome and hemodynamic instability during the course of their illnesses and , therefore , weaning from mechanical ventilation and/or tracheostomy procedures were delayed . it may also explain the large number of tracheostomy procedures performed in our patients , when compared to data published by coquet et al . . in addition , and similar to one study , near half of our patients received vasopressors during their icu stay , which contributed as well to adverse outcomes . these results may be explained by factors related to our patient population and their availability to access medical care . probably , our patients did not seek medical care at the beginning of their illnesses , which likely is related to suboptimal outcomes . the main strength of this study is the prospective nature of its design , with evaluation of all consecutive admissions to icu of patients with hiv infection and respiratory failure . second , the case number is relatively small and it was conducted in a single center . therefore , the results may not be generalizable and applicable to other institutions and hiv populations since clinical practice and demographics may differ across institutions . therefore , the predictors of long - term survival and any impact of antiretrovirals on long - term survival remain unknown . the icu utilization for hiv - infected patients in our institution has not been changed by the advent of haart , and the impact of antiretroviral therapy on the indications and outcome of icu admission remains controversial . our data suggested that the short - term survival of hiv - infected patients admitted to the icu is not dependent on the use of haart and that outcomes are mainly related to the degree of immunosuppression . larger prospective studies are needed to examine the impact of haart and other parameters on the outcomes of patients with hiv infection and respiratory failure admitted to the icu .

background . although access to haart has prolonged survival and improved quality of life , hiv - infected patients with severe immunosuppression or comorbidities may develop complications that require critical care support . our objective is to evaluate the etiology of respiratory failure in patients with hiv infection admitted to the icu , its relationship with the t - lymphocytes cell count as well as the use of haart , and its impact on outcome . methods . a single - center , prospective , and observational study among all patients with hiv - infection and respiratory failure admitted to the icu from december 1 , 2011 , to february 28 , 2013 , was conducted . results . a total of 42 patients were admitted during the study period . their median cd4 cell count was 123 cells/l ( mean 205.7 , range 2.0694.0 ) , with a median hiv viral load of 203.5 copies / ml ( mean 58,676 , range < 20367,649 ) . at the time of admission , 23 patients ( 54.8% ) were receiving haart . use of antiretroviral therapy at icu admission was not associated with survival , but it was associated with higher cd4 cell counts and lower hiv viral loads . twenty - five patients ( 59.5% ) had respiratory failure secondary to non - hiv - related diseases . mechanical ventilation was required in 36 patients ( 85.1% ) . thirteen patients ( 31.0% ) died . conclusions . noninfectious etiologies of respiratory failure account for majority of hiv - infected patients admitted to icu . increased mortality was observed among patients with sepsis as etiology of respiratory failure ( hiv related and non - aids related ) , in those receiving mechanical ventilation , and in patients with decreased cd4 cell count . survival was not associated with the use of haart . complementary studies are warranted to address the impact of haart on outcomes of hiv - infected patients with respiratory failure admitted to icu .

lead ( pb ) exposures have decreased with the removal of pb from gasoline . however , pb exposure and toxicity remains an important public health issue . certain populations in the usa as well as in many developing countries still experience high exposures . an inverse association between blood pb level and cognitive abilities is observed at very low blood pb concentrations , and the pb associated intellectual decrement was steeper at low blood pb levels than at higher blood pb levels [ 13 ] . in adults , it has been shown that even low pb exposures are associated with significant health effects among nonoccupationally exposed populations [ 49 ] . traditionally , blood pb is used as a biomarker to determine pb exposures , but blood pb has a half - life of 30 days and therefore correlates less well with long - term exposure than does bone pb , for which the half - life is several years to decades [ 10 , 11 ] . cd-109 induced k x - ray fluorescence ( kxrf ) technology has been used to measure pb in bone for over two decades and has made significant contributions to the study of associations between long - term cumulative pb exposure and adverse health outcomes [ 4 , 5 , 7 , 1214 ] . however , the system requirement of a radioactive source , long acquisition times , and a sizeable space for the equipment limits this research to very few groups who possess this technology . in a previous study , we demonstrated the validity of a portable xrf system that made use of pb l x - rays to quantify pb in bone . improvements to this portable system 's geometry and detector have been made , which decrease the minimum detection limit and make the device more compatible for use in vivo . the new system was tested with phantoms to determine the minimum detection limit of the device . tests with phantom , goat bone , and cadaver bone samples were used to determine the accuracy of the device in determining bone pb concentrations . pb l x - rays , which have relatively low energies , have greater soft tissue attenuation for the signals and hence the correction for this is a significant issue . to this end , new calibration methods are being explored in this study to establish a more accurate approach to quantify the pb in bone in vivo . kxrf technology is used in this study to validate the results found with the portable xrf device . the setup of the device is the same as that used in previous studies [ 16 , 17 ] . the system uses four 16 mm diameter high - purified germanium ( hpge ) detectors with 10 mm thickness , four feedback resistance preamplifiers , four digital signal processing systems , and a computer . a 135 mci cd source is used to irradiate tibia bone or bone equivalent samples to produce the pb k x - rays . the bone pb measurements were taken for 30 minutes with the hpge detector and then processed with digital electronics . the spectra were analyzed using an in - house peak fitting program and the final pb concentrations were calculated [ 1719 ] . the whole body effective dose from this system was measured to be 0.26 sv for adults . two customized portable xrf devices were used in this project ( xl3 t and xl3 t goldd+ , thermo fisher scientific inc . , billerica , ma ) . the xl3 t device used in our previous study is used in this study for a comparison to determine how the improvements in the device technology impact the measurements . previously the device was equipped with a thermoelectric cooled si pin diode with 8 mm area and 1 mm thickness . the device also has a tube voltage of up to 50 kv , a current of up to 40 a , and various filter combinations . the new device ( xl3t - goldd+ ) has a more compact and optimized geometry . it uses a thermoelectric cooled silicon drift detector with a 25 mm area and 1 mm thickness . the devices were customized so that the voltage of the x - ray tube , the current of the tube , and the filter combinations could be selected to allow the best performance for in vivo measurement of pb in bone . in our experiment , we used a measurement time of 3 minutes . based on our previous study , by adjusting values for increased measurement time and tube current , we estimated the entrance skin dose of the system was 31 msv to a 1 cm area and the whole body effective dose was 3.6 sv . this can be compared to the whole body effective dose for a standard ap chest x - ray of about 100 sv . soft tissue and bone equivalent phantoms were used in this study to determine the sensitivity of the device and to calibrate the system . lucite plate phantoms were used to simulate soft tissue over bone by placing the lucite over the flat surface of the bone phantoms in increments of 1 mm up to 5 mm of lucite thickness . cylindrical pb doped phantoms made of plaster - of - paris were used to simulate bone with pb concentrations ranging from 0 to 100 ppm ( 0 , 5 , 10 , 15 , 20 , 30 , 50 , 75 , and 100 ppm ) . these measurements were made from the flat base of the phantom . in our new calibration method ( i.e. , background subtraction ) , the compton scattering peak was used to determine the background under the pb l x - ray peak and the attenuation of the pb signal . hence , mc simulations were performed to test the differences between plaster - of - paris and bone and lucite and soft tissue in terms of pb over compton signal . no significant differences in xrf spectra were found between plaster - of - paris with lucite and bone with soft tissue . thus , the phantom measurements were used to accurately calibrate the system , correlate the compton peak counts with soft tissue thickness , and calculate the detection limit of the system . four goat bone samples and ten human cadaver tibia bone samples were measured with the device as well . the bones were all vacuum - sealed in plastic bags and labeled for ease of measurements . for goat bone , measurements were made at 0 , 1 , 2 , 3 , 4 , and 5 mm of lucite and for bare cadaver bone , measurements were made using 0 , 1 , 2 , and 3 mm of lucite for comparison between portable xrf devices and kxrf . the cadaver bones measurements did not include 4 and 5 mm of lucite due to the difficulty of adjusting the geometry in these situations . three cadaver bone samples had intact soft tissue over them and were measured through the soft tissue . the cadaver and goat bone samples were taken to give a more realistic sense of the device capabilities for in vivo use by attempting to replicate the difficulties from increased attenuation with lxrf energies and soft tissue thickness . the spectrum was analyzed using a background subtraction method described in detail in our previous study . in summary , the method is focused on deriving two functions that will enable us to estimate the pb concentration . first , we define the relation of the background in the pb peak areas to compton peak area counts for 0 to 5 mm of lucite . second , we define the relation between pb l - x ray signal and compton peak counts for 0 to 5 mm of lucite . the background at 0 ppm will relate to scatter events , which will be the main contribution to background in the spectrum . the compton scattering peak will give a correlation to the amount of scatter events in the spectrum and the background throughout the spectrum . thus , defining a function that will relate the compton peak counts and the 0 ppm background can be feasibly used to determine the background under the pb l - x ray peaks . the net signal will decrease with increasing lucite thickness because of an increase in attenuation of the signal as well as distance from the bone . the compton peak has been shown to accurately correlate with lucite thickness through the increase in scatter events created by additional lucite . since the attenuation and distance will increase directly with lucite thickness , we can correct each pb peak by relating it with compton peak counts . this function can then be used to accurately determine the signal attenuation that occurs in each spectrum . then , one can determine from the spectrum the net counts coming from pb in the sample and relate that to a known signal concentration value to compute the final sample concentration . in our study , as a modification to this method for better applicability for use in vivo , we tried two modifications in addition to the original method . our second method is similar to the background subtraction method . instead of making an adjustment to match the phantom calibration for this calibration we used four goat bones with concentrations of 1 , 13 , 16 , and 31 ppm of pb at varying lucite thicknesses as our calibration standards for the background subtraction method . for our 0 ppm data , we extrapolated from these values for each lucite thickness from the 1 ppm bone and used our highest concentration bone at 31 ppm to replace the 100 ppm phantom in the background subtraction method . for this calibration method , we found the difference in compton peak between actual bone samples and our calibration phantoms for varying lucite thicknesses . these peaks change both with bone versus phantom and with varying lucite thicknesses because of the densities and effective z values of the materials . we were able to apply a fit to this change and using this fit , we can apply the change between phantom and bone to any bone data we take , thus correcting it for use with phantoms . we implemented a traditional peak fitting method primarily for comparisons between our novel calibration methods and the calibration methods used in previous studies of lxrf bone pb measurement systems . the fitting was performed on the pb l and l peaks for phantoms at different lucite thicknesses to determine the signal associated with each concentration . then the same function was used to fit the l and l peaks associated with our cadaver bone and goat bone samples and the corresponding concentration was determined based on the net counts in those peaks corrected for lucite or soft tissue attenuation . in previous studies of lxrf technology , it was concluded that the technology was not suitable for in vivo bone pb measurement due to the significant soft tissue attenuation . we included traditional peak fitting results from our goat bone data to show the comparison to our novel calibration methods . figure 2 shows the resultant portable xrf spectrum from a measurement of an intact human cadaver bone with 1.3 mm soft tissue . as shown in the spectrum , the compton scattering peak comes from the x - ray tube silver characteristic x - rays undergoing compton scattering in our sample . this peak is a significant spectral feature and can be related to the background scattering events throughout the x - ray spectrum as we will demonstrate with our background subtraction calibration method in later results . this spectrum also demonstrates the difficulty in using traditional peak fitting methods , since with more soft tissue , there will be more background and the peaks will become increasingly noisier . the measurements of the lucite covered pb doped phantoms were used to calculate the detection limit . the detection limit was calculated as ( 1)dl=20 ppm=211/,0 ppm2 + 1/,0 ppm2 , where ( 2),0 ppm=100 ppmbkg0 ppm/180 sgross100 ppmbkg0 ppm , where bkg0 ppm is the background count rate under the l or l peak for the 0 ppm phantom and gross100 ppm is the total count rate under the l or l peak for the 100 ppm phantom . table 1 lists the detection limit of the portable xl3 t goldd+ system and the older portable xl3 t system . this comparison was taken at the same x - ray tube settings and filter on each device in order to demonstrate the improvements of the new system . this data was taken for 3 minutes at the same settings used for other measurements . measurements were made to validate the portable xrf system against the standard kxrf systems for in vivo bone pb measurements . phantoms , goat bones , and cadaver bones with 0 , 1 , 2 , and 3 mm of lucite were measured by both systems , with goat bone also being measured at 4 and 5 mm of lucite thickness . table 2 shows the measured phantom pb concentrations at different lucite thicknesses . the correlation ( r - squared ) between the expected concentrations and those measured with portable xrf system ranges from 0.991 to 0.999 for soft tissue thicknesses of 0 to 3 mm , demonstrating a good agreement of pb concentrations determined by kxrf and portable xrf for bare and lucite covered phantoms . tables 3 and 4 demonstrate the ability of the three calibration methods to quantify bare cadaver bone pb values . table 3 shows the pb concentration in bare cadaver bone calculated using the three calibration methods . table 4 shows the bone pb concentrations for cadaver bone covered with 3 mm of lucite . without lucite the calibration methods tend to be fairly similar , but with the introduction of more lucite the bone adjustment method tends to get further from kxrf values by overestimating background levels . bone calibration has a similar correlation , but with only 4 points on the calibration line and the highest point at 30 ppm the actual values tend to deviate from kxrf especially for higher pb concentrations . higher concentration standards are necessary to get visible signal while defining our function to correct for the inverse square and attenuation signal degradation as soft tissue increases . background subtraction was the most reliable calibration method for higher lucite thicknesses and lower pb concentrations , which was determined using the correlation values for the cadaver bone evaluated at different lucite thicknesses . figures 3(a)3(f ) show the comparison of the correlations between goat bone pb concentrations calculated by kxrf and portable xrf at lucite thicknesses of 05 mm , with the pb concentrations for portable xrf being calculated using traditional peak fitting or background subtraction . from the correlations , one can see that traditional peak fitting does fairly well for bare bone or at lower lucite thicknesses , but with higher lucite thicknesses the correlation falls off quickly due to the high background leading to the pb peak being highly distorted especially at low concentrations . the chi - squared values for all the spectral fittings are close to 1 , with the average chi - squared and standard deviation of the chi - squared value for these fits being 1.1 0.4 , which demonstrates that even for poor results the data is accurately represented by fitted function . the data for cadaver bones with different lucite thicknesses analyzed using the background subtraction method is presented in table 5 . the correlations ( r - squared ) between the concentrations obtained from kxrf and those from the portable xrf system range from 0.58 to 0.94 with lucite thicknesses of 0 to 3 mm . the correlations between the pb concentrations obtained from kxrf and portable xrf are worse for the cadaver bones than for the goat bones . this is mainly due to the lack of pb concentration variation among the cadaver bones and geometry stability . also , cadaver bone 6918 is an outlier ( see discussion ) . to test the reliability and reproducibility of the technology for in vivo measurement , only three such cadaver bones were available in our lab , so there are limited data for this test . table 6 shows the pb concentrations from lxrf and kxrf for the three cadaver bones with intact soft tissue . the comparison in table 6 demonstrates the abilities of the device in use through actual soft tissue . using the compton peak to determine soft tissue thickness , which was shown to be comparable to an ultrasound measurement in our previous paper , we found the intact soft tissue thicknesses for our three cadaver bone samples to be 1.3 mm for cadaver bone 7042 , 4.1 mm for cadaver bone 7031 , and 5.6 mm for cadaver bone 7168 . the higher errors for individual measurements and higher standard deviation for grouped measurements are associated with larger soft tissue thicknesses , which is what we expect . this study investigated the detection limit of an improved portable xrf system for in vivo bone pb quantification and validated the system using phantoms , goat bones , and human cadaver bones . the improved system geometry and detector size greatly enhanced the detection limit of the device and the ability of the device to accurately determine the concentration of pb in bone especially at the in vivo situation . the detection limit for the portable xrf device is improved from the previous portable xrf device by a factor of about 2 . through soft tissue thickness of 4 mm the device has the capability of a detection limit of 8 ppm , which is comparable to detection limit of 610 ppm with kxrf bone pb measurement systems in most labs . it is also relevant to point out that this was with a 3-minute measurement time and that time could be increased by a factor of 2 or 3 to lower the detection limit further , while maintaining a reasonable radiation exposure . the main disadvantage of lxrf systems is the lack of penetration of the low energy x - rays and thus at depth , the ability of the system to determine concentration becomes limited . with this system , it is shown that even at depth of 4 mm the portable xrf device now has the capability of obtaining measurements in 3 minutes , which would be equivalent to a kxrf device with a 30-minute measurement . in studies it has been shown that tibia measurement sites with tissue thickness of less than 4 mm can be found on most seniors and about half of the general population [ 15 , 22 ] . one of our target populations for this device is the senior people whose mobility might be confined by their health conditions . another point of clarification is the fact that , with the low penetration depth of lxrf , the kxrf and portable xrf systems are sampling different sites of the bone . kxrf would be sampling the whole bone and lxrf would be sampling the superficial 0.51 mm of the bone . it is not very clear how pb distributes over the layer of tibia bone and the literature on this topic is limited . todd et al . showed higher concentrations of lead at 1 - 2 mm to the surface in bone , while bellis et al . while the bone pb concentrations from kxrf and lxrf are highly correlated in our study , further investigation with larger amount of samples is needed on the comparison for the absolute bone pb concentrations from these two methods . it is also relevant to point out that with measurements of bone lead the goal is a correlation with health effect , which should be reflected in both surface and depth bone measurement sites . the pb concentrations found through kxrf and portable xrf measurements of bare bone show good correlation . in order for our device to determine the in vivo pb concentration bone has different density and effective atomic number compared to our calibration phantoms , which led to differences in its resultant xrf spectrum . our results with cadaver and goat bones show that our calibration methods adequately address these differences as the results are well correlated with kxrf data . in comparison , the traditional peak fitting calibration method results are shown with goat bones , and at larger lucite thicknesses this method is worse than the background subtraction method . it is relevant to point out that other studies exploring the validity of lxrf for pb studies used traditional peak fitting methods and showed that the results were not reliable especially at higher soft tissue thicknesses . the bone adjustment method did correct the compton peak to phantom values for a more equal comparison , but it fails to take into account the balance between the compton peak , background , and signal , and because of this , at higher lucite thicknesses , it exaggerates the problems seen with background subtraction . bone calibration should be the best calibration method in theory , but due to the lack of standard bones with higher pb concentrations , the calibration line for this method tended to produce results that were less accurate than the background subtraction method . the correlation of bone pb concentrations between kxrf and portable xrf is very good for phantoms and goat bones with lucite thickness up to 5 mm , while the r - squared degraded a little for cadaver bones . this is mainly due to the small variation of the pb concentration for these cadaver bones , as well as the difficulty to adjust the geometry of the bare bones . in measuring cadaver bones , the geometry presented issues if not strictly monitored . we found that the cadaver bones were prone to air gaps in the geometry , which led to significant changes in the spectrum caused by the increased distance without significant attenuation . although this effect was visible in the bare bone data , given the geometry of in vivo measurements this effect would not be present as the bone is covered in soft tissue , so there will not be air gaps between the soft tissue and bone in vivo . attenuation by soft tissue is accounted for with our calibration by determining the soft tissue thickness from the compton peak , which in turn corrects for distance , as the gap between the detector and bone is filled with soft tissue . although only three intact cadaver bones were used to test the reproducibility of the system for bone pb quantification and to validate the system in a real in vivo situation , several conclusions can be drawn from these limited data . first , this set of data confirmed the validity of the system for in vivo measurements , especially for the measurements with soft tissue thicknesses less than 5 mm . second , the data confirmed that the thicknesses of the soft tissue significantly affect the uncertainties of the resultant concentrations . the standard deviations from the repeat measurements are lower than the uncertainties for individual measurements , which indicate that the uncertainties for individual measurements may be overestimated . in addition , the detection limit of the measurements calculated from the pb concentration uncertainties ( dl = 2 sigma ) for cadaver bones listed in table 6 would be higher than those listed in table 1 for corresponding soft tissue thicknesses . this is because the uncertainty calculated in table 6 includes the error on the gross count and net count of the signal under the pb l x - ray peak , while the dl calculated in table 2 only includes error associated with the background of a blank phantom covered with the corresponding thicknesses of lucite . nonetheless , this data set shows an excellent agreement of bone pb concentrations for cadaver bones at thickness of 1.3 and 4.1 mm , while the agreement deteriorates at 5.6 mm . in the cadaver bone measurements , there is one bone ( cadaver bone 6918 ) , which we considered an outlier in our dataset . this bone came from a 100-year - old female and presents further challenges with the lxrf device . the spectrum from the bone had a much higher than normal compton peak , which we attributed to the bone appearing more like soft tissue with respect to the spectral features . in general the background subtraction method should overcome slight variations in bone between individuals , as the compton peak will also relate back to the material of the bone . the relationship we had derived between the compton peak and the pb signal broke down for this particular bone . we have not isolated the characteristic that causes this issue but plan to look into bone density effects on the lxrf spectrum through simulation as well as our cadaver bone samples . although bone 6918 has a compton peak that is significantly different , other spectral features show differences that may be able to be exploited to correct the issues with the compton peak in this spectrum . the portable xrf system , now with a significantly lower detection limit , has its main advantages over kxrf with its portability , acquisition times , and ease of use . the new system can achieve a minimum detection limit equivalent to a kxrf measurement even through tissue thicknesses up to 5 mm . the portable xrf system also has the advantage of using an x - ray tube , which can be turned off when it is not in use and is less complicated for radiation license than a radioisotope source . the portable device lends itself for use in epidemiologic studies because of its quick measurement times and portability . the device allows for on - site pb surveys and risk assessments of the environment , while performing exposure assessment of the community members . in the future , the device can be improved by perfecting the data analysis algorithms for pb as well as other metals . monte carlo methods could be used to accurately model the device and the spectrum of in vivo situations . this would help decrease the variability of measurements over different bone densities while also accounting for the tissue thickness over the bone . a main goal for the future of portable xrf technology would be applying it for the detection of other metals in vivo . the device can be used in collaboration with metal epidemiologists and toxicologists to study exposures and health effects of metals . we have validated an advanced portable xrf system for in vivo bone pb measurement and demonstrated the validity of using such a system to accurately quantify pb in bone with soft tissue thickness up to 4 - 5 mm . the detection limit of the device with 4 mm of soft tissue is approximately the same as the detection limit of kxrf systems , and the novel analysis methods provide a better correlation for pb quantification in bone samples . this device now has vast applicability in pb exposure assessment in clinical and research settings .

lead is a ubiquitous toxicant . bone lead has been established as an important biomarker for cumulative lead exposures and has been correlated with adverse health effects on many systems in the body . k - shell x - ray fluorescence ( kxrf ) is the standard method for measuring bone lead , but this approach has many difficulties that have limited the widespread use of this exposure assessment method . with recent advancements in x - ray fluorescence ( xrf ) technology , we have developed a portable system that can quantify lead in bone in vivo within 3 minutes . our study investigated improvements to the system , four calibration methods , and system validation for in vivo measurements . our main results show that the detection limit of the system is 2.9 ppm with 2 mm soft tissue thickness , the best calibration method for in vivo measurement is background subtraction , and there is strong correlation between kxrf and portable lxrf bone lead results . our results indicate that the technology is ready to be used in large human population studies to investigate adverse health effects of lead exposure . the portability of the system and fast measurement time should allow for this technology to greatly advance the research on lead exposure and public / environmental health .

symptomatic grade 1 and 2 hemorrhoids rarely require surgical intervention and are usually treated with less invasive technologies most frequently with rubber band ligation or infrared coagulation . rubber band ligation is associated with frequent post - procedural pain and discomfort,16 as well as other , sometimes very serious , complications.3,714 infrared coagulation causes much fewer complications than rubber band ligation , but produces inconsistent clinical results and is frequently ineffective.2,6,1519 the purpose of this study was to compare histologic findings between the infrared coagulator ( irc 2100 , redfield corporation , rochelle park , nj , usa ) and the het bipolar ligator ( het systems , northvale , nj , usa ) a new bipolar device for tissue manipulation and coagulation . the het bipolar ligator system is comprised of an innovative tissue ligator and an associated temperature monitor . the het bipolar ligator has a unique , constant tissue compression mechanism , a temperature sensor adjacent to the bipolar electrodes , and integral led - based illumination . the het bipolar ligator s unique tissue clamp and bipolar radio - frequency - based tissue coagulation allow compression and ligation of the target tissue with a constant force and predictable energy delivery . when this operator - independent force is applied parallel to the rectal wall , the associated superior hemorrhoid vasculature in the formed tissue fold continuous tissue temperature monitoring during use of the het bipolar ligator provides the operator with objective , real - time feedback about the treated tissue temperature.20 the redfield irc 2100 includes a halogen lamp - based heat generator and an elongated probe , which interfaces with the target tissue and delivers heat by regulating the amount of time the device is activated . this approach does not control the amount of heat applied to the tissue ; however , it controls the length of time the tissue will be exposed to varying temperatures . the irc system does not provide a mechanism for controlling or gauging tissue compression , or a method for measuring the temperature of the treated tissue . additionally , the pressure applied to the treatment site by the irc device is operator dependent . an in vivo single animal , multiple treatment sites study was conducted to compare the histologic effects of the two systems when used on the recto - sigmoid swine colon . the objective of this study was to assess histopathologic changes following treatment with heat delivered to normal colonic tissues by the two devices . since both the redfield irc 2000 and the het bipolar ligator are applied to the normal colonic mucosa of humans during the treatment of internal hemorrhoids , and since the hemorrhoidal tissue is not directly affected by thermal energy , only normal animal colonic tissues were studied . the effects of thermal treatment in the mucosa , submucosa , submucosal vessels , and muscularis layer were evaluated . following receipt of university institutional animal care and use committee approval , the study was carried out on a single female sus scrofa pig weighing approximately 54.5 kg . preanesthesia of intramuscular ketamine ( 14.7 mg / kg ) and intravenous acepromazine ( 1.5 mg / kg ) was given . the pig was connected to a heart rate monitor , oxygen monitor , and ventilator . a longitudinal incision was made on the antimesenteric side of the recto - sigmoid colon and wide access to the colonic mucosa was obtained . three similar segments of the colon were used to study three treatment groups with six areas per group . the het bipolar ligator was used to treat two groups at two different temperatures : 55c ( het55 group ) and 50c ( het50 group ) . both the irc 2100 ( irc ) and het bipolar ligator ( het ) devices were used according to the manufacturer s instructions for use . the het bipolar ligator was connected to a conmed hyfrecator 2000 bipolar radiofrequency generator ( conmed , utica , ny , usa ) with an output coagulation setting at 10 watts . the tissue in the het bipolar ligator was compressed parallel to the bowel wall , and bipolar radiofrequency energy was then delivered until the temperature reached 50c ( across six areas ) or 55c ( across six areas ) . the irc device delivers pulses of infrared light through a small contact tip applicator that is applied to the tissue . the light causes thermal coagulation which results in tissue necrosis.21 the amount of energy delivered is regulated by the amount of time the device is activated . as recommended by the manufacturer , the irc timer was set in between 1.01.5 seconds for the treatment of hemorrhoids . the irc heat pulse was delivered perpendicular to the mucosal surface to each of the six treatment mucosal areas . the temperature of the irc probe was continuously measured during the heat delivery using a digital thermometer ( tektronix dtm920 , tektronix , inc , beaverton , oregon , usa ) . the maximum temperature , observed in all cases at 1.5 seconds , was documented . when all treatment groups were completed , the pig was euthanized , and the recto - sigmoid colon segment removed . all samples were sectioned perpendicular to the mucosa into two pieces and embedded in paraffin blocks . the blocks were sectioned in 4 cuts , and an additional step was performed approximately 150 from the first cut . the thermal damage in the mucosa , submucosa , and muscularis layers was measured as follows : layer thinning was measured by an ocular micrometer comparing the treated area to an adjacent normal area , and was graded on a scale of 0 to + 4 with 0 representing 0% thinning , + 1 representing 25% thinning , + 2 representing 50% thinning , + 3 representing 75% thinning , and + 4 representing total loss of the layer . absolute dimensions were not used as the layer thickness can vary from place to place , and such measurements would not reflect the importance of the percent of layer damage . the percentage measurements used are a standard technique in assessing histological damage , including thermal damage . the mucosa was graded based on the layer thinning , loss of glands and loss of basophilic staining . the submucosa was graded based on the layer thinning and the degree of the vascular injury . the muscularis layer was graded based on the increased eosinophilia , vacuolization and nuclear changes . the values were compared with the standard students t - test.22 collagen damaged was accessed by polarized light microscopy . in the het50 and het55 groups , the treated tissues were also examined at different distances from the bipolar electrodes . the lateral thermal spread outside of the energy application site was evaluated as well . the treatment time and treatment site temperature readings are summarized by study group in table 1 . the treated tissue reached 50c in 47 sec ( het50 group ) and 55c in 69 sec ( het55 group ) . the compressed tissue in both het groups was 12 mm . in the irc group , after 1.5 seconds of treatment , the temperature at the tip of the probe varied from 127.8c to 155.7c ( 149.9c 11.1c ) ; with the final tissue temperature being highly dependent on the pressure applied by the device to the tissue . the gross , comparative changes between the irc , het55 , and het50 groups are shown in figure 1 . table 2 summarizes the comparative mucosal and submucosal changes between the irc , het55 , and het50 groups at sites immediately adjacent to or in contact with the treatment elements . the mucosal and submucosal tissue changes seen in the irc and the het55 groups were similar . both the mucosal and submucosal tissue changes in the het50 group were significantly milder than irc and het55 groups ( p < 0.05 ) . none of the samples displayed loss of tissue architecture or thermal damage of the full thickness of the bowel wall . the lateral thermal spread from the application site into the normal tissue in all cases was minimal . the treated mucosa displayed thinning caused by thermal desiccation and the loss of crypt glands ( figures 2 and 3 ) . some thinning in the het specimens was also a result of compression from the tissue clamp of the device . these effects , with the exception of glandular loss , were significantly less pronounced in the het50 group . the submucosal thinning ( figure 5 ) was similar between the irc and the het55 groups , while the het50 group demonstrated significantly less submucosal thinning ( p < 0.05 ) . in the het55 group , 50% of samples displayed a full thickness submucosal injury without muscle involvement , while none of the het50 samples showed full thickness submucosal thermal injury . vascular injury included congestion and small amounts of hemorrhage , and in some cases , the vessel endothelial cells were affected ( figure 6 ) . there were signs of collagen denaturation present in all irc , het55 , and het50 specimens with all samples showing reduced collagen damage in the areas that were more remote from the source of the heat . this was demonstrated by the polarized photomicrograph showing the normal collagen as bright areas and the denatured as darkened areas ( figure 7 ) . the maximal tissue damage was observed immediately adjacent to the ligation electrodes in the het55 and het50 groups . while hemorrhage and occasional thinning were observed in the mucosa , the thermal effects were prevalent in the submucosa and presented as vascular injury including congestion and hemorrhage . evaluation of folds of tissue between the ligation electrodes demonstrated no difference in vascular damage between the two het groups ( table 3 ) . all samples from the irc group displayed similar thermal damage to the muscularis layer , but this damage was scant and isolated . in the het50 and het55 groups , 75% of samples had no muscular injury , while 25% of the samples presented only with superficial muscularis injury ( figure 8) . lateral thermal spread from the application site into normal tissue was minimal in all samples . the treated mucosa displayed thinning caused by thermal desiccation and the loss of crypt glands ( figures 2 and 3 ) . some thinning in the het specimens was also a result of compression from the tissue clamp of the device . these effects , with the exception of glandular loss , were significantly less pronounced in the het50 group . the submucosal thinning ( figure 5 ) was similar between the irc and the het55 groups , while the het50 group demonstrated significantly less submucosal thinning ( p < 0.05 ) . in the het55 group , 50% of samples displayed a full thickness submucosal injury without muscle involvement , while none of the het50 samples showed full thickness submucosal thermal injury . vascular injury included congestion and small amounts of hemorrhage , and in some cases , the vessel endothelial cells were affected ( figure 6 ) . there were signs of collagen denaturation present in all irc , het55 , and het50 specimens with all samples showing reduced collagen damage in the areas that were more remote from the source of the heat . this was demonstrated by the polarized photomicrograph showing the normal collagen as bright areas and the denatured as darkened areas ( figure 7 ) . the maximal tissue damage was observed immediately adjacent to the ligation electrodes in the het55 and het50 groups . while hemorrhage and occasional thinning were observed in the mucosa , the thermal effects were prevalent in the submucosa and presented as vascular injury including congestion and hemorrhage . evaluation of folds of tissue between the ligation electrodes demonstrated no difference in vascular damage between the two het groups ( table 3 ) . all samples from the irc group displayed similar thermal damage to the muscularis layer , but this damage was scant and isolated . in the het50 and het55 groups , 75% of samples had no muscular injury , while 25% of the samples presented only with superficial muscularis injury ( figure 8) . lateral thermal spread from the application site into normal tissue was minimal in all samples . the goal of therapeutic intervention is to effectively target the pathologic lesion while minimizing injury to collateral tissues . electrocoagulation as a treatment modality was utilized as early as 1867 and explained by dr we kessey in 1934.23 over the past several decades , new developments and techniques to treat hemorrhoids in a less invasive manner have targeted the superior hemorrhoidal vasculature and the associated submucosa above the dentate line in order to minimize patient pain , discomfort , and tenesmuses.2428 both the irc device and the het bipolar ligator intend to produce histologic changes proximal to the internal hemorrhoid , which would lead to an occlusion of the superior hemorrhoid blood supply and formation of moderate scarring in the associated submucosa . to minimize potential collateral tissue damage , a precise capture of the target tissue proximal to the internal hemorrhoid , and predictable energy delivery to both the submucosal vessels and the surrounding connective tissue , are thus highly desirable . in this evaluation there was greater variability in the temperature at the tissue treatment site in the irc group than in the het groups , and the temperature reached in the irc treatment group was significantly higher . with the irc device , the procedure was driven by the manufacturer recommended time setting and operator variability in compression pressure.21 both inevitably impacted the amount of energy delivered to the target site . in contrast , when the het bipolar ligator was used , temperature at the treatment site was monitored continuously , and energy was delivered consistently until the target tissue temperature of 50c55c was achieved . the time of energy delivery during tissue ligation using the het bipolar ligator is a derivative of the targeted tissue temperature . additionally , the device is designed to provide constant consistent compression , which further minimized variability in both the energy delivery to the tissue and consequent tissue changes.29 tissue compression is a known factor affecting energy delivery to targeted tissue.30,31 variability in the tissue compression , therefore , would lead to variability in the energy delivery to the tissue . this was demonstrated in this investigation given that the consistently compressed tissue ( among the het groups ) required much less temperature than non - compressed tissue ( among the irc group ) to cause similar histologic effects . the irc device does not provide a mechanism for predictable or measurable tissue compression , meaning that the tissue compression was operator - dependent . contrary and advantageously , the minimal variability observed within the het treatment groups was a function of the unique design of the het bipolar ligator device . the amount of energy delivered to the tissue correlates with the histological changes.32 the tissue temperature monitoring , therefore , may serve as a useful guideline during the energy delivery to the tissue . the irc device did not offer a mechanism for temperature monitoring and relied strictly on the time of energy delivery for tissue treatment.17 as information on temperature generated by the irc probe was not found in the literature , it was measured directly in this study . the irc generated 149.9c 11.1c during 1.5 seconds of activation in this hemorrhoid treatment model . in contrast , the het bipolar ligator was designed to allow temperature monitoring of the treated tissue . this affords standardization of the treatment by specifically exposing the target tissue to a constant 55c temperature to achieve the desired therapeutic effect . both devices created similar histological changes in the submucosal vessels and submucosa layer of the target tissues . the irc achieved these changes in 1.5 seconds at much higher temperature , while the het bipolar ligator achieved the desired tissue changes using temperatures that were 3.0 and 2.7 times lower in het50 and het55 groups , respectively . it appears that the constant tissue compression accompanying the tissue ligation using the het bipolar ligator reduced the amount of energy required to produce similar histological effects . recent discovery of targeted metal nanoparticles in selective tissue hyperthermia and cytotoxicity may play an interesting role in facilitating a minimally - invasive chemical ligation of the targeted small vessels.3335 in the irc and both het groups , signs of tissue desiccation were observed , including some loss of intercellular water and associated pyknotic and hyperchromic changes in the nuclei . changes such as loss of normal tissue architecture and complete cellular protein denaturation were not observed in any of the samples . histologically , cells showed lighter hematoxylin and eosin staining due to the loss of cell nuclei and intracellular protein ; such cells are sometimes referred to as ghost cells . white cell infiltration and capillary growth into the damaged area will occur from the surrounding normal tissue . this regenerative process will also lead to fibrosis.28 in contrast , a more significant heat - related injury typically produces more significant volume of tissue damage , and the adjacent tissue would be unable to regenerate , usually leading to ulceration.36,37 while the overall histological changes created by the tissue ligation using the het bipolar ligator at 50c ( het50 ) appeared to be milder than the ligation at 55c ( het55 ) , the submucosal changes in the treated folds of tissue between the ligation electrodes were substantially equivalent between the groups . this finding suggests that the efficacy of the het device operating at 50c and 55c is similar . however , from a clinical standpoint , patients exposed to a lower temperature ( 50c ) may experience less discomfort . the thermal injury in all het specimens was limited to the areas compressed between the bipolar electrodes with no or minimal collateral tissue damage outside of the tissue that was in contact with the electrodes . since the operator controls the position of the electrodes , the tissue compression within the unique het clamp is constant , and therefore the tissue is treated to the defined temperature . the energy delivery to the target tissue by the het bipolar ligator is highly consistent and predictable , minimizing collateral tissue damage . the demonstrated recognizable architecture , the lack of full thickness damage , the intact tunica muscularis , and the minimally damaged blood supply suggest that little ulceration would occur in tissues treated with the het bipolar ligator at 55c or 50c . the intact residual blood supply would thus promote routine fibroblast infiltration and active fibrosis in the area of the thermal desiccation.3638 tissue compression is a known factor affecting energy delivery to targeted tissue.30,31 variability in the tissue compression , therefore , would lead to variability in the energy delivery to the tissue . this was demonstrated in this investigation given that the consistently compressed tissue ( among the het groups ) required much less temperature than non - compressed tissue ( among the irc group ) to cause similar histologic effects . the irc device does not provide a mechanism for predictable or measurable tissue compression , meaning that the tissue compression was operator - dependent . contrary and advantageously , the minimal variability observed within the het treatment groups was a function of the unique design of the het bipolar ligator device . the amount of energy delivered to the tissue correlates with the histological changes.32 the tissue temperature monitoring , therefore , may serve as a useful guideline during the energy delivery to the tissue . the irc device did not offer a mechanism for temperature monitoring and relied strictly on the time of energy delivery for tissue treatment.17 as information on temperature generated by the irc probe was not found in the literature , it was measured directly in this study . the irc generated 149.9c 11.1c during 1.5 seconds of activation in this hemorrhoid treatment model . in contrast , the het bipolar ligator was designed to allow temperature monitoring of the treated tissue . this affords standardization of the treatment by specifically exposing the target tissue to a constant 55c temperature to achieve the desired therapeutic effect . both devices created similar histological changes in the submucosal vessels and submucosa layer of the target tissues . the irc achieved these changes in 1.5 seconds at much higher temperature , while the het bipolar ligator achieved the desired tissue changes using temperatures that were 3.0 and 2.7 times lower in het50 and het55 groups , respectively . it appears that the constant tissue compression accompanying the tissue ligation using the het bipolar ligator reduced the amount of energy required to produce similar histological effects . recent discovery of targeted metal nanoparticles in selective tissue hyperthermia and cytotoxicity may play an interesting role in facilitating a minimally - invasive chemical ligation of the targeted small vessels.3335 in the irc and both het groups , signs of tissue desiccation were observed , including some loss of intercellular water and associated pyknotic and hyperchromic changes in the nuclei . changes such as loss of normal tissue architecture and complete cellular protein denaturation were not observed in any of the samples . histologically , cells showed lighter hematoxylin and eosin staining due to the loss of cell nuclei and intracellular protein ; such cells are sometimes referred to as ghost cells . white cell infiltration and capillary growth into the damaged area will occur from the surrounding normal tissue . this regenerative process will also lead to fibrosis.28 in contrast , a more significant heat - related injury typically produces more significant volume of tissue damage , and the adjacent tissue would be unable to regenerate , usually leading to ulceration.36,37 while the overall histological changes created by the tissue ligation using the het bipolar ligator at 50c ( het50 ) appeared to be milder than the ligation at 55c ( het55 ) , the submucosal changes in the treated folds of tissue between the ligation electrodes were substantially equivalent between the groups . this finding suggests that the efficacy of the het device operating at 50c and 55c is similar . however , from a clinical standpoint , patients exposed to a lower temperature ( 50c ) may experience less discomfort . the thermal injury in all het specimens was limited to the areas compressed between the bipolar electrodes with no or minimal collateral tissue damage outside of the tissue that was in contact with the electrodes . since the operator controls the position of the electrodes , the tissue compression within the unique het clamp is constant , and therefore the tissue is treated to the defined temperature . the energy delivery to the target tissue by the het bipolar ligator is highly consistent and predictable , minimizing collateral tissue damage . the demonstrated recognizable architecture , the lack of full thickness damage , the intact tunica muscularis , and the minimally damaged blood supply suggest that little ulceration would occur in tissues treated with the het bipolar ligator at 55c or 50c . the intact residual blood supply would thus promote routine fibroblast infiltration and active fibrosis in the area of the thermal desiccation.3638 the combination of constant tissue compression and temperature - guided energy delivery achieves desirable histological changes between 50c and 55c . the unique design of the het bipolar ligator provides precise delivery of energy to the target tissue , as well as real - time temperature monitoring and well - gauged , constant tissue compression . these mechanisms facilitate accurate therapeutic energy delivery to the target tissues between 50c and 55c while minimizing the collateral muscular damage . in this comparative histological study , while both devices achieved similar results , the het bipolar ligator achieved the desired histologic changes with less muscular damage , using focused delivery of radio frequency energy at a much lower temperature than irc . therefore , the use of the het bipolar ligator could provide significant clinical advantages , potentially enhance patients tolerance of the ligation procedure , and minimize the chance of heat - related complications and collateral tissue damage . the conclusions of this study need to be considered with caution due to the anatomical differences between humans and pigs . specifically , the differences in colonic layer thickness , tissue metabolism , and reactions to heat between human and pig tissue could potentially influence the study results . to make clinically meaningful conclusions , it is important to continue studying these new devices in a well - designed , outcome - based clinical study .

backgroundseveral minimally invasive technologies are available to treat common soft tissue lesions including symptomatic hemorrhoids . the use of energy to deliver heat and coagulate target lesions is commonly practiced . this study compares the histologic effects produced on intestinal tissues by two energy - based systems which employ different approaches of heat delivery.methodstwo heat delivery systems were evaluated in vivo in a single porcine subject : infrared coagulator and bipolar tissue ligator utilizing constant tissue compression and temperature guidance . eighteen treatment sites divided into three groups of six were assessed . treatment site temperature was measured and the effects of thermal treatment in the mucosa , submucosa , submucosal vessels , and muscularis layer were scored . lateral thermal spread beyond the energy application site was also assessed.resultstreatment site temperatures were much lower in the bipolar ligator group than in the infrared coagulator group . the mucosal and submucosal tissue changes observed in tissues treated with infrared energy and bipolar energy at 55c were similar . both the mucosal and submucosal tissue changes with bipolar energy at 50c were significantly less.conclusionboth devices achieved similar histologic results . however , the unique design of the bipolar ligator , which allows consistent capture , constant compression , and temperature monitoring of target tissue , accomplished the desired histologic changes with less muscular damage at much lower temperatures than the infrared coagulator . the use of bipolar ligation could offer clinical advantages such as reduced patient pain and a minimized chance of heat - related collateral tissue damage .

during restorative procedures for a carious tooth , dental pulp is exposed to a variety of stimulations such as heat stress produced by cavity preparation , and hypoxia produced by local anesthesia . when composite resin is used as a restorative material , dentists sometimes note inflammatory responses of dental pulp , which are considered to be caused by microleakage at the interface between cavity walls and filled composite resin . other studies have suggested that unpolymerized resin monomers remaining on the cavity floor may irritate dental pulp cells directly or indirectly through dentin , resulting in inflammatory responses . it has previously been reported that apoptosis of pulp cells was induced during pulp wound healing after cavity preparation , and that apoptosis of pulp cell line was induced by heat stress and hypoxia . apoptotic cells induced in dental pulp disappear during pulp wound healing processes , suggesting cytotoxic effects of external stimulations via residual dentin and the recovery of dental pulp viability . however , it is largely unknown whether dental pulp cells adversely affected by resin monomers can recover their viability . in the present study , we examined effects of resin monomer , 2 , 2-bis [ 4-(2-hydroxy-3-methacryloxypropoxy ) phenyl ] propane ( bis - gma ) , on the viability of human dental pulp cell line . lsc2 cells , a dental pulp cell line derived from human dental pulp , were maintained in dulbecco 's modified eagle 's medium ( dmem ; invitrogen corp . , carlsbad , ca , usa ) containing 10% heat - inactivated fetal calf serum ( fcs ) , 100 g / ml of streptomycin , and 100 u / ml of penicillin , and then incubated in a humidified atmosphere of 5% co2 at 37c . lsc2 cells ( 2x10 cells / well ) were cultured in 96 well plate with a medium containing 5% fcs for 24 h , then the medium was exchanged to that containing 1% fcs . several different concentrations ( 0.01 , 0.02 , 0.03 , and 0.04 mmol / l ) of bis - gma ( polyscience inc . , warrington , pa , usa ) diluted by dimethylsulfoxide ( dmso ; wako pure chemical inc , osaka , japan ) were added to the culture medium . at 48 h after exposure to bis - gma , cell proliferation was examined using the 5-bromo-2'-deoxyuridine ( brdu ) assay ( the biotrak cell proliferation elisa system version 2 ; ge healthcare uk ltd , uk ) . cell viability was analyzed by measuring optical density ( od ) using a test wavelength of 540 nm and a reference wavelength of 620 nm with a multiscan biochromatic microplate reader ( multiscan jx ; thermo fisher scientific k.k . , lsc2 cells were exposed to bis - gma for 48 h , then suspended in a hypotonic solution ( 0.1% triton x-100 , 1 mmol / l tris / hcl , ph 8.0 , 3.4 mmol / l sodium citrate , 0.1 mmol / l edta ) and stained with 5 g / ml of propidium iodide , after which cell - cycle distribution was analyzed with a facscalibur flow cytometer epics xl ( beckman coulter inc . , lsc2 cells were exposed to bis - gma in a chamber slides for 48 h , then fixed with 1% glutaraldehyde for 30 min , washed with 1x phosphate buffered saline ( pbs ) , and stained with 5 g / ml of hoechst dye 33342 ( life technologies co. , ca , usa ) . cells detached from the coverslips into the medium were also recovered by centrifugation and stained . nuclei were observed under a fluorescence microscope ( olympus bx50/bx - fla / dp70 ; olympus co. , tokyo , japan ) . after exposure of lsc2 cells to bis - gma for 48 h , the wells were rinsed by pbs , and added a medium containing 10% fcs without bis - gma . after the culture of surviving cells in a humidified atmosphere of 5% co2 at 37c for 96 h , cell proliferation and cell cycle were analyzed . dentin discs ( 1 mm thick , minimum diameter : 6 mm ) were cut from freshly extracted sound human third molars using a low speed diamond - coated saw ( isomet ; buehler , lake bluff , il , usa ) under water coolant , then etched for 60 s with 35% phosphoric acid to completely open dentinal tubules , rinsed with ultrasonication for 3 min in distilled water , and placed in the pfa tube ( sanplatec , osaka , japan ) . the lower chamber was filled with distilled water and the upper chamber with 0.2 mmol / l of bis - gma . after standing in a humid chamber at room temperature for 2 weeks , the distilled water was collected and mixed with chloroform to isolate bis - gma . bis - gma was detected using isocratic high - performance liquid chromatography ( hplc ) ( waters , milford , ma , usa ) with a reverse - phase column ( -bondashere c18 , 5 m , 300 a column , jlc010032y ) ( waters ) at 280 nm . the peak area of bis - gma shown by hplc tracing was analyzed using the waters 2487 dual absorbance detector ( waters ) , and the amount of bis - gma was calculated from the calibration curve of known concentrations from control experiments . statistical analysis of the data was performed using one - way anova followed by a multiple - comparison sheffe 's test with statview 5.0 software ( sas software inc . , cary , nc , usa ) . lsc2 cells , a dental pulp cell line derived from human dental pulp , were maintained in dulbecco 's modified eagle 's medium ( dmem ; invitrogen corp . , carlsbad , ca , usa ) containing 10% heat - inactivated fetal calf serum ( fcs ) , 100 g / ml of streptomycin , and 100 u / ml of penicillin , and then incubated in a humidified atmosphere of 5% co2 at 37c . lsc2 cells ( 2x10 cells / well ) were cultured in 96 well plate with a medium containing 5% fcs for 24 h , then the medium was exchanged to that containing 1% fcs . several different concentrations ( 0.01 , 0.02 , 0.03 , and 0.04 mmol / l ) of bis - gma ( polyscience inc . , warrington , pa , usa ) diluted by dimethylsulfoxide ( dmso ; wako pure chemical inc , osaka , japan ) were added to the culture medium . at 48 h after exposure to bis - gma , cell proliferation was examined using the 5-bromo-2'-deoxyuridine ( brdu ) assay ( the biotrak cell proliferation elisa system version 2 ; ge healthcare uk ltd , uk ) . cell viability was analyzed by measuring optical density ( od ) using a test wavelength of 540 nm and a reference wavelength of 620 nm with a multiscan biochromatic microplate reader ( multiscan jx ; thermo fisher scientific k.k . , lsc2 cells were exposed to bis - gma for 48 h , then suspended in a hypotonic solution ( 0.1% triton x-100 , 1 mmol / l tris / hcl , ph 8.0 , 3.4 mmol / l sodium citrate , 0.1 mmol / l edta ) and stained with 5 g / ml of propidium iodide , after which cell - cycle distribution was analyzed with a facscalibur flow cytometer epics xl ( beckman coulter inc . , lsc2 cells were exposed to bis - gma in a chamber slides for 48 h , then fixed with 1% glutaraldehyde for 30 min , washed with 1x phosphate buffered saline ( pbs ) , and stained with 5 g / ml of hoechst dye 33342 ( life technologies co. , ca , usa ) . cells detached from the coverslips into the medium were also recovered by centrifugation and stained . nuclei were observed under a fluorescence microscope ( olympus bx50/bx - fla / dp70 ; olympus co. , tokyo , japan ) . after exposure of lsc2 cells to bis - gma for 48 h , the wells were rinsed by pbs , and added a medium containing 10% fcs without bis - gma . after the culture of surviving cells in a humidified atmosphere of 5% co2 at 37c for 96 h , cell proliferation and cell cycle were analyzed . dentin discs ( 1 mm thick , minimum diameter : 6 mm ) were cut from freshly extracted sound human third molars using a low speed diamond - coated saw ( isomet ; buehler , lake bluff , il , usa ) under water coolant , then etched for 60 s with 35% phosphoric acid to completely open dentinal tubules , rinsed with ultrasonication for 3 min in distilled water , and placed in the pfa tube ( sanplatec , osaka , japan ) . the lower chamber was filled with distilled water and the upper chamber with 0.2 mmol / l of bis - gma . after standing in a humid chamber at room temperature for 2 weeks , the distilled water was collected and mixed with chloroform to isolate bis - gma . bis - gma was detected using isocratic high - performance liquid chromatography ( hplc ) ( waters , milford , ma , usa ) with a reverse - phase column ( -bondashere c18 , 5 m , 300 a column , jlc010032y ) ( waters ) at 280 nm . the peak area of bis - gma shown by hplc tracing was analyzed using the waters 2487 dual absorbance detector ( waters ) , and the amount of bis - gma was calculated from the calibration curve of known concentrations from control experiments . statistical analysis of the data was performed using one - way anova followed by a multiple - comparison sheffe 's test with statview 5.0 software ( sas software inc . , cary , nc , usa ) . the viability of lsc2 cells decreased after exposure to bis - gma in a dose - dependent manner ( figure 1a ) . there was no significant difference ( p>0.05 ) in viability between 0 and 0.01 mmol / l of bis - gma , whereas significant decreases of cell viability were observed after being cultured with 0.02 , 0.03 , and 0.04 mmol / l of bis - gma ( p<0.01 ) . the effects of exposure to bis - gma on cell cycle progression of lsc2 cells are shown in figure 1b . exposure to bis - gma reduced the number of the cells in g1 , s , and g2/m phases in a dose - dependent manner , with a concomitant increase of those in the sub - g1 population , implying cell death such as apoptosis . cytotoxic effects of 2 , 2-bis [ 4-(2-hydroxy-3- methacryloxypropoxy ) phenyl ] propane ( bis - gma ) on lsc2 cells . cell cycle of lsc2 cells after exposure to bis - gma morphological changes of lsc2 cells following exposure to bis - gma are shown in figure 2 . the structures of those cells exposed to 0.01 mmol / l of bis - gma were nearly the same as those of the control , whereas the rounding and the detachment were observed in cells exposed to 0.04 mmol / l of bis - gma ( figure 2a ) . fluorescent microscopic analysis revealed that the nuclei of adhesive cells had normal structures , whereas those of cells that had fallen into the medium after exposure to 0.04 mmol / l of bis - gma showed fragmentation , a typical characteristic of apoptotic cells ( figure 2b ) . morphological changes of lsc2 cells promoted by 2 , 2-bis [ 4-(2-hydroxy-3-methacryloxypropoxy ) phenyl ] propane ( bis - gma ) . phase - contrast microphotographs of lsc2 cells after exposure to bis - gma . original magnification , x100 . original magnification , x400 following exposure to bis - gma , surviving cells attached to the bottom of the well were cultured in normal condition without bis - gma ( figure 3a ) . the proliferation of surviving cells following exposure to 0.01 and 0.02 mmol / l of bis - gma was not different after 24 h of cell culture in normal condition , and then increased at 48 and 96 h , while surviving cells exposed to 0.03 mmol / l of bis - gma showed proliferation at 96 h. in contrast , exposure to 0.04 mmol / l of bis - gma showed great cytotoxicity on the viability of the cells , and proliferation was not recovered during the culture in the normal condition . the recovery of cell - cycle progression in surviving cells exposed to 0.01 , 0.02 , and 0.03 mmol / l of bis - gma was also observed ( figure 3b ) . when bis - gma - exposed cells were cultured in normal condition without bis - gma for 96 h , the cell cycle distribution pattern of bis - gma - insulted cells returned to that prior to exposure to bis - gma . survival of lsc2 cells against 2 , 2-bis [ 4-(2-hydroxy-3-methacryloxypropoxy ) phenyl ] propane ( bis - gma ) . recovery of viability of lsc2 cells that survived after exposure to bis - gma . mmol / l ) ; 0 ( black circle ) , 0.01 ( white circle ) , 0.02 ( white rectangle ) , 0.03 ( black triangle ) , and 0.04 ( white triangle ) . cell cycle of lsc2 cells that survived after exposure to bis - gma in the penetration model , bis - gma penetrated through 1 mm thick dentin disc . from the calibration curve of known concentrations of bis - gma , the average concentration of penetrated bis - gma was 0.0043 calibration curve of known concentrations of 2 , 2-bis [ 4-(2-hydroxy-3-methacryloxypropoxy ) phenyl ] propane ( bis - gma ) and concentrations of bis - gma that penetrated 1 mm dentin discs . white rectangles indicate known concentrations of control bis - gma for the calibration curve . the viability of lsc2 cells decreased after exposure to bis - gma in a dose - dependent manner ( figure 1a ) . there was no significant difference ( p>0.05 ) in viability between 0 and 0.01 mmol / l of bis - gma , whereas significant decreases of cell viability were observed after being cultured with 0.02 , 0.03 , and 0.04 mmol / l of bis - gma ( p<0.01 ) . the effects of exposure to bis - gma on cell cycle progression of lsc2 cells are shown in figure 1b . exposure to bis - gma reduced the number of the cells in g1 , s , and g2/m phases in a dose - dependent manner , with a concomitant increase of those in the sub - g1 population , implying cell death such as apoptosis . cytotoxic effects of 2 , 2-bis [ 4-(2-hydroxy-3- methacryloxypropoxy ) phenyl ] propane ( bis - gma ) on lsc2 cells . cell cycle of lsc2 cells after exposure to bis - gma morphological changes of lsc2 cells following exposure to bis - gma are shown in figure 2 . the structures of those cells exposed to 0.01 mmol / l of bis - gma were nearly the same as those of the control , whereas the rounding and the detachment were observed in cells exposed to 0.04 mmol / l of bis - gma ( figure 2a ) . fluorescent microscopic analysis revealed that the nuclei of adhesive cells had normal structures , whereas those of cells that had fallen into the medium after exposure to 0.04 mmol / l of bis - gma showed fragmentation , a typical characteristic of apoptotic cells ( figure 2b ) . morphological changes of lsc2 cells promoted by 2 , 2-bis [ 4-(2-hydroxy-3-methacryloxypropoxy ) phenyl ] propane ( bis - gma ) . phase - contrast microphotographs of lsc2 cells after exposure to bis - gma . original magnification , x100 . following exposure to bis - gma , surviving cells attached to the bottom of the well were cultured in normal condition without bis - gma ( figure 3a ) . the proliferation of surviving cells following exposure to 0.01 and 0.02 mmol / l of bis - gma was not different after 24 h of cell culture in normal condition , and then increased at 48 and 96 h , while surviving cells exposed to 0.03 mmol / l of bis - gma showed proliferation at 96 h. in contrast , exposure to 0.04 mmol / l of bis - gma showed great cytotoxicity on the viability of the cells , and proliferation was not recovered during the culture in the normal condition . the recovery of cell - cycle progression in surviving cells exposed to 0.01 , 0.02 , and 0.03 mmol / l of bis - gma was also observed ( figure 3b ) . when bis - gma - exposed cells were cultured in normal condition without bis - gma for 96 h , the cell cycle distribution pattern of bis - gma - insulted cells returned to that prior to exposure to bis - gma . survival of lsc2 cells against 2 , 2-bis [ 4-(2-hydroxy-3-methacryloxypropoxy ) phenyl ] propane ( bis - gma ) . recovery of viability of lsc2 cells that survived after exposure to bis - gma . mmol / l ) ; 0 ( black circle ) , 0.01 ( white circle ) , 0.02 ( white rectangle ) , 0.03 ( black triangle ) , and 0.04 ( white triangle ) . ( b ) . in the penetration model , bis - gma penetrated through 1 mm thick dentin disc . from the calibration curve of known concentrations of bis - gma , calibration curve of known concentrations of 2 , 2-bis [ 4-(2-hydroxy-3-methacryloxypropoxy ) phenyl ] propane ( bis - gma ) and concentrations of bis - gma that penetrated 1 mm dentin discs . white rectangles indicate known concentrations of control bis - gma for the calibration curve . in the present study , it was found that exposure to high concentrations of bis - gma induced an increase in cell number in the sub - g1 population in cell - cycle , and the cell death with typical structures of apoptosis , suggesting that direct exposure to a high concentration of bis - gma induced apoptosis in human dental pulp cells . it was also examined whether lsc2 cells exposed to bis - gma can recover their viability after the removal of bis - gma . we cultured surviving cells after exposure to bis - gma in the normal medium without bis - gma , and found that the cells exposed to 0.01 , 0,02 , and 0.03 mmol / l of bis - gma showed the recovery of the cell proliferation , whereas those exposed to 0.04 mmol / l of bis - gma did not recover their proliferation . in addition , cell - cycle of surviving cells after exposure to 0.01 , 0.02 , and 0.03 mmol / l of bis - gma returned to the same level as the control . these results suggest that even after the exposure to bis - gma , lsc2 cells have an ability to recover their viability when the concentration of bis - gma is low , whereas the cells exposed to high concentration of bis - gma lose such recovery ability . another finding of the present study was that the average concentration of bis - gma that penetrated 1 mm thick dentin was 0.0043 mmol / l , which was lower than the concentration used in the present study . these results are consistent with previous studies , and suggest that an adequate volume of residual dentin thickness would be able to suppress the penetration of a harmful concentration of bis - gma . taken together , the findings of the present study suggest that dental pulp cells have the tolerance against lower concentrations of resin monomers , and that polymerized composite resin on the sufficient thickness of residual dentin may not be harmful to dental pulp . these results produced evidence that bis - gma has cytotoxic effects , though dental pulp exposed to lower concentrations is able to recover their viability when bis - gma is removed .

objectivein the present study , we examined whether 2 , 2-bis [ 4-(2-hydroxy-3methacryloxypropoxy ) phenyl ] propane ( bis - gma ) has effects on lsc2 cells , human dental pulp cell line.material and methodsthe viability , cell cycle , and morphology of lsc2 cells were analyzed after exposure to several different concentrations of bis - gma . the recovery of viability of bis - gma exposed cells was also analyzed in the condition without bis - gma . further , penetration of bis - gma to dentin disc was examined using isocratic high - performance liquid chromatography.resultsthere was a concentration - dependent decrease in cell proliferation and an increase in cell number in the sub - g1 population after exposure to bis - gma . furthermore , the cells showed typical characteristics of apoptotic cells after the exposure to high concentration of bis - gma . in contrast , cells exposed to lower concentrations of bis - gma recovered their viability after being cultured without bis - gma . we also found that bis - gma is capable of penetrating 1-mm - thick dentin discs , though the penetrated concentration was lower than that showing cytotoxicity.conclusionthese results suggest that bis - gma has cytotoxic effects , though dental pulp exposed to lower concentrations is able to recover their viability when bis - gma is removed .

cancer has been a major area of research for several years and information is now available about how tumor cells evade the immune system of the body , and how they effectively get immortal . chemo- and radio - therapies are currently the main cancer therapies for treatment/ control of the localized as well as invasive cancers . however , new treatment strategies are being studied so that a safer and a more effective cancer therapy may be developed . recent studies have shown that cancer cells in case of several cancer types show high epidermal growth factor ( egf ) receptor expression on their surface and this is responsible for metastasis as well as self - induced proliferation . besides metastasis , cancer cells induce angiogenesis to obtain nutrition , and to ensure survival and ability to proliferate . based on these findings , attempts are being made to target egf and/ or vegf ( vascular endothelium growth factor ) receptors in order to reduce the rate of tumor progression , and eventually stop the tumor cells from becoming cancerous . studies have demonstrated reduced cell growth , and degeneration of tumors upon treatment with anti - egf receptor antibodies . however , most studies show positive results in vitro but may fail to be equally effective in vivo . this is possibly due to the interaction between different sub - systems of a body , which play a crucial role in tumor growth and development of cancer but could not be taken into account during in vitro experiments because of the complexity of the system . therefore , it is important to relate the in vitro experiments to the in vivo situations to be able to predict the efficacy of a treatment strategy . various models have been developed to study cancer development and tumor growth [ 3 - 6 ] . some of these models have studied the tumor growth before it becomes invasive or during the early stages of invasion ; others analyze the stages in tumor development . several models are based on the spheroid model or other related models to analyze the dynamics of antibody - drug therapy . these models consider the diffusion of an antibody - drug into the tumor mass followed by the binding of the antibody to the tumor cell , and subsequent internalization of the antibody - drug complex . however , these models study the dynamics of an individual tumor without considering the effect of other tumors growing simultaneously in the body , i.e. the dynamics of the process as a whole is not considered . thus , the previously developed models give a microscopic picture , which may not be useful in studying the efficacy and design of cancer therapies as the therapies act on a macroscopic scale . the kinetic modeling may be expected to be useful in the identification of the critical parameters that should be targetted in therapy . in addition to that , the requirement for a combination therapy may also be revealed from the kinetic modeling of the complete system . a kinetic model based on interactions between different compartments of a body has been developed . thus , the kinetics of the process has been studied rather than the kinetics of a single entity . the model has thereafter been analyzed using a set of representative parameter values to demonstrate the utility and the applicability of this model . we have also shown how one can study and compare various treatment strategies using the developed kinetic model . cancer starts with a tumor growth , which may be followed by metastasis leading to spreading of the cancerous cells in various regions of the body . these metastasizing cells are carried away to the different regions through bloodstream where they may attach to a tissue and proliferate . however , the tumor cells entering the bloodstream may also be cleared through the body by the immune system or any drug action , or death by apoptosis due to the requirement of the cells to attach to the tissue at a new site and adapt to the new environment . thus , only a very small fraction of cells that escape from a primary tumor survive and initiate a new tumor . cell death and unsuccessful cell attachment are thus taken into account as plasma clearance in the model . tranformed cells are exchanged between a primary tumor and the plasma , which have been treated as two interacting but independent compartments . the cells present in plasma may either be cleared or may attach to another tissue and start growing into a new solid tumor . in order to model the cell growth within solid tumors , population dynamics model has been used , where t0 is the equilibrium tumor cell concentration in the tumor and r is the specific growth rate . however , in physiological situation , t0 may be variable as the tumor cells are able to procure more nutrition by carrying out angiogenesis ( assuming no spatial limitations for tumor growth ) . thus , dttum / dt = rttum(1-ttum / t0 ) - kf1ttum + kr1tplas ... ( 1 ) dtplas / dt = kf1ttum - kr1 tplas + n. ( - kf2tplas + kr2tnew ) - ctplas ... ( 2 ) dtnew / dt = rtnew(1 - tnew / t0 ) + kf2tplas - kr2tnew ... ( 3 ) ttum = cell concentration of the original tumor tplas = cancer cell concentration in the plasma tnew = cell concentration of new and developing tumor n = number of new tumors being developed simultaneously and all kf 's and kr 's are the corresponding rate constants of the steps shown in figure 1 . all equations take into account the cell growth , loss into plasma due to metastasis , and attachment of the cells from plasma to the tissue . in the model , , , and these factors represent the effect a specific cancer therapy has on the different parameters . in the absence of any therapy , these factors are all equal to unity as the values are relative to the case when no therapy is being applied . moreover , cells are cleared out by means of immune response and apoptosis due to the inability of cells to attach to the new tissue . for example , by choosing the parameter values for the developing tumor , we can consider the initial stages when it does not metastasize and thereafter after a certain cell concentration , the parameter values may be changed to incorporate metastasis . in fact , once the new tumor has sufficient cell concentration , it can itself be considered as a source tumor and the cancer cell growth reanalyzed . on the other hand , this assumption may be valid if the cell concentration limit for the cell to start metastasizing is orders of magnitude less than the steady state concentration it attains . thus , this kinetic model is of a very general nature and a simplified form of the physiological situation , as there exists a far greater interaction between different compartments than what has been modeled . to study the dynamics of cancer growth , we have used certain representative values for the model parameters as tabulated in table 1 . all simulations have been carried out using mathematica 4 ( wolfram research ) . in the graphs this is important as during drug treatment or any other therapy , it is useful to know the concentration profile as a function of time such that different therapies my be compared . representative parameter values used for simulating cancer cell growth . for the simulation of the cancer growth , the drug efficacy parameters have all been taken as one in the absence of any cancer therapy , as mentioned earlier . however , depending on the therapy , different values of , , and have been taken . drug efficacy parameters for simulating different cancer therapies the parameter values are mostly based on some experimental observations for various cancer types . we have taken the potential doubling time ( tpot ) for the ( human ) tumor mass under consideration as ten days , though some tumors may have a slower growth rate . it is known that metastasis reduces the effective doubling time such that the observed doubling time may be few weeks to few months . incorporating this information , we obtain the rate constant for metastasis for our model ( kf1 and kr2 ) . for the analysis , cells are unable to attach to the endothelial tissue easily , the rate constants for attachment of cells to the existing tumor or to a new tumor has been taken as nearly 1% of the rate of metastasis . based on a metastasis experiment done in mouse model , the initial conditions for the simulations as well as the rate of plasma clearance have been chosen . in the experiments that have been previously carried out , a primary tumor was developed , which has been assumed to have a cell concentration of 3 10 cells/ ml based on in vitro studies as well as the number of cells implanted in the mouse model . thereafter , 2 10 tumor cells were injected into vein , and metastasis of the tumor cells was observed . assuming that tumor cells injected into the plasma at the beginning of the experiment correspond to the steady state cell concentration in the plasma , the rate constant for plasma clearance has been obtained . this may be valid due to the fact that the plasma volume is large , tumors may be localized or may not invade several tissues , and eventually steady state is reached so that the cells released by existing tumor either result in new tumors or are cleared out from the body . the variation in cell concentrations in the existing tumor , and the developing tumor in the absence of any cancer therapy has been shown in figure 2(a ) . different therapies considered are reduction in cell growth as well as metastasis by egf receptor inhibitors , and reduction in specific growth rate and increase in clearance rate by activating immune response against cancer cells using therapies like adoptive therapy [ 15 - 22 ] . figures 2(b ) and 2(c ) depict the cell growth in the presence of the two drug therapies- egfr inhibitors and adoptive therapy respectively . growth of existing tumor and new tumor : a ) in the absence of any therapy ; b ) egfr inhibitor therapy ; c ) adoptive therapy ; and d ) antibody - drug therapy . a single the same analysis may be applied to all the existing tumors at any given time to study the kinetics of cancer progression in the absence and the presence of various cancer therapies . cell growth in the absence of any cancer therapy has been depicted in figure 2(a ) . the existing tumor cells grow and attains a steady - state concentration . at the same time , the tumor cells escape from the primary tumor and form a secondary tumor at a new site . eventually , both the primary as well as the secondary tumors reach the same steady state cell concentration . thereafter , the primary and the secondary tumors can result in formation of new tumors , and thus the cancer spreads to various regions of the body . in case of egfr inhibitor therapy , the effective value of specific growth rate ( r ) and rate of metastasis ( kf1 and kr2 ) decrease , and this subsequently results in slower tumor growth of the existing as well as the developing tumors ( figure 2(b ) ) . however , unless the inhibitor efficacy is sufficient enough to decrease the rate of cell growth to less than the rate of cell clearance from plasma , cancer cells can not be eradicated from the body and the effect is merely to delay the cell growth ( which in this case is by a factor of ~4 ) . this extension may be helpful if there is any subsequent and/ or parallel treatment . the reduction in the cell release rate may in fact result in higher steady state cell concentration in the primary tumor thereby , partially or totally overcoming the effect of the reduction in the cell release rate constant . however , if the egfr inhibitor therapy is combined with other treatment(s ) that can further reduce the rate of cell growth , then tumor degeneration may be observed . in contrast to anti - egfr treatment , the model predicts that the adoptive therapy may lead to a nearly complete elimination of new as well as existing tumors . the parameters affected by adoptive therapy are r and c , i.e. rate of cell growth decreases and rate of plasma clearance of cells increases , as the immune response destroys the tumor cells in the tumor masses as well as in the plasma . as seen in figure 2(c ) , a small reduction in specific rate of cell growth and an increase in plasma clearance can result in complete removal of cancer cells from existing tumors in a matter of few months and it also ensures that the new tumor masses are not able to reach the high concentrations . these new tumor masses also degenerate progressively and thus , the cancerous cells are completely eliminated in a few months . since , this form of treatment is relatively fast compared to any combination therapy involving egfr inhibitor therapy , this may even be used for cancers in advanced stages . however , activation of immune response against cancer cells itself is a big challenge . this is so because cancer cells are able to escape immune surveillance by shedding the antigen peptides on their surfaces , and by releasing blocking factors , which can neutralize the nk cells . in this direction , various strategies are being explored like adoptive therapy , dendritic cell vaccine and enhancement of nk cell activity through il-2 . one way to overcome this limitation is to activate an immune response against the blocking factors themselves . in that way , all the blocking factors may be neutralized so that the tnf/ il-1 or nk can destroy the cancer cells in the tumor as well as in the plasma . after activating the immune response against the blocking factors , nk cell activity may be enhanced by priming them with il-2 depending on the state of cancer and the nk cell activity . lastly , therapies like antibody therapy with antibody - drug targeted against the cancer cells , and anti - angiogenesis therapy decrease the rate of cell growth keeping rate constant for cell release from the tumor unchanged . in that case , this may be explained by the fact that the balance between cell growth and cell clearance is disturbed and this may result in smaller tumors and/or eradication of tumor masses . the degeneration of tumor mass with antibody therapy assumes that the antibody is accessible to all the tumor cells , which is not the case in physiological situation . in this way , we observe that cancer growth can be modeled by considering the cell growth and metastasis as interaction between various compartments . using this model , various cancer therapies may be compared for their efficacies , and may be focussed to result in a better and an effective therapy . however , we would like to point out that these results are based on the parameter values selected for the analysis and therefore , the true efficacy of a therapy can be realized only after carrying out analysis with the corresponding parameter values . in addition , the results depend on the efficacy of a therapy , i.e. the values of the drug efficacy factors determine the efficacy of a therapy . the actual effect of any therapy will depend on the drug efficacy parameters but from this analysis , we infer that the reduction in specific growth rate of cells is primary , and the rate of cell release from the tumor masses should not be decreased substantially for the success of the treatment strategy . thus , these results should not be taken as a means to accept or reject a therapy , but should rather be used for improving and designing the cancer therapies . cancer growth has been modeled as the growth as an existing tumor and metastasis resulting in interaction between the existing tumor and plasma viewed as two independent but interacting compartments . the cells present in plasma , thereafter attach to another tissue and grow into a new tumor mass . the new tumor mass increases in size due to cell growth and cell uptake from plasma . however , once the cell concentration reaches a steady state concentration , this tumor mass also becomes a source for new tumor masses , i.e. it forms another stage for cancer growth . further , two different cancer therapies- egfr inhibitor therapy and adoptive therapy- were analyzed using this kinetic model . the results point to the importance of targeting the specific growth rate as well as the plasma clearance rate in the system . thus , this model helps study the efficacy of the cancer treatment therapies , and also helps determine the critical factors , which may be targeted . however , these results are strongly dependent on the parameter values , which should be appropriately taken and analyzed for specific case ; but this model is useful in focussing and improving a cancer therapy in order to make it more effective . sj would also like to thank alireza khademhosseini for critical comments during the preparation of the manuscript .

a kinetic model has been developed to study cancer growth . cancer growth has been considered as interaction between various independent but interacting compartments . the model considers cell growth and metastasis resulting in the formation of new tumor masses . using certain representative parameter values , cell growth has been modeled in the absence and the presence of various cancer therapies . based on this analysis , the critical parameters involved in cancer development have been identified . this model may thus be useful in studying and designing a cancer therapy using the data obtained from specific in vitro experiments .

facioscapulohumeral muscular dystrophy ( fshd ) is a common inherited muscular dystrophy presented clinically with slowly progressive weakness and wasting of facial and limb muscles and rare bulbar muscle involvement . we present herein a known case of fshd presented with recent onset of severe bulbar symptoms and was found to have myasthenia gravis ( mg ) based on electrodiagnostic study , elevated level of acetylcholine receptor antibody and dramatic improvement with choline esterase inhibitor agents . clinical presentation , electrodiagnostic and pathologic findings of this patient are described . a 70-year - old man presented to our department with complaint of 15-day history of progressive difficulty in chewing and dysartheria . he had a 50-year history of slowly progressive asymmetrical weakness of proximal upper limb muscles . examinations revealed reduction in forces of bilateral orbicularis oculi muscles , weakness and wasting of bilateral triceps muscles especially on right side and bilateral winging of scapula more prominent on right side . the legs and pelvic girdle muscles had normal forces . edrophonium test was performed and dysartheria and chewing difficulty showed dramatic improvement but had no effect on limb weakness . muscle biopsy showed myopathic changes with invariability in muscle fiber size , intramuscular infiltration of chronic inflammatory cells , mostly lymphocytes , few hyaline fibers and prominent fat infiltration . our patient 's bulbar symptoms showed dramatic improvement following administration of choline - esterase inhibitor agents . a 70-year - old man presented to our department with complaint of 15-day history of progressive difficulty in chewing and dysartheria . he had a 50-year history of slowly progressive asymmetrical weakness of proximal upper limb muscles . examinations revealed reduction in forces of bilateral orbicularis oculi muscles , weakness and wasting of bilateral triceps muscles especially on right side and bilateral winging of scapula more prominent on right side . the legs and pelvic girdle muscles had normal forces . edrophonium test was performed and dysartheria and chewing difficulty showed dramatic improvement but had no effect on limb weakness . muscle biopsy showed myopathic changes with invariability in muscle fiber size , intramuscular infiltration of chronic inflammatory cells , mostly lymphocytes , few hyaline fibers and prominent fat infiltration . our patient 's bulbar symptoms showed dramatic improvement following administration of choline - esterase inhibitor agents . it presents clinically with slowly progressive weakness and wasting of facial and shoulder girdle muscles and sometimes involvement of lower extremities . the clinical severity is wide ranging from asymptomatic individuals to wheel - chair dependent patients . any unusual changes in course of disease or development of unusual symptoms should raise the possibility of concomitant disease . sansone et al . reported a 69-year - old known case of fshd who presented with sudden deterioration of limb weakness and development of bulbar symptoms and was found to have mg based on repetitive nerve stimulation , elevated level of acetylcholine receptor - binding antibody and dramatic improvement following immunomodulator administration . reported a 50-year - old man with a 35 year history of fshd who presented with acute progressive weakness of lower extremities three weeks prior to admission . the patient was found to have mg based on decrement response on repetitive nerve stimulation , elevated level of acetylcholine receptor - binding antibody and improvement after thymectomy and administration of corticosteroid . in another report , mcgonigal et al . presented a 56-year old newly diagnosed myasthenic patient who found to have 40-year history of progressive foot drop and although our case is a rare coexistence of fshd and mg , low prevalence of both diseases , may raise the possibility of the presence of other etiologies . theoretically , it is related to breaking of immune tolerance to achrs as a result of muscle fiber degeneration . patients with genetic myopathies may occasionally develop antibodies to achr . while these antibodies may not have pathogenic effects , their production is likely to be a consequence of sensitization to achr secondary to muscle fiber damage , rather than through an immune process in thymus . muscle histopathological examination in some cases of fshd shows inflammatory changes , disproportionate to muscle fiber necrosis but the presence of mononuclear infiltration does not affect disease progression and the patients do not benefit from prednisone treatment . to the best of our knowledge , there are limited reports of concomitant occurrence of mg and fshd in literatures . although the association of mg with fshd in our patient could be an incidental finding , it may raise the possibility of innate immune response , i.e. autoinflammation in development of achr antibodies in genetic myopathies or it may suggest immune mechanisms in pathogenesis of fshd . moreover , our experience may warrant concomitant neuromuscular disorder in patients with unusual symptoms of fshd .

facioscapulohumeral muscular dystrophy ( fshd ) is a common inherited muscular dystrophy presented clinically with slowly progressive weakness and wasting of facial and limb muscles and rare bulbar muscle involvement . we present herein a 70-year - old man who was a known case of fshd with complaint of 15-day history of progressive difficulty in chewing and dysarthria and was found to have myasthenia gravis . related literatures have been also reviewed .

the south american sea lions ( otaria flavescens , carnivora : otariidae ) are common pinnipeds living along the eastern and western coasts of south america and are generally found in peru , chile , argentina , and south brazil [ 16 ] . along the chilean coastal shores , more than 200 colonies of free - ranging sea lions a vast amount of data on feeding ecology , reproduction , life history parameters , and population dynamics of these otariid species was published [ 716 ] . several studies have also addressed aspects of the helminth fauna of south american sea lions , comprising single species records , taxonomy , and population studies of some parasitoses [ 5 , 1422 ] . however , very little is known on protozoan parasite infections of these free - ranging marine mammals . although their natural habitat is the marine environment , several pinniped and cetacean species are found in rivers containing fresh water . as such , a stable population of south american sea lions has been established within the city of valdivia , chile , resulting in permanent colonization for 20 years now . these animals have adapted extremely well not only to the fresh water of the river but also to human activities in the river calle - calle , such as regular ship- and boat - trafficking , rowing , kayaking , and sealing activities . sea lion colony is allocated approximately 7 km upstream from the ocean shore and animals mainly feed on fish captured by themselves in the river ( mainly carps , trouts , and salmons ) or on remains of the local fish market . this unusual urban colony of south american sea lions consists of more than 72 individuals and is exclusively composed of males . the age of these animals varies from 2 to 15 years but some animals might be even older . given that o. flavescens need to rest after swimming and diving activities , the sea lions in valdivia utilize river floats , riverside piers , and footways along the river promenade as recreation areas with all of them being allocated in close proximity to inhabitants , tourists , domestic pets , or the local fish market . since some of these animals behave rather aggressive towards humans , animals , or even vehicles and additionally tend to expand their territory into the city center , the local city authorities have established a sea lion task force which should prevent the animals from harming humans and withhold them to premises alongside the river shore . however , given that these animals nowadays represent a tourist attraction , direct contacts of humans with these animals or their faeces as well as faecal contamination of the river water or the terrestrial environment can not be avoided . the present study therefore aimed to identify the actual gastrointestinal fauna in these free - ranging sea lions within their natural habitat in the river of the city of valdivia , chile , and to gain some insights in their potential zoonotic impact on public health issues . south american sea lions ( o. flavescens ) were studied along the shores of the river calle - calle of the city of valdivia , chile . the study area encompassed 3 km and comprised river floats , riverside piers , footways along the river promenade , and the local fish market with all of them being allocated in close proximity of humans and domestic pets ( figures 1(a ) , 1(b ) , 1(c ) , and 1(d ) ) . whenever defecation occurred , scat samples were immediately collected and transferred into 2 ml plastic tubes ( eppendorf ) . parasitological analyses were conducted at the institute of parasitology , justus liebig university giessen , germany . coproscopical analyses were performed by using the standard sodium acetate acetic acid formalin ( saf ) technique . the saf technique was used for the detection of parasite eggs , cysts , sporocysts , and oocysts within faecal material in marine mammals as described elsewhere . additionally , a carbol fuchsin - stained faecal smear ( cfs ) was carried out for the detection of cryptosporidium oocysts . moreover , coproantigen elisas ( prospect , oxoid ) were performed for the detection of cryptosporidium and giardia antigens in faecal samples . the parasitological identification of eggs and cysts was based on morphological criteria referring to previous reports [ 24 , 26 , 27 ] . all sampling procedures were conducted in accordance with institutional ethic commission of university austral of chile and the current chilean animal law . giardia - positive sea lion samples were further analyzed for the presence of g. intestinalis dna by conventional and nested pcr detecting the beta - giardin gene ( assemblage c ) . genomic dna was extracted from sea lion faecal material using the dna extraction stool kit ( qiagen ) according to the mammalian faecal protocol . briefly , 1 ml of ethanol - fixed faeces was lysed in asl buffer containing 30 glass beads ( 4 mm diameter ) , under permanent stirring conditions . the dna was thereafter purified using an anion exchange column ( qiagen ) and eluted in 100 l of distilled water . for the conventional g. intestinalis - pcr , the following specific oligonucleotide sequences were used : the forward oligonucleotide -giardin g7f : 5-aagcccgacgacctcacccgcagtcg-3 and the reverse oligonucleotide -giardin g759r : 5-gaggccgccctggatcttcgagacgac-3 . the pcr was performed in a total volume of 25 l containing 5 l faecal dna sample , 5 l faecal dna ( 1 : 100 ) , 1 l g7f oligonucleotides ( 10 m ) , 1 l g759r oligonucleotides ( 10 m ) , dntps 0.5 l ( 10 m ) , 0.5 l taq - polymerase ( 1 u/l ; peqlab ) , and 14.5 l h2o . the following thermocycle profiles were used : 95c for 5 min , 35 cycles at 95c for 30 s , 65c for 45 s , and 72c for 1 min and 30 s followed by a final extension step at 72c for 5 min and a final hold at 20c . pcr amplificates were visualized in gelred - stained 2% agarose gels ( biotium incorporation ) . in addition , a g. intestinalis - nested pcr was performed . for the nested pcr the following forward oligonucleotide sequences of giarf were used : 5-gaacgagatcgaggtccg-3 and reverse oligonucleotide sequence of giarr : 5-ctcgacgagcttcgttgtt-3 . the following thermocycle profiles for the nested pcr were used : 95c for 5 min , 35 cycles at 95c for 30 s , 50c for 40 s , and 72c for 1 min and 30 s followed by a final extension step at 72c for 5 min . pcr amplificates were visualized using gelred - stained 2% agarose gels as described above ( biotium incorporation ) . parasitological analyses of faecal samples of south american sea lions revealed 8 different protozoan ( 4 ) and metazoan ( 4 ) taxa . the metazoan parasites consisted of trematodes ( one species ) , cestodes ( one species ) , and nematodes ( two species ) . a complete list of the parasite stages and their prevalence is given in table 1 . the most prevalent metazoan parasites found in this urban sea lion colony were anisakidae gen . the most prevalent protozoan parasites were cryptosporidium ( 10% ) , giardia , and isospora showing the same prevalence ( 5.3% ) . balantidium infections were detected at lower prevalence ( 2.5% , table 1 ) . within the metazoan endoparasites , the nematodes were the most rich in species ( two species ) followed by cestodes and trematodes with one species each . referring to parasite genus level these parasitological findings included four new host records ( cryptosporidium , giardia , balantidium , and otostrongylus ) for o. flavescens . to our best knowledge , the genus balantidium had only been described in fin whales ( balaenoptera physalus ) from the north atlantic . all other protozoan parasites have already been reported for other marine mammals [ 4 , 5 , 17 , 19 , 20 , 24 , 30 ] . some of the protozoan ( 3 ) and metazoan ( 2 ) endoparasite genera detected in sea lions bear an anthropozoonotic potential , such as cryptosporidium , giardia , balantidium , diphyllobothriidae gen . although sea lion faecal samples were immediately fixed in 70% ethanol after collection in the field in order to avoid dna degradation , this goal was not successfully achieved . thus no adequate giardia dna was possible to be extracted for further detailed molecular identification . common collection methods for analyses of gastrointestinal parasites of wild sea lions generally rely on sections of accidentally stranded animals or on dead animals obtained from marine zoos [ 5 , 14 ] . several studies are restricted to the helminth parasite fauna of south american sea lions and include several single species records [ 4 , 5 , 15 , 16 , 1822 , 31 ] . conversely , by obtaining fresh faecal samples from resting sea lions , this record reveals unique insights into the actual gastrointestinal parasite fauna of free - ranging sea lions within their natural habitat . in the present survey , 8 different parasite taxa were detected in individual sea lion faecal samples covering a respectable range of parasitic taxa . the parasitological diagnosis based on morphological criteria revealed as quite a challenge since very little data on parasitic eggs , larvae , cysts and oocysts for sea lions are available in literature . thus , the photo galleries provided here might supply a supportive tool for future parasitological research activities on sea lions and other marine mammals since many of the parasites described here infect a wide range of marine mammals , such as sea otters , seals , sea elephants , baleen , and toothed cetaceans . the most prevalent parasitic stages found in the current study were eggs of anisakidae gen . sp . however , owing to undistinguishable egg morphologies , characterization on species level was not possible . based on parasite frequencies , these eggs most probably belong to the genera contracaecum , pseudoterranova , or anisakis since these parasite species appear to be quite common in south american sea lions [ 5 , 31 ] . thus , south american sea lions are known as definitive hosts for the zoonotic nematodes anisakis spp . , contracaecum ogmorhini , and pseudoterranova cattani [ 5 , 31 ] . ascarids parasitize either freely in the stomach or firmly attached , often as clusters , to the gastric mucosa . mucosal penetration via larvae and adults can cause severe ulcers , gastritis , and perforation . moreover , allergic reactions against epitopes of anisakis simplex major allergen ( ani s1 ) have been reported to occur in humans after the reexposure to these parasites [ 35 , 36 ] . consistent to our findings , at least three different diphyllobothriid cestodes have previously been recorded in chile , two freshwater species ( diphyllobothrium latum and d. dendriticum ) and one marine species ( i.e. , adenocephalus pacificus ( diphyllobothrium pacificum ) ) [ 37 , 38 ] and also in the intestine of stranded sea lions from patagonia , but at a higher prevalence ( 26.8% ) . diphyllobothriasis in sea lions generally is innocuous , but debilitation or even death of parasitized hosts might result in cases of high parasitic burdens . in addition , diphyllobothriasis also represents an important fish - borne zoonosis worldwide [ 3943 ] . trematode infections in the pancreas and liver occur in almost all marine mammals by members of the genera campula , zalophotrema , oschmarinella , and orthosplanchus . additionally , also the genera apophallus , ascocotyle , ogmogaster , and pocitrema have been reported as intestinal parasites of otariids [ 5 , 44 ] . these may induce necrosis of the parenchymal tissue , chronic fibrotic hepatitis , enteritis , and even meningoencephalitis by aberrant trematode migrations . furthermore , the trematode genera pricetrema and nanophyetes have been reported to parasitize sea lions in the northern hemisphere . whilst pricetrema resides in the liver , nanophyetes infects the small intestine . infections with the crenosomatid nematode otostrongylus in pinnipeds are generally associated with respiratory clinical manifestations , primarily in young animals . interestingly , some sea lions of the current study showed strong coughing episodes , thereby expelling vast amount of mucus . based on the current findings this might indicate the presence of clinical otostrongylosis within the valdivian colony . the parasitological findings of the study also included four new parasite host reports for o. flavescens , namely , cryptosporidium , giardia , balantidium , and otostrongylus , thereby providing a broader insight into the spectrum of parasitoses of this marine species . in addition , the protozoan parasites ( i.e. , cryptosporidium , giardia , and balantidium ) clearly bear zoonotic potential and are considered as typical water - borne / food - borne diseases . in consequence , the urban sea lion colony in valdivia may function as relevant reservoir for these protozoans since the animals reside at the shore of the river and in close proximity to humans , pet animals , and especially in direct contact to the products of local fish market . within the genus balantidium , b. coli is the only species of trichostome ciliates nowadays considered as pathogenic for mammals [ 48 , 49 ] . consistent with these findings , balatidium infections in free - ranging fin whales ( balaenoptera physalus ) from the north atlantic ocean were recently diagnosed , indicating that this terrestrial disease is circulating in the marine environment . more importantly , b. coli infections have been demonstrated in humans and pigs in chile [ 50 , 51 ] . as seen for balantidiasis , giardiasis and cryptosporidiosis have an almost cosmopolitan distribution . cryptosporidium and giardia are two common aethiological parasites of infectious enteritis in humans and animals [ 52 , 53 ] . these enteric protozoans are usually transmitted by the faecal - oral route , following the ingestion of infective stages ( oocysts or cysts ) . moreover , marine mammals are well known as final hosts of giardia and cryptosporidium [ 24 , 5458 ] . keeping in mind that the urban sea lion colony is close to populated riversides and that some touristic activities , such as photos with sea lions or kayaking with sea lions , are becoming more popular , sea lions may become infected by human excretions or vice versa . as seen for giardiasis , cryptosporidium infections can cause severe diarrhoea in terrestrial mammals ; nonetheless , very little is known on the pathogenesis of cryptosporidiosis within the marine ecosystems [ 24 , 55 ] . an essential component of the control of these diseases , from a public health perspective , is a better understanding of the sources and routes of transmission in different geographical regions . unfortunately , the current knowledge and understanding of the ecto- and endoparasite fauna ( especially protozoan species ) in free - ranging sea lions are still very scarce . although some parasitoses , such as hookworm ( e.g. , uncinaria hamiltoni ) or ascarid ( contracaecum ogmorhini , pseudoterranova cattani , and anisakis spp . ) , infections are discussed as pathogenic species for sea lions , the total parasite fauna of marine vertebrates has unfortunately not obtained sufficient attention , so far [ 24 , 59 ] . in conclusion , this study adds some new anthropozoonotic parasite records to free - ranging sea lions and calls for more integrated research to avoid the exposure of humans and domestic animals with these parasites . in particular , regular monitoring programs should be established by local authorities for public health issues and for sea lion populations living in close proximity to human beings .

the present study represents the first report on the gastrointestinal endoparasite fauna of a free - ranging urban colony of south american sea lions ( otaria flavescens ) living within the city of valdivia , chile . a total of 40 individual faecal samples of south american sea lions were collected during the year 2012 within their natural habitat along the river calle - calle and in the local fish market of valdivia . coprological analyses applying sodium acetate acetic formalin methanol ( saf ) technique , carbol fuchsin - stained faecal smears and giardia / cryptosporidium coproantigen elisas , revealed infections with 8 different parasites belonging to protozoan and metazoan taxa with some of them bearing anthropozoonotic potential . thus , five of these parasites were zoonotic ( diphyllobothriidae gen . sp . , anisakidae gen . sp . , giardia , cryptosporidium , and balantidium ) . overall , these parasitological findings included four new parasite records for otaria flavescens , that is , giardia , cryptosporidium , balantidium , and otostrongylus . the current data serve as a baseline for future monitoring studies on anthropozoonotic parasites circulating in these marine mammals and their potential impact on public health .

gastroduodenal artery ( gda ) aneurysm , a type of visceral aneurysm , is extremely rare . ct angiography is the gold standard for its management , which serves as both a diagnostic and a therapeutic modality . alternatively , gda aneurysm can be managed either by surgical vessel ligation and sac excision or stenting . here , we present a case of a 20 year old male who suffered blunt abdominal trauma . on initial evaluation using abdominal contrast enhanced computed tomography ( cect ) , the patient showed pancreatic contusion and retroduodenal air with suspected duodenal injury . emergency laparotomy revealed duodenal wall and pancreatic head contusion with mild hemoperitoneum and no evidence of perforation . but on postoperative day 5 , the patient developed upper gastrointestinal hemorrhage which was further revealed to be induced by gda aneurysm . a 20 years male presented to emergency department with alleged history of road traffic injury . on initial assessment as per atls protocol , primary survey was normal except for positive fast and tenderness in the right hypochondrium with mild guarding . after initial resuscitation , the patient was shifted for cect of the abdomen and pelvis , which revealed multiple contusions of the pancreatic head and retroduodenal air with suspected duodenal injury . preoperatively the patient had mild hemoperitoneum with contusions in the head of pancreas and no duodenal perforation . the patient was shifted to icu for monitoring in the postoperative period , where he had a single episode of hematemesis and melena on the postoperative day 3 . he was managed with fluids and medical treatment for upper gastrointestinal hemorrhage , and remained hemodynamically stable . on day 5 , the patient had a similar episode of hematemesis and melena again with a sudden drop in hemoglobin from 114 g / l to 88 g / l . however , the patient remained hemodynamically stable and all the other blood parameters were within normal limits . abdominal mri was done to evaluate any evolution in pancreatic and duodenal injury . to our surprise , mri revealed gda pseudoaneurysm with free passage of contrast into the duodenum ( fig . 1 ) . superior mesenteric artery ( sma ) was then scanned to rule out collaterals supplying the aneurysm , which showed sma in normal territory and no collaterals to the aneurysm ( fig . 2 ) . permanent angioembolization was done by coils across the possible origin of aneurysm in main hepatic artery . after embolization there was no distal flow in the hepatic artery and gda ( fig . the postoperative period was uneventful and the patient was discharged 4 days after the angioembolization . till the six months of follow - up the patient did not have any episode of hemetemesis or melena . most of the gda aneurysms seen are associated with pancreatitis , ethanol abuse , cholecystectomy , various congenital disorders like marfan 's syndrome , polyarteritis nodosa , fibromuscular dysplasia and liver cirrhosis.1 , 2 , 3 gda aneurysm in setting of trauma has not been described yet to the best of our knowledge . the most common presentation of gda aneurysm is gastrointestinal hemorrhage ( normally into duodenum ) secondary to rupture ( 52% ) , followed by abdominal pain ( 46% ) . rarer presentations include retroperitoneal and intraperitoneal bleeding , occasionally leading to obstructive symptoms like gastric outlet obstruction , bleeding into pancreatic duct or common bile duct and features of obstructive jaundice . angiography is the gold standard diagnostic test as it can serve as a therapeutic modality as well , allowing therapeutic intervention with highest sensitivity ( 100% ) . magnetic resonance angiography has also been reported to be as effective as visceral angiography in the diagnosis of abdominal vascular lesions . tradiotinal surgical intervention includes resection or ligation of aneurysm or bypass surgery . with the advancement in intervention radiology hur et al in a study of 16 patients with gda aneurysms following pancreaticoduodenostomy concluded endovascular trapping of the hepatic artery as a safe and effective procedure . thus , the endovascular option being a less aggressive approach offers a good therapeutic alternative to surgical intervention , especially in patients with multiple comorbidities . visceral ischemia and end organ infarction , end - organ thrombosis , and late - term vessel recanalization are the major complications of angioembolization . others include coil / stent migration , intra - procedural aneurysm dissection , or rupture , access artery pseudoaneurysms and contrast - induced nephropathy . gda aneurysms have an up to 40% mortality rate following rupture , which depends on the site and severity of bleeding . highest mortality is seen following rupture into the duodenum ( 21% ) . hence gda aneurysms should be managed promptly once identified , before any catastrophe occurs . in conclusion , though gda aneurysm is usually seen after chronic pancreatitis and atherosclerotic disease , it should also be considered as a differential in post - traumatic upper gastrointestinal bleeding , especially when there is pancreaticoduodenal injury . ct angiography is the gold standard but abdominal mri is also effective in the diagnosis of gda aneurysm , as in our case .

aneurysm of gastroduodenal artery ( gda ) is rare . most reported cases are due to pancreatitis and atherosclerosis ; however , those following pancreatic trauma have not been reported . we encountered gda aneurysm in a patient of blunt abdominal trauma , who had pancreatic contusion and retroduodenal air on contrast enhanced computed tomography of abdomen . emergency laparotomy for suspected duodenal injury revealed duodenal wall and pancreatic head contusion , mild hemoperitoneum and no evidence of duodenal perforation . in the postoperative period , the patient developed upper gastrointestinal hemorrhage on day 5 . repeat imaging revealed gda aneurysm , which was managed successfully by angioembolization . this case highlights , one , delayed presentation of gda aneurysm after blunt pancreatic trauma and two , its successful management using endovascular technique .

neisseria gonorrheae ( ng ) and neisseria meningitides are gram - negative bacterial pathogens responsible for gonorrhea and meningococcal meningitis , respectively . for these bacteria , phagocytosis , cellular invasion , is induced by the binding of opacity - associated ( opa ) proteins to host receptors . opa proteins are eight - stranded -barrel integral outer membrane proteins with four extracellular loops ( figure 1a ) . high sequence diversity is observed in regions of the extracellular loops of opa variants ( table s1 , supporting information ) , which is predominantly a result of recombination events between genes of the same isolate ( 70% ) and import of genes from other isolates ( 16% ) . the three variable regions within the extracellular loops , hypervariable 1 and 2 ( hv1 and hv2 ) and the semivariable ( sv ) regions , engage host receptors to induce phagocytosis and determine the specific host receptors engaged . the regions vary in length and do not comprise the entire extracellular loop : sv is 310 amino acids , hv1 is 2431 amino acids , and hv2 is 4551 amino acids . there are 26 sv , 96 hv1 , and 127 hv2 different sequences in the 338 distinct opa alleles sequenced ( http://www.neisseria.org ) . this sequence diversity likely plays a beneficial role in helping neisseria to evade host immune responses ; however , it poses a challenge in that highly varied loop sequences must engage a common set of receptors to mediate cellular invasion . the determinants of opa receptor interactions are of the utmost importance for understanding neisserial pathogenesis and the innate immune response . opa proteins also provide a means of foreign body cellular entry through specific human receptors that can be exploited synthetically for pharmaceutical and technological purposes . opahs bind to heparansulfate proteoglycans ( hspgs ) directly and indirectly to integrin receptors via an hspg - mediated interaction . the more abundant class , opacea , bind to the nonglycosylated face of the carcinoembryonic antigen - related cellular adhesion molecule ( ceacam ) ig n - domain . although all ceacam receptors contain this domain , opa proteins only bind to ceacam1 , 3 , 5 , and 6 , and most selectively bind to only a subset . the ceacam n - domain residues that interact with opacea have been identified : y34 and i91 are essential for all opacea interactions , and an additional seven nearby residues are implicated in binding depending on the particular opacea . the specificity - determining residues on opacea are predominantly in the hv1 and 2 regions , and the hv sequences are concomitant : chimeric opa proteins with an hv1 and hv2 region from two opa proteins that bind the same receptor do not bind . toward understanding the molecular recognition required to gain entry into human cells , we report the structure of opa60 , which binds ceacam1 , 3 , 5 , and 6 . structure determination of membrane proteins is challenging and even more so for proteins that have large portions of both soluble and membrane - embedded regions . thus , opa proteins presented some methodological obstacles in both nmr resonance assignments and structure calculation and refinement . to overcome these obstacles , we employed a hybrid method that used the restraints determined with solution nuclear magnetic resonance ( nmr ) spectroscopy in detergent micelles in conjunction with molecular dynamics ( md ) simulations in a lipid bilayer . this approach preserved the structural features that were well - determined spectroscopically but employed a more physical sampling method ( molecular dynamics versus simulated annealing ) and more detailed treatment of solvation and electrostatics to better define regions that either are flexible or remained underdetermined from the spectroscopic data alone . opa60 is a canonical eight - stranded -barrel with extensive ionic interactions inside the barrel . three of the extracellular loops are longer than those found in any -barrel structure previously determined . the hv regions within these loops are dynamic on the nanosecond time scale and are predominantly disordered . however , the loops are compact and interact with each other weakly such that long - lived specific intraloop interactions are not observed . the diverse and dynamic nature of the loop structural ensemble is likely required for highly variable opa loop sequences to bind a common receptor and also for a single opa protein to bind a variety of host receptors . ( a ) opa60 is an eight - stranded -barrel with long extracellular loops ( 60% of the protein ) . charged residues within the -barrel region are colored green and in the periplasmic turns colored blue . aromatic residues located near the headgroup region of the bilayer are colored yellow . the semivariable region ( sv ) is colored orange , and the hypervariable ( hv ) regions 1 and 2 are colored red . residues in the loops that have nmr assignments have gray circles and regions demonstrating transient helices in the md simulations have black outlined font . ( b ) the n , h trosy - hsqc is labeled with the nmr backbone assignment for opa60 in dpc micelles . ( c ) sample strips from the n - noesy spectrum indicating intra- ( solid lines ) and interstrand ( dashed lines ) noes observed . residues in -strand 4 ( black ) , -strand 3 ( blue ) and -strand 5 ( red ) are labeled . ( d ) nmr n , h trosy - hsqc spectra of opa60 in nanodiscs containing dmpc lipids with peaks labeled with the nmr backbone assignment . the spectra shown were recorded at 800 mhz and at 40 c ( b , c , and d ) or 10 c to resolve only loop resonances ( d ) . all nmr samples were deuterated with labile protons back exchanged and the protein concentrations were 750 m for dodecylphosphocholine samples and 500 m for nanodiscs preparations . the gene for opa60 with n- and c - terminal fusion tags was subcloned into pet28b from the orginal pex vector provided ( martine bos , ultrech university ) and transformed into bl21(de3 ) e. coli . cells were grown in d2o ( 99.8% ) minimal media containing 4 g / l c(99%)-glucose and 1 g / l n(99%)-ammonium chloride ( cambridge isotopes laboratory ) at 310 k until an od600 of 0.8 expression was induced with 1 mm isopropyl--thio - d - galactoside for 8 h. cells were lysed after resuspension in 50 mm tris hcl and 150 mm nacl ( lysis buffer ) . cell debris from the lysate was removed via centrifugation at 12000 g for 30 min . the pellet was resuspended in lysis buffer with the addition of 8 m urea overnight and centrifuged again at 12000 g for 30 min . the soluble fraction was added to a co - immobilized metal affinity chromatography column and washed with 15 cv of 20 mm sodium phosphate , ph 7.8 , 150 mm nacl , 20 mm imidazole , 8 m urea followed by a 5 cv elution ( 20 mm sodium phosphate , ph 7.0 , 150 mm nacal , 680 mm imidazole ) . the eluted protein fraction was concentrated to 200 m and rapidly diluted 20-fold with 20 mm tris hcl , ph 8.0 , 500 mm nacl , and 4.5 mm n - dodecylphosphocholine ( dpc ; anatrace ) . after 5 days of room temperature incubation , the protein was fully folded as assessed with sds page gel shift analysis . the sample was then concentrated and dialyzed against 3 4 l of 20 mm sodium phosphate , ph 6.2 , and 150 mm nacl for 1 h each . final nmr samples were concentrated to 400800 m and contained 110150 mm dodecylphosphocholine ( dpc ) as measured by comparing sample detergent intensities with standard concentrations . opa60 was reconstituted into nanodisks according to established protocols using plasmid for msp1d1h5 generously provided by gerhard wagner ( harvard university ) . msp1d1h5 was purified and assembled in 20 mm tris / hcl ph 7.5 , 100 mm nacl , and 5 mm edta buffer with the appropriate amount of dry lipid / detergent to obtain a mixture of msp1d1h5/dmpc / sodium cholate with a molar ratio of 1:50:100 . opa60 refolded in dpc was added to the mixture , and the opa60/msp1d1h5 ratio was adjusted to 1:4 . the mixture was incubated at 4 c for 1 h , and detergent was removed with 0.5 g of washed biobeads sm-2 ( biorad ) per ml of assembly mixture . h. biobeads were pelleted by centrifugation , and the decanted supernatant was concentrated and purified on a superdex 200 gel filtration column equilibrated with 20 mm sodium phosphate , ph 6.5 , 50 mm nacl , and 5 mm edta . fractions corresponding to the main peak were pooled and concentrated using an amicon centrifugal filter unit of 30 kda mwco ( millipore ) . the nmr sample consisted of 0.5 mm h , n opa60 in msp1d1h5 nanodiscs with d54 - 1,2-dimyristoyl - sn - glycero-3-phosphocholine ( dmpc ; avanti polar lipids ) , in gel filtration buffer supplemented with 10% ( v / v ) d2o . nmr spectra were collected on bruker avance spectrometers operating at proton frequencies of 600 and 800 mhz equipped with bruker 5 mm txi cryoprobes and recorded at 313 k. spectra were processed with topspin and assigned using cara ( cara.nmr.ch ) . the assignment strategy for opa60 is published and mapped onto the n , h - trosy - hsqc in figure s1 ( supporting information ) . through these strategies , complete nitrogen , hydrogen , c , c , and co resonances were assigned for residues 114 , 2931 , 5171 , 9597 , 109110 , 118140 , 157 , 159178 , 190212 , 231 , and 233238 ( figure 1b ) with only c , c , and co resonances for the seven assigned prolines and a lack of c assignment for two additional resonances ( y71 and i97 ) . further , the entire side chain carbon and hydrogen assignments were obtained for residues 159178 through tocsy and cosy assignment using a corresponding synthetic peptide . additionally , nine aromatic side chain protons ( y10 , f62 , w65 , f125 , f131 , y134 , f200 , y236 , and f238 ) were observed due to incomplete protein deuteration and dynamics of the side chain and assigned using the n - noesy spectrum . resonance assignments were achieved for 92% of the -barrel region ( as defined by the md refined structure ) and 27% of the extracellular loops . the nspa structure was not used for the assignment or identification of restraints . the talos+ program was used to obtain backbone dihedral angle restraints . assigned noe peak heights were measured and binned into strong , medium , or weak interactions . observed inter--strand noes are schematically indicated in figure 1a . thirty - one additional noes between backbone hn protons and aromatic side chain protons were also included . these were assigned upper limits of 3.5 , 5.0 , and 6.5 . in most cases , hydrogen bonding partners could be unambiguously assigned based on noe patterns ( a representative strip is shown in figure 1c ) , and two distance restraints were used with upper limits of 2.5 and 3.5 for hno and no , respectively . additionally , planar restraints ( 32 4 as a square potential ) were used to represent the lipid bilayer . the extracellular loops are very long , and without the bilayer restraint they sampled conformers that would be embedded into or transverse the bilayer ( figure s1 , supporting information ) . the restraint distance was chosen on the basis of ( 1 ) the hydrophobic thickness of porb a -barrel membrane protein from ng for which there is a crystal structure and ( 2 ) the residues that were observed to have noes with detergent headgroup choline protons ( figure s2 , supporting information ) . starting at 3000 k , 5000 steps of high - temperature annealing was used to fold the initial extended structure . twenty of the lowest overall violation energies of the 300 calculated structures with selected for further md simulations . all simulations were performed using gromacs 4.5 and the charmm36 forcefield for protein and lipid interactions . as detailed below , distance and dihedral restraints derived from the nmr data and used in xplor simulated annealing runs were enforced throughout the molecular dynamics simulations . simulations were run under npt conditions using the velocity - rescaling thermostat at 300 k with a time - constant of 0.1 ps and semi - isotropic pressure coupling using the parrinello all covalent bonds were constrained using lincs , and long - range electrostatics were computed every step using particle mesh ewald ( pme ) . a lipid bilayer of 512 dmpc molecules ( bilayer thickness , 34 1 ; hydrophobic thickness , 2326 ) was constructed using the charmm - gui membrane builder tool and solvated with approximately 40000 tip3p waters . ions were added to obtain a system with 150 mm nacl and no net charge . the dmpc bilayer was equilibrated prior to protein insertion with a resulting area per lipid headgroup of 0.60 nm , close to the experimentally determined value ( 0.606 0.005 nm ) . the system dimensions were approximately 12.5 nm ( sides ) and 11 nm ( height ) . each of the 20 lowest energy structures from xplor simulated annealing was independently inserted in the equilibrated membrane using the gromacs tool g_membed , removing approximately 1520 lipids in the process . each system was then energy minimized for 1000 steps using the steepest descents method . twenty production runs , one per protein structure , were then carried out for 100 ns using a time step of 2 fs . noe - based distance restraints and talos+-derived dihedral restraints as used in the xplor simulated annealing calculations were imposed using spring potentials with force constants of 1000 kj / mol / nm and 1000 kj / mol / rad , respectively . to perform the c density analysis , all trajectories were aligned to a single consistent reference structure using a rigid - body fit where the objective function was calculated only on the sheet and turn residues . then the density was calculated on a 3d - grid with the mdanalysis toolkit , using a grid - spacing of 0.1 nm . clustering was performed using the gromacs tool g_cluster , which was extended to include the k - means algorithm . the 20 most - populated , and therefore lowest free energy clusters , were selected , and the minimum energy structure from each was reported to form the hybrid refinement ensemble . the c rmsd within each cluster ranged from 1.52 to 4.22 and 1.87 to 5.33 for the entire protein and the extracellular loops , respectively ( table s2 , supporting information ) . the rmsd for the extracellular loops between clusters is much greater with the pairwise rmsd for the minimum energy structures ranging from 5.60 to 29.3 ( table s3 , supporting information ) . only contacts greater than 1.5 kt estimated free energy difference between the md and xplor structures additional analysis of contact between the hv1 and hv2 loops was performed by generating contact maps between these residues , where a contact was defined using a 6 interatomic distance cutoff . contact probabilities and lifetimes were computed using these contact maps , and highly contacting structures ( defined as > 50 simultaneous contacts ) were further analyzed via agglomerative single - linkage clustering using euclidean distance on the hv1-hv2 contact maps , yielding 10 clusters of hv1hv2 contacts . side chain restraints were not included in the md / nmr hybrid refinement . xplor ensemble without side chain noes had a mean global backbone rmsd for the -sheet residues of 1.04 0.15 . chemical shifts were calculated on all snapshots in the md simulations and for the xplor structures using the sparta+ software . the calculated shifts were then averaged for each atom , and the average values compared to the experimentally determined shifts ( which were corrected for deuterium and trosy induced shifts ) , where existing . in all cases , the errors reported are those used by sparta+ ( 0.92 ppm for c and 0.49 ppm for hn , respectively ) . relaxation rates were measured using two - dimensional n h trosy - based experiments recorded at 600 mhz and 313 k. nmr data were processed and fit with nmrpipe . h bond vector ci(t ) = < i ( 0 ) , i ( t ) > , averaged across all trajectories , where i is the n h bond vector for the ith residue . ( a ) -barrel and periplasmic turns are colored black ; extracellular loop 1 , green ; loop 2 , blue ; loop 3 , red ; loop 4 , magenta . ( b ) differences in carbon chemical shifts compared to random coil values are plotted ; ( c c ) = /3(ci1 + ci data for turns are colored red , for -strands blue , for loops green , and for the n - terminus gray . the chemical shifts have been deposited in the biomagresbank under the bmrb accession no . atomic coordinates for the xplor and md / xplor refined 20 conformers representing the structure of opa60 have been deposited in the pdb ( pdb i d : 2mlh and 2maf , respectively ) . opa60 is an eight - stranded -barrel with four extracellular loops ( figures 1a and 2a ) . the combination of a stable , membrane - inserted -barrel domain and long unstructured loops complicated assignment and structure determination . strategies for the assignment of the protein included trypsin cleavage , peptide synthesis , and assignment at various temperatures . hn noes , h - bond restraints , and backbone dihedrals calculated with talos ( table 1 ) . the backbone rmsd of the barrel region for the 20 lowest energy structures is 0.96 ( table 1 and figure 2a ) . the structure calculations were complicated by the long unstructured loops ; in the initial calculation , extracellular loops adopted unreasonable conformations that spanned the membrane embedded region with excursions to the periplasmic side of the protein ( figure s1 , supporting information ) . to address this problem , planar restraints were introduced at a distance of 32 4 ( see the experimental section for details ) . most of loops 1 , 2 , and 4 were not assigned ( figure 2b ) because the resonances were not observed ( although spectral overlap contributed ) . lowering the temperature broadened -barrel and some loop peaks beyond detection simplifying the spectra to only the most dynamic regions of the loops and facilitating 27 loop resonances to be assigned . to further assign the functionally important hv2 region , a synthetic 20 amino acid peptide was synthesized and had nearly complete spectral overlap with the full - length protein allowing 17 loop resonances to be assigned . there are two likely phenomena that contribute to the line broadening of the resonances that are not observed : ( i ) conformational exchange and ( ii ) structural heterogeneity . the former was speculated to contribute to the lack of assignments in ompx and ompa in dihexanoylphosphatidylcholine ( dhpc ) and dpc micelles , respectively . the missing resonances ( approximately half of the extracellular loops ) corresponded to residues that connect the ordered -barrel and the flexible central region of the extracellular loops . some of the missing loop resonances of ompx were resolved when the micelle was replaced with nanodiscs containing dmpc and 1,2-dimyristoyl - sn - glycero-3-phosphoglycerol ( dmpg ) . to better refine the structure , each of the 20 structures of the nmr ensemble was subjected to molecular dynamics simulations in a dmpc lipid bilayer . an interval of 100 ns of simulation was selected for the structure refinement because t1 and t2 relaxation data indicated the hv regions were dynamic on the nanosecond time scale . in addition , the chemical shifts and cd indicated the hv and extracellular loops , respectively , are random coil . although the solution nmr structure was determined in dpc , the simulations were sought in a more biologically relevant membrane environment . there are several pieces of evidence that suggest opa60 has the same structure in dmpc as in dpc . cd spectra of opa60 in dpc and dmpc small unilamellar vesicles ( liposomes ) indicate the protein structure is approximately 50% random coil 50% -strand ( figure s3 , supporting information ) . in addition , opa60 in nanodiscs with dmpc have -barrel chemical shifts that are superimposable with the dpc spectra ; however , a few barrel resonances corresponding to residues on strands 3 , 6 , and 8 are missing in the nanodisc spectrum ( figure 1d ) . to further refine the solution nmr structure , 100-ns md simulations were performed on each of the 20 lowest energy nmr structures embedded in a dmpc lipid bilayer ( bilayer thickness , 34 1 ; hydrophobic thickness , 2326 ) . the resulting ensemble from the md simulations has a backbone rmsd of 1.19 for the -barrel region ( figure 3a and table 1 ) . the md ensemble is composed of the minimum energy structure from each of the 20 lowest free energy clusters and captures 2937 of the 4000 snapshots ( table 1 ) . the c rmsd within each cluster ranged from 1.524.22 and 1.875.33 for the entire protein and the extracellular loops , respectively ( table s2 , supporting information ) . the rmsd for the extracellular loops between clusters is much greater than within clusters , with the pairwise rmsd for the minimum - energy structures ranging from 5.60 to 29.3 ( table s3 , supporting information ) . although sampling is not sufficient to achieve full convergence , principal components analysis of the loop conformations across all simulations yields good overlap in the subspace of the two largest principal components ( figure s4 , supporting information ) , indicating that at least in terms of the highest amplitude loop motions , individual simulations sampled overlapping rather than isolated regions of conformation space . thus , the ensemble from the 20 lowest energy clusters represents the loop structural diversity observed in the trajectories . the -barrel of opa60 is similar in sequence to nspa ( figure s5 , supporting information ) ; however , the overall fold from the md / nmr refinement is most similar to ompa and ompx ( table s4 , supporting information ) . similar to the 12 eight - stranded -barrels deposited in the protein data bank , the strands have a right - handed twist ( figure 3a ) and an aromatic belt around the circumference of the barrel at the lipid headgroup regions ( figure s6 , supporting information ) . a significant ionic network exists inside the barrel ( figure s7 , supporting information ) ; however , there is no observable pore through the length of the barrel . the ionic residues on the extracellular side of the barrel ( figure s7 , supporting information ) are excluded from solvent in many of the clusters , and those on the periplasmic surface ( figure s7 , supporting information ) are accessible to solvent in all the clusters . this ionic network may contribute to the significant stability observed for opa60 ; the barrel remains intact after cleavage with trypsin and boiling in sds loading buffer . basic residues ( figure s8 , supporting information ) are clustered in the extracellular loops near the barrel domain . these residues may interact with the negatively charged lipooligosaccharide outer leaflet of the outer membrane as was observed for fhua and one of the lps interactions identified with oprh . however , specific interactions between the barrel and lps were not detected by chemical shift perturbation when lps ( los , which is in the outer leaflet of the neisseria outer membrane , is not commercially available ) was titrated into the opa60dpc micelle ( data not shown ) . colored by density ( probability of a c atom within an grid cell ) from yellow ( 2 10 ) to blue . the gray planes show the average positions of the phosphorus atoms in the lipid head groups . contact map for the ensemble calculated with xplor in detergent ( a ) and for the md in lipid ( b ) . the contact map is rendered as a contour plot of contact probability , with evenly spaced contours from 10% to 100% contact probability in each ensemble colored from dark blue to dark red . contacts were defined as two atoms from respective residues approaching within 5 . comparison of the amide proton ( c ) and c ( d ) chemical shifts calculated from the xplor ensemble and the md ensemble . positive values indicate the chemical shifts calculated from the md ensemble agree better with the observed chemical shifts , and negative values indicate chemical shifts calculated from the xplor ensemble agree better with the observed chemical shifts . the md - refined ensemble has several more inter - residue contacts than the xplor refined ensemble ( figure 4a , b ) . these new contacts are primarily intraloop : 11 are across the periplasmic side of the -barrel and 17 extend the strands on the extracellular side ( only the 76 contacts that represented a greater than 1.5 kt estimated free energy difference between the md and xplor structures were considered ) . consequently , the most probable loop conformations in the md refined structures are much more compact than the xplor refined structures ( figure 3a ) with most of the loop density above the -barrel ( figure 3b ) . this decrease in loop volume can be quantified via the protein radius of gyration , which decreases from the initial structure over the course of 19 of the 20 trajectories ( figure s9 , supporting information ) . the accuracy of the structural representation of the xplor and md refined ensembles can be evaluated by comparing chemical shifts calculated from the ensemble structures via semiempirical shift prediction methods against the experimental chemical shifts ( exp ) . parts c and d of figure 4 compare the deviations between the experimentally measured values ( exp ) of c and hn opa60 chemical shifts and the xplor and md predictions . most residues do not show differences between xplor and md larger than the sparta+ reported prediction accuracy . however , for carbon shifts , which primarily depend on backbone dihedral angles , the md predictions agreed better with the experimental shifts indicating that the md ensemble is an accurate representation of the observed opa60 structure . the -barrel provides the scaffolding for the functional extracellular loops , which are disordered and sample a diverse ensemble of conformers . the nmr and md dynamics data ( figure 5 ) indicate that hv2 and three extracellular loops ( l1l3 ) , respectively , are dynamic on the nanosecond time scale . because the opa detergent complex has a large overall correlation time , t1 values are highly sensitive to backbone nanosecond motions . opa t1 values decrease significantly at the n - terminus , periplasmic turns , and extracellular loops 1 and 3 compared to the -strands ( figure 5a ) , indicating these regions have high amplitude motions in the nanosecond time scale . several of the -strands have a general trend of increased dynamics toward the n- and c - terminal ends of the strands ( although most have order parameters greater than 0.85 ) , which was previously reported for other -barrel membrane proteins investigated with nmr . t2 changes are much more difficult to interpret since values increase with nanosecond motions and decrease with s ms motions . nonetheless , the opa60 t2 values ( figure 5a ) are consistent with the interpretation of the t1 values . thus , on the basis of the nmr data , the hv2 region of opa has a high amplitude of motion on the nanosecond time scale . these nmr data are consistent with those observed with md , which provides a more comprehensive understanding of the motions of the loops . although sampling of loop conformations was not globally converged , the rank order of backbone dynamics showed good convergence . assessed at 20 ns , the spearman was 0.975 between the mean autocorrelation function value and the fifth percentile of sampled trajectories , while the between the mean and the 95th percentile was 0.988 . a gradient is observed for the md - derived backbone nh bond vector time autocorrelation function for each of the three longer extracellular loops ( l1l3 ; figure 5b ) , with loop regions furthest from the barrel more dynamic than the regions closest to the barrel . based on the md simulations , the sv , hv1 , and hv2 regions are moving within the nanosecond time regime and with a high amplitude of motion . ( a ) n t1 and n t2 relaxation values for opa60 plotted versus sequence and secondary structure . n t1 values that are less than 80% ( dotted line ) of the value predicted ( 1 s ) for a 20 ns overall correlation time have s values less than 0.85 ( e = 10 ps ) . data for turns are colored red ; -strands , blue ; and loops , green . ( b ) cartoon representation of opa60 with residues colored according to the n h orientational time autocorrelation function calculated from md trajectories . as might be expected from the extensive nanosecond - time scale dynamics , the loops do not maintain long - lived structural features . however , they do form considerable intraloop contacts , which are captured more readily by the hybrid refinement strategy than simulated annealing alone ( figure 4a , b and 6a ) . there are several contacts within each loop that are observed in the md refined structures . of the 76 contacts above 1.5 kt estimated free energy difference between the xplor and xplor / md ensembles , 31 are within each of the three loops ( l1l3 ) . contacts between hv1 and hv2 are of most interest since they are both required to bind to ceacam receptors . common contacts between the two regions are observed ( figure 6a ) ; however , these contacts have short lifetimes ( figure 6b ) . throughout the simulations these contacts are frequent but short - lived . based on this observation , the 4000 snapshots were reclustered based on the hv1 and hv2 regions and analyzed in terms of contacts and representative structures ( figure s10 , supporting information ) . recurrent contacts were observed between residues in the range 153165 ( hv1 ) and 8695 ( hv2 ) as well as 171180 ( hv1 ) and 9498 ( hv2 ) . not surprisingly , there are several hydrophobic residues that mediate these hv1hv2 interactions ( figure s10 , supporting information ) . the recurring contact patterns were observed in loop conformations that were globally quite different and across multiple independent md simulations from different starting structures , suggesting robust formation of transient yet frequent interactions . these observations are broadly consistent with the primary nmr data in that long - lived interactions or persistent structure in hv1 and hv2 were not observed on the basis of chemical shift ( figure 2b ) and the lack of nonsequential noes in assigned regions . ( b ) average contact lifetimes are plotted for each contact , with each contour line representing a 5-ns lifetime increment . comparison of these panels shows that hv1hv2 contacts are relatively frequent but short - lived . in addition to the contacts observed , the sv , hv1 , and hv2 regions each sample helical conformers in a small fraction of the 4000 snapshots of the 20 trajectories ( figure s11 , supporting information ) . other secondary structures , such as ppii and 310 helices , were less abundant in these regions ( figure s11 , supporting information ) . the existence of these lowly populated structures is difficult to probe with traditional nmr methods ; however , for populated secondary structure elucidation , carbon chemical shifts ( figure 2b ) are typically used . for the data obtained , only a few residues in the sv and hv2 regions indicate -helical structure ( positive values ) , but overall the values indicate the dominant population is random coil which is consistent with the md results . the lack of any long - lived discrete structure in the extracellular regions of opa60 is compatible with the degree of sequence variability that still confers binding to host receptors ( table s1 , supporting information ) . it would be surprising should such extreme variability result in a single stable structure . despite the structural plasticity of the extracellular loops , opa proteins must still bind a common set of receptors . depending on the hypervariable sequences in the extracellular loops , opa proteins bind selectively to the n - domain of ceacam1 , 3 , 5 , and/or 6 but do not bind the n - domains of ceacam4 , 7 , and 8 . using mutagenesis , residues y34 and i91 of ceacam n - domains ( figure s12 , supporting information ) were identified to be essential for the opa - receptor interaction . several other residues ( 27 , 28 , 29 , 32 , 39 , 44 , and 89 ) dictate the different opa ceacam selectivity reported ( figures s12 and s13 , supporting information ) . the total exposed surface area of these identified residues is approximately 440 and is composed of both hydrophobic and polar moieties , which can easily be complemented by the hydrophobic and polar groups in the hv regions of opa proteins ( table s1 , supporting information ) . beyond the enthaplic interactions , the dynamics and conformations of the extracellular loops are important to the molecular recognition event . the extracellular loops are intrinsically disordered yet are sampling a restricted volume such that there are interactions between the loops on the nanosecond time scale . md simulations further suggest recurrent yet transient interaction patterns between specific regions of the loops . thus , the loops adopt an intermediate state that is not folded but is also not lacking in interactions ; the state of the loops may be best described as premolten globule or both hv1 and hv2 are required ; the hv regions are concomitant since chimeric opa proteins with an hv1 and hv2 region from two opa proteins that bind the same receptor do not bind receptor . state may be a mechanism to retain disorder yet provide conformers in which hv1 and hv2 are in proximity and competent to interact with ceacam . the small ceacam binding surface ( figure s13 , supporting information ) and the requirement of both opa hypervariable regions suggest that a large folding event of the extracellular loops is unlikely upon binding suggesting that the binding mechanism is more likely conformational selection rather than induced fit . however , there is a plethora of commentary on the similarities and differences of these two binding mechanisms with the prevailing idea that binding reactions could have elements of both mechanisms . in addition , sequences are selected for function not mechanism ; therefore , the mechanism of different opa proteins may vary . since opa ceacam interactions differ among variants and receptors , the dynamic nature of the loops maximizes the likelihood that a sequence will engage the receptor by increasing conformer sampling and the potential binding modes for receptor engagement . we report the structure of opa60 , a neisserial outer membrane protein that induces host phagocytosis of the bacterium through specific receptor interactions . this eight - stranded -barrel protein possesses three extracellular loops ( greater than 34 residues ) that are longer than any -barrel structures yet reported and required a membrane restraint in the xplor structure calculation . to understand the structure and dynamics of the loops , we employed a hybrid xplor / md refinement where nmr - derived restraints were used in 20 100 ns md simulations to obtain a structure of opa60 in a dmpc membrane . the hybrid - refined structure is consistent with the initial xplor structure but has an increase in loop structure and compactness . although there is little secondary structure evident in either the md simulations or the primary nmr data , there are many short - lived contacts between the loops on the nanosecond time scale due to extension of the -strands . we hypothesize that the observed dynamic ensemble is critical for maximizing the conformations of a highly variable region of opa to engage receptors . that is , a high degree of plasticity is required to tolerate the diverse sequences in these regions and sample conformers competent to engage receptors . it remains to be shown , however , whether different opa variants engage a single receptor ( e.g. , ceacam1 ) via similar or different loop structures . the structure of several opa variants in complex with an identical receptor will elucidate whether the binding modes are indeed convergent or divergent .

the structure and dynamics of opa proteins , which we report herein , are responsible for the receptor - mediated engulfment of neisseria gonorrheae or neisseria meningitidis by human cells and can offer deep understanding into the molecular recognition of pathogen host receptor interactions . such interactions are vital to understanding bacterial pathogenesis as well as the mechanism of foreign body entry to a human cell , which may provide insights for the development of targeted pharmaceutical delivery systems . the size and dynamics of the extracellular loops of opa60 required a hybrid refinement approach wherein membrane and distance restraints were used to generate an initial nmr structural ensemble , which was then further refined using molecular dynamics in a dmpc bilayer . the resulting ensemble revealed that the extracellular loops , which bind host receptors , occupy compact conformations , interact with each other weakly , and are dynamic on the nanosecond time scale . we predict that this conformational sampling is critical for enabling diverse opa loop sequences to engage a common set of receptors .

neuroendocrine tumors are a group of epithelial neoplasms with a predominantly neuroendocrine differentiation that can affect almost any organ system ( klimstra et al . , 2010 ) . although these are typically associated with the gastrointestinal tract , pancreas and lung , gynecological cases have also been reported in the cervix , endometrium and ovaries ( fisseler - eckhoff and demes , 2012 , kupryjaczyk , 1997 , chun , 2015 ) . amongst these , primary ovarian carcinoid tumors are exceptionally rare , accounting for approximately 0.1% of all ovarian and 0.5% of all carcinoid neoplasms ( modlin and sador , 1997 , modlin et al . , 2003 ) . only 15% of these reportedly exist in pure form , with the remainder featuring teratomatous components such as struma ovarii or dermoid cysts ( fisseler - eckhoff and demes , 2012 , kupryjaczyk , 1997 , talerman , 1984 ) . this report describes an unusual case of primary ovarian neuroendocrine tumor arising in association with a mature cystic teratoma , and highlights the diagnostic challenges posed by these neoplasms in the absence of a consensus ovarian classification system . a 65-year - old caucasian woman with a bmi of 28.8 kg / m was referred as a fast - track patient for an abdominal mass and raised ca 125 following a three - week period of refractory diarrhea . transvaginal ultrasound revealed a large , complex and predominantly vascular cystic mass extending from the pelvis to the umbilicus which was 14 14 9 cm in size . the uterus was retroverted with an endometrial thickness of 15 mm and no evidence of vascular enhancement . subsequent ct abdomen and pelvis with contrast confirmed a large pelvic mass with a small volume of ascites but no convincing metastases . her past medical history included carcinoid heart disease with moderate to severe left ventricular dysfunction and severe tricuspid regurgitation . preoperative hematological profile was within normal limits ; ca 125 was 248 units / ml and cea 3.0 ng / ml . urinary 5-hydroxyindoleacetic acid ( 5-hiaa ) and chromogranin a were both raised at 771 mg/24 h and 338.6 g / l , respectively . the patient underwent a total abdominal hysterectomy , bilateral salpingo - oophorectomy , pelvic and para - aortic lymphadenectomy and omentectomy for presumed carcinoid syndrome . postoperative recovery was uneventful and she was discharged after 7 days with routine follow - up . macroscopically , the right adnexa was enlarged , nodular and cystic , and measured 130 130 85 mm . it weighed 654 g and had a smooth , intact capsular surface incorporating the fimbrial end of the fallopian tube . slicing of the specimen revealed cystic areas filled with hemorrhagic fluid , a tan colored solid component and mucinous elements . the allied uterus , cervix , left tube and ovary , and all lymph nodes were broadly unremarkable and of dimensions and appearances within normal limits , other than for the incidental finding of a fibroid in the anterior uterine wall . approximately 90% of the main tumor was composed of an insular pattern resembling a carcinoid tumor with nested areas , together with foci of tumor cells arranged in groups and trabeculae ( fig . the nuclei in these areas exhibited mild to moderate pleomorphism and no mitotic figures were identified . the remaining 10% of the tumor consisted of sheets and groups of cells with highly pleomorphic and bizarre nuclei within a vascular stroma . this component also contained foci of coagulative tumor cell necrosis and contained 23 mitoses per 10 high power fields . other focal elements of the tumor were reminiscent of a mature cystic teratoma ( dermoid cyst ) , featuring cyst - like spaces , variably lined by flat or cuboidal cells , and areas showing duct - like structures resembling skin appendages . some of these cystic spaces were also lined by histiocytes and foreign body - type giant cells . immunohistochemistry was performed for chromogranin , synaptophysin and cd56 given that these have previously been shown to be expressed in ovarian neuroendocrine tumors ( chun , 2015 ) . further staining was conducted for -catenin and ki-67 ( kim et al . , 2015 , rindi et al . , 2006 , rindi et al . , 2007 , bosman et al . , 2010 ) . the organoid arrangements of tumor cells identified were consistent with the neuroendocrine differentiation growth patterns described for these lesions ( klimstra et al . , 2010 ) , and the staining patterns were similarly supportive . according to the who classification of pulmonary and thymic lesions ( klimstra et al . , 2010 , travis , 2004 ) , low to intermediate grade lesions can be classified into typical and atypical subtypes ( with high grade lesions being either small or large cell carcinomas ) . the former should have fewer than 2 mitoses per 10 high power fields ( hpfs ) and no necrosis . however , as underscored by a previous case report ( kim et al . , 2015 ) , there is a dilemma when attempting to apply this distinction to ovarian carcinoid tumors . given their rarity , there are currently no widely accepted diagnostic criteria to cover their subclassification or sufficient patient outcome data to relate to atypical or aggressive histological appearance . nevertheless , based on the presence of > 2 mitotic figures/10 hpfs and the presence of necrotic foci , this tumor fell into the category of atypical ( intermediate grade ) carcinoid by applying the criteria set for pulmonary / thymic lesions . importantly , necrosis per se is not diagnostic of neuroendocrine carcinoma ; rather , a ki-67 index of > 20% is diagnostic ( rindi et al . , 2006 , rindi et al . , 2007 , bosman et al . , 2010 ) , which is in excess of what was recorded for this lesion . in the gastrointestinal tract , a neuroendocrine tumor with features such as those described for this lesion would be considered to be a well - differentiated neuroendocrine tumor , grade 2 ( intermediate grade ) . on balance , we elected to favor this classification system , resulting in a final diagnosis of well - differentiated neuroendocrine tumor , grade 2 ( intermediate grade ) , arising in association with a mature cystic teratoma / dermoid cyst . a previous case report of a similar lesion noted strong nuclear immunoreactivity for -catenin ( kim et al . , 2015 ) , an aberrant staining pattern of aggressive histology associated with both pulmonary neuroendocrine carcinomas and atypical carcinoid tumors ( pelosi et al . , moreover , this is also described to be an independent predictor of lymph node spread in patients with atypical pulmonary carcinoid tumors . in the present case , there was no nuclear -catenin accumulation and all four identified lymph nodes were negative for tumor . although primary ovarian carcinoid tumors ' surgical management reportedly has a good outcome for organ - confined disease ( davis et al . , 1996 ) , these remain poorly studied entities ( gardner et al . , 2011 ) and as such , attentive follow up was recommended and the case was referred to the neuroendocrine multidisciplinary team . importantly , clinicoradiological correlation was recommended to exclude the possibility of any other primary site given the obvious impact of metastatic disease on prognosis ( chun , 2015 ) . , this case highlights the diagnostic challenges posed by ovarian carcinoid tumors due to the lack of established diagnostic criteria available for these rare entities and the need to develop a unified system to which ovarian neuroendocrine tumors can be aligned . further cases and the accumulation of additional clinical outcome data would further contribute to improving the accuracy of future prognostic prediction .

primary ovarian carcinoid tumors are exceptionally rare entities accounting for approximately 0.1% of all ovarian neoplasms . this report describes a primary ovarian neuroendocrine tumor arising in association with a mature cystic teratoma in a 65 year - old woman . macroscopically , the unilateral adnexal tumor was composed of cystic , solid and mucinous elements which resolved into a dual component lesion histologically . the majority of the tumor displayed an organoid architecture with mild to moderate pleomorphism and no discernible mitotic activity , while approximately 10% consisted of sheets and groups of cells with highly pleomorphic nuclei , necrosis and occasional mitoses . features of a mature cystic teratoma were seen very focally . immunohistochemistry revealed strong , diffuse positivity for cd56 and synaptophysin . chromogranin immunonegativity was noted and there was an absence of nuclear -catenin accumulation . ki-67 index was 1012% . although there is no established diagnostic framework for primary ovarian carcinoid tumors , this case was diagnosed as a well - differentiated neuroendocrine tumor , grade 2 ( intermediate grade ) , arising in association with a mature cystic teratoma / dermoid cyst . this case highlights the need to develop ovarian diagnostic criteria in this area .

oral metastatic lesions from distant tumors are uncommon and mainly involve the bony structures , whereas metastases to soft tissues are extraordinarily rare . in most patients , the primary tumor is already known before the oral metastatic lesions appear . this particular case is worth reporting , because of extremely rare nature of simultaneous metastasis of pancoast tumor to the adrenal gland and gingiva and in spite of the wide spread metastasis the patient did not present with any other symptoms , except for the presence of recurrent gingival lesions . the clinical appearance in this case was of a gingival mass that could have been misdiagnosed as a hyperplastic lesion associated with a tooth or as a reactive process . in many cases diagnosis can be difficult and requires patient 's remote medical history , biopsy for histological diagnosis as well as some target organs investigations , such as brain , lungs , liver , to establish the correct diagnosis . a 49-year - old male patient reported to the department of oral and maxillofacial surgery , government dental college , with a painless sessile growth of size 2 3 cm on the lingual attached gingiva in relation to mandibular incisors of one month duration . clinically , only four mandibular anterior teeth were present which includes left incisors , right central incisor , and canine . clinical examination revealed the lesion to be soft to firm in consistency with no bleeding on probing . grade 3 mobility of anterior teeth was found except for the left lateral incisor which was grade i mobile . radiographic examination showed extensive bone resorption in relation to the mandibular anterior ( left lateral incisor to right lateral incisor ) region with a floating tooth appearance [ figure 1 ] . a tentative diagnosis of pyogenic granuloma was suggested by the oral surgeons as there were no other symptoms . extensive bone resorption in relation to the mandibular anterior teeth region with a floating tooth appearance after 20 days of initial excision , the patient came back with the presentation of recurrent lesion at the same site , i.e. on the gingiva in mandibular anterior teeth region in the extracted area [ figure 2 ] along with two other similar looking lesions on palate close to the alveolus on the left side in the molar region and in the maxillary tuberosity area on the same side [ figure 3 ] . on examination , the lesions were reddish in color , firm in consistency and bleeding on probing was also noticed . submandibular and sublingual lymph nodes were enlarged and fixed , but no involvement of the supraclavicular , superficial and deep cervical lymph nodes . patient was also complaining of weight loss and dull radiating pain in the left shoulder . recurrent lesion on the gingiva in mandibular anterior teeth region after the extraction lesion on palate close to the alveolus on the left side in the molar region and in the maxillary tuberosity area on the same side on routine investigation , hemoglobin was seen to be reduced by two units over a period of 20 days . orthopantomogram revealed bone loss in the edentulous alveolus in relation to lower left and right incisor area and left canine premolar region [ figure 4 ] orthopantomogram revealing bone loss in the edentulous alveolus in relation to lower left and right incisor area and left canine premolar region all the three lesions were excised in the department of oral surgery and sent for histopathological examination . on gross examination , there were three bits of firm consistency ; light brown in color . size of the larger bit was 1.5 1 1 cm , and two smaller bits of size each 1.5 0.5 0.5 cm . two out of three bits showed stratified squamous epithelium overlying connective tissue , which appeared torn at places which was thought to be a processing error . underlying connective tissue showed some atypical cells with granular cytoplasm which were arranged in alveolar pattern . large atypical hyperchromatic cells and some clear cells with nucleus pushed to one side were also seen . none of the cells had any resemblance to the native cells of oral mucosa [ figure 5 ] . h and e photomicrograph of magnification 40 showing large atypical hyperchromatic cells and some clear cells with nucleus pushed to one side patient underwent human immunodeficiency virus ( hiv ) test to rule out any hiv associated tumor , but it came negative . other differential diagnoses were malignant minor salivary gland neoplasm , angioproliferative lesions , melanoma , and lymphoma . to rule out malignant salivary gland neoplasm , immunohistochemistry using p63 immunohistochemical ( ihc ) markers , leucocyte common antigen ( lca ) and cd 34 , were used to rule out lymphoma and angioproliferative lesions , respectively . to rule out metastasis from lung and thyroid , thyroid transcription factor ( ttf-1 ) immunohistochemistry for cytokeratin showed focal positivity [ figure 6 ] which suggested metastasis from kidney or gastrointestinal tract ( git ) or lung . immunohistochemistry for cytokeratin showing focal positivity , 4 ultrasound of chest and abdomen showed hyperechoic bilateral adrenal mass . x - ray chest suggested a homogenous opacity in the apex of left lung [ figure 7 ] with destruction of underlying ribs . x - ray was suggestive of pancoast tumor . computerized tomography ( ct ) scan of chest and abdomen with iv contrast revealed an irregularly and poorly enhancing soft tissue density mass in the apico - posterior segment of left upper lobe of size 6.2 5 cm [ figure 8 ] . left apical soft tissue mass lesion measuring approx 7 5 cm with destruction of the adjoining posterior aspect of left rib was also noted . well defined poorly enhancing areas were noted in the aorto pulmonary window and left hilum suggestive of enlarged lymphnodes of size 3 2 cm . a left suprarenal mass was noted of size 5 3.2 cm [ figure 9 ] . x - ray chest suggesting a homogenous opacity in the apex of left lung the ct scan of chest revealing an irregularly and poorly enhancing soft tissue density mass in the apico - posterior segment of left upper lobe the ct scan of abdomen revealing a left suprarenal mass ct scan of head and neck region revealed destruction of left alveolar process with enhancing soft tissue component . there was destruction of floor of the left maxillary sinus with soft tissue component into it [ figure 10 ] . multiple enlarged bilateral level ia , ib , and ii nodes were noticed . a cyst measuring 1.3 1 cm the ct scan of head and neck region revealed destruction of left alveolar process and floor of the left maxillary sinus with soft tissue component infiltrating into it the lesion was diagnosed as bronchoalveolar carcinoma of the left lung metastasized to bilateral adrenal glands and multiple sites in the oral cavity . metastasis to the oral cavity is very rare and represents only 1% of all neoplasm in the oral cavity . in 25% of the cases , oral metastases are found to be the first sign of the metastatic spread ; and in 23% of the cases , it is the first indication of an undiscovered malignancy at a distant site . oral soft tissues were less frequently affected than the jaw bones ( 1:2.5 ) . in the oral soft tissues , the major primary sites presenting oral metastases were the lung , kidney , liver , and prostate for men ; breast , female genital organs ( fgo ) , kidney , and colo - rectum for women . in men , the lung was the most common primary site affecting both the jawbones and oral mucosa ( 22% and 31.3% , respectively ) followed by the prostate gland in the jawbones ( 11% ) , and kidney in the oral soft tissues ( 14% ) . in women , the breast was the most common primary tumor affecting the jawbones and soft tissues ( 41% and 24.3% , respectively ) , followed by the adrenal and fgo to the jawbones ( 7.7% ) , and fgo to the soft tissues ( 14.8% ) . lung carcinoma normally metastasizes to the gingiva and kidney primary metastasizes to the jaw . according to van der waal , in most patients , this particular case is worth reporting , because , in spite of the wide spread metastasis , the patient was unaware of the symptoms , except for the presence of recurrent oral lesions . thus , a retrograde diagnosis had to be made by an experienced pathologist with the aid of all supporting investigations . pancoast tumor is a tumor in the apico - posterior part of the lung . it represents fewer than 5% of all primary lung cancers . pancoast tumor can invade the brachial plexus , pleura , or ribs , causing shoulder and upper extremity pain and weakness or atrophy of the ipsilateral hand and horner 's syndrome ( ptosis , miosis , enophthalmos , and anhidrosis ) . more than 95% of pancoast tumors are non small cell carcinomas , most commonly squamous cell carcinomas ( 52% ) , or adenocarcinomas ( 23% ) , and large cell carcinomas ( approximately 23% ) . bronchoalveolar carcinoma , a subtype of adenocarcinoma , is a malignant neoplasm of the lung arising from the epithelium of the bronchus or bronchiole in which patient presents with a variety of clinical manifestations like cough , weight loss , chest pain , and dyspnea . in this particular case , patient did not have any of these features except for occasional dull radiating pain in the shoulder . brain , adrenal gland , liver , and bone are the common distant sites for a pancoast tumor to be metastasized . to the best of our knowledge , there have been no previous reports of metastases of pancoast tumors to the gingiva . though the adrenal gland is one of the common sites of metastasis of pancoast tumor , it usually occurs unilaterally . however , bilateral adrenal metastases are seen in 10% of all lung cancer patients ; of these 23% occurs at the time of initial presentation of non - small cell lung cancer . studies have shown that bilateral adrenal neoplasms are almost always metastatic tumors rather than primary , and clinical presentation varies with tumor type . hence , there was a need for search of a primary elsewhere in the body in our case , even after locating bilateral mass in the adrenal gland . to the best of our knowledge , as of now , there is no case report of simultaneous metastasis to both adrenal glands and oral cavity from a lung primary with the first symptoms noted clinically in the oral cavity and the patient being apparently healthy . as the prognosis of the metastatic lesions to the oral cavity is very poor , combination chemotherapy to alleviate the symptoms is the only preferred therapeutic modality . because of its rarity , the diagnosis of metastatic lesions in the oral cavity is very often missed . so , diligent clinical and histopathological investigations should be done to diagnose the metastatic lesion and its origin .

metastatic lesions to the oral region are uncommon and account for approximately 1% of all malignant oral tumors . in 25% of the cases , oral metastases are found to be the first sign of the metastatic spread ; and in 23% of the cases , it is the first indication of an undiscovered malignancy at a distant site . metastases to oral soft tissues are even less frequent than jaw bones . because of its rarity , the clinical presentation of a metastatic lesion in the oral cavity can be deceiving , leading to a misdiagnosis of a benign process ; therefore , in any case where the clinical presentation is unusual , especially in patients with a known malignant disease , a biopsy is mandatory . here , we are presenting a rare case of multiple secondary tumors in the attached gingiva in an otherwise apparently healthy patient with no other symptoms of the primary tumor . it subsequently led to the diagnosis of pancoast tumor ( bronchoalveolar carcinoma ) metastasizing simultaneously to multiple sites in the oral cavity and bilateral adrenal glands .

a 55-year - old man weighing 50 kg and 160 cm tall , non - smoker , presented with cough with expectoration and shortness of breath on exertion since 3 years . he also gave a history of intermittent diarrhoea and flushing of the face and upper body since 3 months , precipitated by alcohol intake . computerised tomographic scan ( ct scan ) of the chest revealed a large , lobulated , heterogeneous enhancing mass lesion measuring 8.6 cm 5.5 cm in left suprahilar region extending to left upper lobe bronchus [ figure 1 ] . computerised tomographic scan chest showing tumour ct guided biopsy on histopathology showed a typical lung carcinoid . his pulmonary function testing , arterial blood gas ( abg ) analysis and breath holding time showed a predicted post - operative forced expiratory volume in 1 s of 54% , pao2 of 93 mmhg , paco2 of 40 mmhg and breath holding time of 24 s. urinary 5-hydroxy-3-indole acetic acid was 50 mg / day ( n 10 mg / day ) . a diagnosis of left upper lobe bronchial carcinoid with carcinoid syndrome was made . pre - operative preparation with subcutaneous ( sc ) octreotide 100 g tds relieved symptoms of flushing and diarrhoea . after 10 days premedication included tablet midazolam 7.5 mg and tablet ranitidine 150 mg at night and 1 h before surgery . on the morning of surgery , inj.octreotide 250 g was given as an intravenous ( iv ) bolus followed by continuous infusion of 100 g / h . an epidural catheter was sited at t7-t8 interspace and after test dose of 3 ml 2% lignocaine , 50 g fentanyl in 6 ml saline was administered . under local anaesthesia drugs for emergency use , octreotide , phenylephrine , glyceryl trinitrate , esmolol , corticosteroids and antihistaminics were kept handy . anaesthesia was induced with fentanyl 100 g , propofol 75 mg with preadministration of inj . using vecuronium , portex right sided double lumen tube ( dlt ) 37 fg was inserted and position was confirmed by fibreoptic bronchoscope . oxygen saturation ( spo2 ) , end tidal co2 ( etco2 ) , invasive blood pressure , ecg , central venous pressure , temperature and airway pressures were monitored throughout the procedure . anaesthesia was maintained with vecuronium and sevoflurane 1 - 1.2% in o2/n2o mixture . during surgical manipulation of tumour , there was sudden rise in peak airway pressure ( p peak ) from 20 to 35 cm h2o then to 42 cm of h2o and etco2 , from 36 to 47 mmhg , then to 50 mmhg , accompanied by fall in spo2 from 97% to 80% . octreotide 250 g iv bolus was given and infusion rate was increased to 200 g / h and inj . clinical improvement was seen within 60 s of octreotide bolus and within 5 min respiratory parameters were normal . during extubation was uneventful and post - operative analgesia was provided with bupivacaine and fentanyl . in the recovery room patient 's spo2 remained 97% on room air with no evidence of wheezing . octreotide infusion was tapered off over next 24 h. histological examination of resected specimen confirmed diagnosis of carcinoid tumour . carcinoids are neuroendocrine tumours derived from enterochromaffin cells and are capable of secreting bioactive substances , most importantly serotonin , histamine and kinin peptides . in 1907 bronchial carcinoids are foregut tumours and approximately 70% are located in major bronchi and accounting for 0.5 - 2.5% of all lung malignancies . carcinoid syndrome occur secondary to the systemic release of mediators either as a result of metastasis in the liver or from lung . it is the respiratory and cardiovascular effects with which an anaesthesiologist should be familiar with because of their severity . bronchoconstriction which may present as wheezing and paroxysmal coughing can be life - threatening and requires prophylactic treatment . anaesthetic considerations in patients with carcinoid syndrome include prevention of mediator release , avoiding triggering factors and preparation for the management of peri - operative carcinoid crisis . hence , inhibition of tumour activity rather than antagonism of released peptides / amines are the mainstay of management , using octreotide . octreotide described as endocrine cyanide " has the ability to prevent peptides / amine release from gastroenteropancreatic system . the protocol from north american neuroendocrine tumour society consensus guidelines for the diagnosis and management of neuroendocrine tumours of the thorax are widely used . for any functional carcinoid tumour the patient should be started on octreotide . for major procedures , a pre - operative iv bolus of 250 - 500 g , followed by a continuous infusion of 100 - 500 g / h during the procedure , has been suggested . the infusion is then weaned by 50% daily for a few days until it can be safely discontinued and is sometimes supplemented by a dose of long - acting depot somatostatin analogue . based on available evidences , we decided to start octreotide 100 g sc thrice daily for 10 days pre - operatively in the present case . these factors may be grouped as physiological ( stress , anxiety , light plane of anaesthesia , hypercapnia , hypothermia , hypertension or hypotension ) , mechanical ( tracheal intubation / extubation , tumour handling ) , or pharmacological ( catecholamine and histamine releasing drugs ) . therefore , general anaesthesia should be induced cautiously using slow , titrated dose of drugs that have minimal haemodynamic effect . a carcinoid crisis caused by excessive tumour mediator release may occur anytime peri - operatively and it may manifest as hypotension or hypertension , flushing , tachycardia or bradycardia , bronchospasm and complete vasomotor collapse . therefore , vigilant monitoring is a must and drugs for its management should be kept ready before induction . bronchospasm during carcinoid crisis should never be treated with conventional drugs such as 2 adrenergic agonists , theophylline and epinephrine as they may further stimulate tumour mediator release . octreotide , corticosteroids , inhaled ipratropium bromide and antihistamines can be used safely , as during the current report . however , successful administration of both epidural and spinal anaesthesia in carcinoid syndrome has been reported . the use of epidural analgesia is only advised in carcinoid patients who have been optimized pre - operatively with octreotide and provided that local anaesthesia is administered in a graded manner with careful haemodynamic monitoring . in continuation of octreotide in the post - operative period depends on resectability and metastasis of tumour . although tumour was completely resected , we continued it in the post - operative period for 24 h as a prophylactic measure . pre - operative optimisation with octreotide should be carried out in diagnosed cases of carcinoid syndrome . receptor antagonists which inhibit vasoactive amines i.e. , octreotide , and h1 and h2 receptor blockers are required inspite of the fact that octreotide provides only prophylaxis against carcinoid crisis and may not prevent overwhelming release of mediators during tumour handling . however , the dose and duration of pre - operative octreotide therapy needs to be well - defined to prevent peri - operative complications .

carcinoid tumours pose a great challenge to anaesthesiologist , especially if carcinoid syndrome is present . we report peri - operative management of a patient with carcinoid syndrome who underwent upper lobectomy . pre - operative optimisation for 10 days before surgery with injection octreotide and administration on the day of surgery as per guidelines was followed ( north american neuroendocrine tumour society guidelines ) . our main goals were to prevent mediator release , avoidance of triggering factors and management of peri - operative carcinoid crisis . during tumour handling patient developed carcinoid crisis which was effectively treated with intravenous bolus octreotide and increasing rate of infusion .

during the past two decades , ideas of health have transformed from ones that focus on illness to ones that consider patient well - being and quality of life ( qol ) . currently , qol instruments that integrate clinical and economic indices serve as a key to understanding treatment outcomes . as spilker defines , " ' quality of life ' in clinical medicine represents the functional effect of an illness and its consequent therapy upon a patient as perceived by the patient . " although randomized controlled trials ( rct ) generally have the objective of evaluating intervention effect ( i.e. drug , modes of care ) , significant results are not recognized unless a suitable measure of outcome is evaluated [ 5 - 7 ] . accordingly , the use of qol assessment as an outcome measure in rcts continues to gain attention . while the consort statement establishes methods of rct reporting , this guideline does not address problems with qol instruments , standards of cultural validity and psychometric validity . sanders et al . studied qol assessment in clinical trials , indicating a high frequency of incomplete responses and a poor quality of reporting . findings described a need for specified standards of qol assessment and reporting . in 2002 , international standards for qol assessment and reporting in clinical trials were proposed . but the fact that qol is culturally influenced reminds us that standardization based on information having originated from english - speaking countries may be impetuous . accordingly , an examination of qol - assessment methodology in rcts collected from nations worldwide could provide valuable insight into the ramifications of standardization . a pubmed - based bibliographical search found approximately 4000 reports on clinical trials published in japanese between 1987 and 2001 ; this ranks second to german - published reports in number of non - english articles . when limited to rcts , the number of articles published between 1995 and 1999 from japanese institutions ranks top amongst asian nations and within the top ten internationally . an understanding of how qol is assessed in japanese clinical trials , particularly in rcts , will contribute to the ongoing international discussion . in this literature study , we calculate the number of rct reports that refer to qol and compare japanese publication trends with international ones . we then examine all reports published from japan as of 1970 . based on our findings , we point to several imperative problems concerning the assessment and reporting of qol that require further examination . we first located articles reporting rcts by using " randomized controlled trial " as a pubmed " publication type s [ pt ] " tag this was also used as a denominator to gain relative frequency . second , we identified rcts that referred to qol by using the free text term " quality of life " . for each year , we calculated the proportion of reports mentioning qol among all rcts . third , we looked for rcts published from japan that referred to qol as follows : " randomized controlled trial " [ pt ] and " quality of life " [ text ] and ( japan [ address ] or japanese [ language ] ) . results were not only examined by frequency of reporting , but were also assessed by hand in more detail . reviews , editorials , meeting abstracts , letters and publications without abstracts were excluded from the latter analysis . items of evaluation were based on previous reports and included : ( 1 ) type of subjects ' condition ; ( 2 ) type of intervention tested ; ( 3 ) type of qol measures used and whether they were validated ; ( 4 ) whether qol was defined conceptually ; ( 5 ) reasons why qol measurement was introduced into the trial ; ( 6 ) whether qol was categorized as the primary or secondary endpoint and description of ( 7 ) the response rate and , lastly , ( 8) respondents of qol measurements . two of the authors independently evaluated all articles for eligibility and resolved disagreements by consensus . in cases when information on qol - instrument validation was not mentioned in the article , we verified what type of validation study was conducted by checking references . counting the number of articles with " publication type " and a free text term , frequency of referring to qol was found to increase over time worldwide from 0 ( 0% ) between 19701974 to 1930 ( 4.4% ) between 20002003 . this trend was similar among reports published from japan with 0 ( 0% ) in 19701974 to 27 ( 1.8% ) in 20002003 ( figure 1 ) . prevalence of reporting on qol in randomized controlled trials during 19702003 65 rct reports referring to qol , published from japan during 19702003 , were found . we excluded 19 of these for not being rcts ( n = 4 ) , not having an abstract ( n = 1 ) , not reporting on qol ( n = 12 ) or for being a review article ( n = 2 ) . the remaining 46 reports ( 32 were written in english and 14 were written in japanese ) met our criteria and were evaluated ( table 1 [ see additional file 1 ] ) . table 2 and table 3 show the distributions of conditions and interventions examined and the language of each article . the most commonly studied condition was oncology ( 15 english reports ; 11 japanese reports ; 26 in total , 56.5% ) , followed by cardiovascular disease ( 5 english reports ; 0 japanese reports ; 5 in total ( 10.9% ) ) and urologic disorders ( 4 english reports ; 2 japanese reports ; 6 in total , 13.0% ) , with other diseases occurring in less than 10% of reports ( table 2 ) . the largest number of interventions was with drugs ( 18 english reports ; 11 japanese reports ; 29 in total , 63.0% ) followed by modes of care ( 9 english reports ; 3 japanese reports ; 12 in total ( 26.1% ) ) ; other interventions were studied in less than 5% of reports ( table 3 ) . subjects studied in 46 randomized controlled trials reporting on qol * health is defined here as the effect of captopril during exercise type of intervention studied in 46 randomized controlled trials reporting on qol qol was the primary endpoint in 10 reports ( 7 english reports ; 3 japanese reports ; 10 in total , 21.7% ) . authors defined qol in 15 reports ( 9 english reports ; 6 japanese reports ; 15 in total ( 32.6% ) ) ( table 4 ) . of these , authors of 9 reports ( 5 english reports ; 4 japanese reports ; 9 in total , 19.6% ) described their own definitions of qol . after 1999 , there were no rct reports in japanese that had investigators ' definition of qol . definition of qol in 46 randomized controlled trials reporting on qol a rising trend was recognized for reports describing why they performed a qol assessment ( table 5 ) prior to 1993 , 2/7 reports ( 1 english report ; 1 japanese report ; 2 in total ( 28.6% ) ) ; between 19941998 , 6/14 reports ( 2 english reports ; 4 japanese reports ; 6 in total ( 42.9% ) ) ; between 19992003 , 11/25 reports ( 11 english reports ; 0 japanese reports ; 11 in total ( 44.0% ) ) . description of why qol assessment was conducted in 46 randomized controlled trials reporting on qol table 6 shows the methods for assessing qol in each respective report . authors used established qol instruments in 12 reports ( 11 english reports ; 1 japanese report ; 12 in total ( 26.1% ) ) and modifications of established instruments in six reports ( 2 english report ; 4 japanese report ; 6 in total ( 13.0% ) ) . seven reports ( 5 english reports ; 2 japanese reports ; 7 in total ( 15.2% ) ) used non - established qol instruments but quoted references that described their methodology . eight reports ( 3 english reports ; 5 japanese reports ; 8 in total ( 17.5% ) ) developed original instruments . except for three qol scales in which each was used in two reports , a different scale was used in all reports . type of evaluated measure used in 46 randomized controlled trials reporting on qol of the remaining articles , 10 reports ( 9 english reports ; 1 japanese report ; 10 in total ( 21.7% ) ) assessed only symptoms or performance status , and three reports ( 2 english report ; 1 japanese report ; 3 in total ( 6.5% ) ) used methods that were unclear . 30 reports ( 21 english reports ; 9 japanese reports ; 30 in total , 65.2% ) specified response rate or number of respondents . lastly , 35 reports described respondents ( 26 english reports ; 9 japanese reports ; 35 in total ( 76.1% ) ) . health status , functional status , and qol are three concepts often used interchangeably to refer to the same domain of " health . " health - related qol continues to be used as a measure of outcome in clinical trials . in the present study , we performed a literature search using pubmed in aims of comparing qol assessment frequencies between international and japanese rct reports . our study , however , has the following limitations . a text - based search was unable to locate articles that do not contain the term , " quality of life , " in the abstract , title or as a medical subject headings ( mesh ) . nonetheless , given that there currently exists no japanese database of rct - related articles in japanese , pubmed serves as the best descriptive database for our study . while the number of rct reports referring to qol published from japan drastically increased after 1982 , this does not necessarily mean that a similar shift exists in relative frequency of total rcts . the relative frequency of japanese rcts rose in parallel with a rising global trend during the 1980s . in the following decade , however , this trend began to diverge from the international one . although japanese interest in qol measures rose in the 1990s , doubt persisted among clinicians and researchers concerning the validity of subjective measures using interviews and questionnaires . at that time , moreover , little research had focused on the methodologies of qol assessment . this study 's findings , we surmise , reflect this history of qol in japan . among the 46 reports examined , a total of 10 studies used qol as a primary endpoint . previous systematic reviews have criticized researchers for not using a suitable health - related qol instrument as an outcome measure when studying the effect of intervention [ 21 - 25 ] . nevertheless , it is still unclear as to whether this reflects a lack an applicable scale or the misuse of available scales . 27 of the 46 reports did not describe their reasons for using qol as an outcome measure . it remains unclear whether this derives from a lack of description concerning outcome measures or insufficient discussion on the need for qol assessment . it has also been suggested that sponsors and researchers may measure health - related qol in aims of raising the image of a clinical trial or of the drug under investigation . this study was unable to address this issue because all reports lacked a description of sponsorship and/or conflict of interests . further studies are needed to investigate and delineate the respective objectives and significance of qol assessment within each field . gill et al . conducted a systematic review of 75 reports published between 1987 and 1991 containing the term qol in the title and reported that only 15% of reports defined qol . editorial procedures and limited word count , however , may possibly be associated with why qol measurement selection and its definition were not mentioned . this lack of consistency indicates the need for studies to describe their definition of qol at least when using their own instrument . moreover , 50% of studies that used an established instrument did not provide a definition of qol . this was probably due to the assumption that qol assessment is equivalent to an establishment scale . this point to the need for defining qol and for better description as to why an established scale was selected in the present study , all 15 ( 32.6% ) reports that defined qol were published after 1993 . furthermore , only four of the 10 reports that used qol as their primary endpoint actually provided a definition . a total of nine reports ( 19.6% ) that measured symptoms and/or reported the assessment of performance status ( ps ) in terms of qol did not clearly define qol . only one report published in 1993 used the measure of ps in defining qol assessment . while the percentage of reports that assessed symptoms and ps in terms of qol was 36% ( 5 out of 14 reports ) before the mid-1990s , this percentage dropped to 15.6% ( 5 out of 32 reports ) in later years . in light of this , we surmise that , since 1997 , qol instruments that use a symptom index have slowly been replaced with subjective qol instruments . that is , qol instruments that measure subjective qol have gained acceptance and the concept of qol has shifted from being objective to subjective . clinical researchers can use qol instruments to measure treatment efficacy , yet questions remain as to how qol instruments should be selected , used , and how findings should be interpreted . we based our current review on the methods discussed in the reports by gill et al . and this review also took the following items into consideration when examining instrument selection : whether the instrument was disease - specific or comprehensive , whether or not established instruments were tested for validity , whether or not modification of an established instrument was tested for validity , whether or not instruments were tested for validity despite the citation of references , and whether or not instruments were original ( developed by the investigators themselves ) . most of reports of this review used different disease - specific scales , and this may be due to the wide range of diseases studied in such a limited sample of rct reports . a serious problem apparent in several studies is the use of original instruments that have not been tested for validity or reliability . previous studies have also indicated the need to consider issues of cultural phenomenon , spirituality and level of medical - service satisfaction in the context of qol assessment . qol , being a multifactorial concept , should not be assessed with only one generic instrument or with only one disease - specific instrument , but rather with multiple instruments . . found that respondents are not clearly described in approximately 30% of rct reports that use qol . the reason may be a lack of interest in the issue of respondents ' criteria and/or a mere insufficiency in reporting procedures . suggests that qol assessment may have more validity when conducted by the patient him / herself than when conducted by medical providers . we found 18.8% ( 6 out of 32 articles ) of english reports and 35.7% ( 5 out of 14 articles ) of japanese reports did not describe respondents . further exemplifies that rct articles written in english provide a clearer description than similar reports written in a non - english language . our findings show that 34.4% ( 11 out of 32 articles ) of english written reports as opposed to 35.7% ( 5 out of 14 articles ) of japanese - written reports showed neither response rate nor number of respondents . these insufficiencies in description highlight a need for better reporting standards . needless to say , a study 's quality and validity are most easily assessed by information described in a report [ 32 - 34 ] . japanese interest in qol assessment used in rcts may remain at low levels when compared internationally . our findings suggest insufficient discussion on when to use qol as an outcome measure , how to select an appropriate scale and how to accurately use that scale . further discussion is needed from the standpoints of study objective and psychometrics on the use of qol assessment as an outcome measure in rcts and on assessment methodology . before doing so , however , it is essential to establish a clear definition of qol within each field for each particular disease . contemporary research concerning qol assessment in rcts has demonstrated the need for international minimal standards including , but not limited to , scale selection , minimal standards of psychometric validity , and agreed standards of cultural validity . we are confident that by collecting data from nations worldwide , a basis for further discussion on an international framework for qol will foster . by participating in this discussion , researchers will subsequently gain the tools necessary to improve their nation 's domestic handling of qol assessment . all authors were responsible for planning , conducting and reporting this work and approved the final manuscript .

backgroundstandardization of quality of life ( qol ) assessment and reporting in clinical trials is an imperative issue . while english - speaking countries have led this movement in standardization , there persists to be a limited amount of information from non - english - speaking including japan . in this study , we bibliographically analyze the reporting of randomized controlled trials ( rct ) conducted in japan that used a qol instrument.methodsa pubmed search of reports published between 19702003 followed by an examination of qol reporting and its frequency of use in rcts published from japan.resultspercentages of qol reporting in rcts have increased between 19702003 both worldwide ( 0% for 19701974 to 4.4% for 20002003 ) and in japan ( 0% to 1.8% for the identical periods ) . we found and evaluated 46 rct reports published from japan ( 32 in english , 14 in japanese ) . the most commonly studied clinical condition was cancer ( 26 , 56.5% ) and the most common intervention was drug therapy ( 29 , 63.0% ) . qol was used as the primary endpoint in 10 studies ( 21.7% ) . authors used established qol instruments in 12 studies ( 26.1% ) , developed original instruments in 8 studies ( 17.5% ) and assessed the symptoms or performance status in 10 studies ( 21.7% ) . authors conceptually defined qol in only 6 studies ( 13.0% ) . neither response rate nor number of respondents for questionnaire surveys was specified in 16 studies ( 34.8% ) ; furthermore , 11 studies ( 23.9% ) did not describe respondents ' attributes.conclusionsfindings on relative frequency suggested that japanese authors of rct reports have less interest in qol instruments than other international researchers in western europe and north america . examination of rct reports published from japan revealed that there were several points to be improved in reporting qol instruments . this study highlights the need to define qol measures specific to clinical specialty and to examine methodology for assessing and reporting qol .

furthermore , hyperglycaemia or impaired glucose tolerance is present in more than a third of acs patients without known diabetes . both in patients with diabetes and in non - diabetics with evolving myocardial infarction ( ami ) hyperglycaemia is associated with increased in - hospital death ( rr 1.7 [ 1.22.4 ] ) , and with other adverse events including cardiac arrest , cardiogenic shock and pulmonary oedema [ 24 ] . also in patients with stable coronary artery disease , diabetes is associated with a higher 1-year incidence of death and cardiovascular events ( 13.0% vs. 5.6% ) . hyperglycaemia has been recognised both as mediator and as marker of adverse outcomes in acs patients . elevated glucose levels can reflect the severity of disease when it results from elevated catecholamine and cortisol levels . also , the presence of additional conditions such as infection or sepsis may further disturb carbohydrate metabolism and glucose levels . insulin resistance due to pre - existent diabetes ( recognised or unrecognised ) amplifies the stress - related effects on glucose levels . in patients with ami , hyperglycaemia is associated with higher free fatty acid concentrations , insulin resistance and impaired myocardial glucose metabolism , resulting in an increased oxygen consumption and consequently a more severe ischaemic state . also , insulin may improve myocardial glucose utilisation by reducing free fatty acid concentrations due to its inhibitory effect on lypolysis . finally , insulin has antithrombotic , anti - inflammatory and vasodilative properties [ 810 ] . in critically ill patients insulin therapy improves outcome [ 1113 ] . also in hyperglycaemic patients with ami , glucose - lowering insulin therapy is associated with reduced mortality at 7 and 30 days when compared with standard treatment ( 11.6% and 15.8% vs. 16.5% and 22.1% , respectively ) . similarly , in patients with stable coronary disease , treatment of hyperglycaemia is associated with a reduction in cardiovascular events at 1 year ( hr 0.22 [ 0.050.97 ] ) . the results of these observational studies have led to three randomised controlled trials investigating the effect of intensive glucose - lowering insulin therapy ( igl ) on outcomes in hyperglycaemic diabetic , and non - diabetic patients admitted with ami [ 11 , 16 , 17 ] . a different concept of insulin treatment in acs was tested in the form of a glucose insulin potassium infusion ( gik ) . polarising solution to prevent arrhythmias and avoid further ischaemic damage in unselected patients with acute myocardial infarction [ 18 , 19 ] . from 1960 onwards , different ( randomised ) trials have been done , utilising gik infusion and later insulin therapy in acs patients . the purpose of this manuscript is to provide an overview of the evidence for implementing igl or gik in acs patients by comparing trials with regard patient characteristics , reperfusion treatment , study protocols and outcomes . a systematic pubmed search was performed to identify all clinical trials with insulin ( both igl and gik were included ) in patients with unstable angina or ami . glucose. all abstracts were screened ; when fitting the criteria the manuscript was obtained and reviewed . hazard ratios and confidence intervals for short - term mortality were recalculated to facilitate comparisons . after screening , 20 gik and three igl trials were reviewed . for the gik studies performed before 1994 , a review article was selected that provided a thorough analysis of these trials . from 1994 to 2004 the three igl studies are discussed separately and in more detail as they are more relevant to current clinical practice . table 1overview of randomised controlled trialsstudy nameinclusion% pci / thrombolysis / cabg / nonelocationperiodfath et al . meta - analysis of nine rcts implementing gik in ami0/1/0/99multicentre , international19651987pol - gikchest pain and ecg changes < 24 h , iddm excluded0/60/0/40multicentre ( 16 ) , poland19941995krljanac et al . stemi patients0/100/0/0belgrade , serbia20002001gips iami presenting within 24 h of symptoms91/0/4/5zwolle , the netherlands19982001gips iistemi < 24 h , heart failure excluded93/2/0/5multicentre ( 7 ) , the netherlands20032004create - eclastemi presenting within 12 h of symptom onset9/74/0/7multicentre ( 470 ) , international ( > 10)19982004oasis 6stemi presenting within 24 h ( later 12 h)31/45/0/24multicentre ( 447 ) international ( 41)20032004digami iami < 24 h and diabetes or glucose of > 11.0 mmol / l0/50/0/50multicentre ( 19 ) , sweden19901993digami iiami < 24 h , type 2 diabetes or > 11.0 mmol / l42/36/0/22multicentre ( 44 ) international ( 6 , europe)19982003hi-5ami < 24 h with diabetes or > 7.8 mmol / l35/32/0/33multicentre ( 6 ) australia20012004rct randomised controlled trial , iddm insulin dependent diabetes mellitus , ami acute myocardial infarction , stemi st - elevation myocardial infarction , gik glucose insulin potassium infusion , ecg electrocardiogramtable 2study outcomes and glycaemic parametersstudytreatmentpatientsglucose target range ( mmol / l)glucose level ( mmol / l sd)mortality ( % ) long - term mortalityadmission1624 h3040 dayshr(95% ci)p%monthspfath et al . control4010.04.0gik783.00.30 ( 0.01.4)0.0810.0120.18gips icontrol4648.58.15.88.2gik4767.011.08.57.74.80.83 ( 0.51.4)0.506.5120.32gips iicontrol4458.3 2.51.83.9gik4448.5 2.82.91.61 ( 0.73.8)0.275.3120.33create - eclacontrol10,1079.07.59.7gik10,0889.08.6101.03 ( 0.91.1)0.45oasis 6control13746.710.4gik13747.61.13 ( 0.91.5)0.3610.86nsdigami icontrol31415.7 4.211.7 4.115.626.1insulin 24 h + 3 months sc3067.01015.4 4.19.6 3.312.40.79 ( 0.51.2)ns18.6120.027digami iistandard practice30612.9 4.610.0 3.67.517insulin 24 h + 3 months sc4747.010.0 + fasting 5.07.012.8 4.59.1 3.07.51.00 ( 0.61.7)ns15insulin 24 h4737.010.012.5 4.49.1 2.87.51.00 ( 0.61.6)ns1212nshi-5control11411.1 3.59.0 2.87.17.9insulin/dextrose1264.010.010.8 4.18.3 2.24.40.62 ( 0.21.8)0.426.160.62sd standard deviation , hr hazard ratio , ci confidence interval , ns not statistically significant , gik glucose insulin potassium , sc subcutaneous insulinmean glucose over 24 hcardiac mortalitymortality at 3 months overview of randomised controlled trials rct randomised controlled trial , iddm insulin dependent diabetes mellitus , ami acute myocardial infarction , stemi st - elevation myocardial infarction , gik glucose insulin potassium infusion , ecg electrocardiogram study outcomes and glycaemic parameters sd standard deviation , hr hazard ratio , ci confidence interval , ns not statistically significant , gik glucose insulin potassium , sc subcutaneous insulin mean glucose over 24 h mortality at 3 months in the prethrombolytic era , two observational studies using historical controls showed inconsistent results for the beneficial effect of insulin treatment in diabetic ami patients . clark et al . reported a reduced incidence of arrhythmias and death in the patients treated with igl . did not find a difference in mortality in diabetics treated with an insulin infusion protocol , though they did find a higher mortality rate in diabetic vs. non - diabetic ami patients . between 1990 and 2004 three randomised controlled trials were performed including patients with ami and known diabetes or hyperglycaemia at admission [ 11 , 16 , 17 ] . patients from europe [ 11 , 16 ] and australia were included ; study size varied from 240 to 1253 patients . igl was administered via an intravenous insulin regime for at least 24 h to achieve glucose levels of < 10.0 admission glucose levels ranged from 10.8 to 15.7 mmol / l . at 24 h , glucose levels were reduced in both igl and control groups , but more so in the igl groups . the difference in glucose levels between patients allocated to igl or conventional treatment ranged from 2.1 mmol / l ( digami-1 ) to 0.7 mmol / l ( the average over 24 h in hi-5 ) . long - term mortality was lower in two of the three studies , but this was statistically significant in digami-1 only . however , mortality was lower in the control group of digami-2 . digami-1 was the first randomised trial for igl , and showed a 29% relative reduction ( 18.6% vs. 26.1% ) in 1-year mortality among hyperglycaemic or diabetic ami patients treated with igl during the first 12 h and continued subcutaneously for 3 months . interestingly , about half of this difference in mortality was achieved in the first 3 months , while additional benefit occurred at longer follow - up . in this trial , the second digami trial was designed to verify whether normalisation of serum glucose ( target 57 mmol / l for fasting glucose ) would further improve outcome . three treatment regimens were compared : igl 24 h , igl 24 h continued for 3 months subcutaneously and a control group . no significant difference was seen in survival in the igl groups compared with conventional management ( p = 0.203 ) while , unexpectedly , the highest survival rate was observed in the control group . most importantly , patients were less ill and admission glucose levels were lower in digami-2 than in digami-1 and the investigators did not succeed in normalising glucose levels in the igl groups . additionally , in digami-2 , 14% of the patients in the control group also received insulin infusion during hospital admission , the overall admission glucose levels were lower and reperfusion treatment was given more often ( 78% vs. 50% ) . the more recent hi-5 study did find a lower mortality at 3 and 6 months in favour of igl , which is consistent with digami-1 , although this was not statistically significant . there was also a lower incidence of heart failure during admission ( 12.7% vs. 22.8% ; p = 0.04 ) and reinfarction within 3 months ( 2.4% vs. 6.1% ; p the mortality rates in the hi-5 study were markedly lower than those in the digami studies . this can be explained by the younger population ( 62 vs. 68 years ) , by inclusion of non - diabetic subjects ( 48% ) , increased use of reperfusion therapy ( 67% vs. 50% ) and overall improved care in the more recent time period ( 20012004 vs. 19901993 ) of the hi-5 vs. the digami study . mmol / l or higher , and aimed for values between 7 and 10 mmol / l . after the landmark trial from leuven , in an intensive care population , found a 10.4% absolute difference in mortality in favour of igl targeted to 4.46.1 mmol / l , many subsequent trials used similar targets . the most recent nice - sugar trial even reported a higher incidence of death , similar to digami-2 . since hypoglycaemic episodes occurred more frequently in the igl group , this resulted in a modification in the acc / aha guidelines to use 10 mmol / l as a threshold for initiating treatment in stemi patients . mmol / l ; however , these were established before the nice - sugar results appeared . the fear for hypoglycaemia in certain igl protocols seems grounded , and prevention requires frequent measurement or a wider glucose target range . . evaluated insulin protocols in 24 studies ( including six with ami patients ) . the best results were found using a dynamic scale protocol for continuous intravenous insulin infusion , combined with frequent blood glucose measurement and taking into account changes in glucose levels rather than single values . computerised insulin protocols exist [ 2628 ] and can improve protocol compliance and glycaemic regulation ; however , frequent measurements are required . more recently , a closed loop system for glycaemic regulation in the intensive care unit was developed using a continuous glucose sensor . also , the reliability of continuous glucose sensors needs further improvement and validation is necessary in the ami patient population , particularly in patients with heart failure and hypoperfusion of the skin and subcutaneous tissues . a meta - analysis by fath et al . of gik trials done in the pre - reperfusion therapy era ( before 1988 ) showed a lower mortality in the gik - treated group than in controls ( 16% vs. 21% ; p = 0.004 ) however , in later trials that included patients receiving reperfusion therapy ( either by thrombolysis or mechanical ) , gik did not show beneficial effects . the early randomised trials which were performed between 1994 and 2004 varied in size from 118 to 20,195 and included patients in europe [ 32 , 34 , 35 ] and other continents [ 36 , 37 ] . reperfusion therapy was given as thrombolysis or primary percutaneous coronary intervention in less than 1% to 100% of patients enrolled . all - cause mortality at 3040 days was higher in the gik group in four of the six studies ( reaching statistical significance in pol - gik ) . in three studies that reported longer follow - up ( 612 months ) the mortality difference in favour of the control group remained ( also reaching statistical significance in pol - gik ) . two studies showed a trend in cardiac and all - cause ( gips ) mortality in favour of gik , though these did not reach statistical significance . because in gips a subgroup of patients without heart failure had a lower 30-day mortality in the gik - treated group ( 1.2% vs. 4.2% , p = 0.01 ) , gips-2 was set up excluding patients with symptoms of heart failure , but this study did not show any beneficial effect of gik . the differences in effect of gik between the earlier and more recent studies can be explained by improvements in treatment ( including reperfusion , antiplatelet and -blocker therapy ) , which is reflected in the lower mortality rates of the studies performed after 1987 . in the more recent studies all 3040 day mortality rates were below 16% , which is the mortality rate of gik - treated patients in the meta - analysis of trials from 1965 to 1987 . a parallel could be drawn between gik and the administration of intravenous magnesium in ami patients ; promising beneficial effects in smaller studies were not confirmed in the large randomised magic trial and the concept was abandoned . in the prethrombolytic era , two observational studies using historical controls showed inconsistent results for the beneficial effect of insulin treatment in diabetic ami patients . clark et al . reported a reduced incidence of arrhythmias and death in the patients treated with igl . in contrast , gwilt et al . did not find a difference in mortality in diabetics treated with an insulin infusion protocol , though they did find a higher mortality rate in diabetic vs. non - diabetic ami patients . between 1990 and 2004 three randomised controlled trials were performed including patients with ami and known diabetes or hyperglycaemia at admission [ 11 , 16 , 17 ] . patients from europe [ 11 , 16 ] and australia were included ; study size varied from 240 to 1253 patients . igl was administered via an intravenous insulin regime for at least 24 h to achieve glucose levels of < 10.0 admission glucose levels ranged from 10.8 to 15.7 mmol / l . at 24 h , glucose levels were reduced in both igl and control groups , but more so in the igl groups . the difference in glucose levels between patients allocated to igl or conventional treatment ranged from 2.1 mmol / l ( digami-1 ) to 0.7 mmol / l ( the average over 24 h in hi-5 ) . long - term mortality was lower in two of the three studies , but this was statistically significant in digami-1 only . however , mortality was lower in the control group of digami-2 . digami-1 was the first randomised trial for igl , and showed a 29% relative reduction ( 18.6% vs. 26.1% ) in 1-year mortality among hyperglycaemic or diabetic ami patients treated with igl during the first 12 h and continued subcutaneously for 3 months . interestingly , about half of this difference in mortality was achieved in the first 3 months , while additional benefit occurred at longer follow - up . in this trial , the second digami trial was designed to verify whether normalisation of serum glucose ( target 57 mmol / l for fasting glucose ) would further improve outcome . three treatment regimens were compared : igl 24 h , igl 24 h continued for 3 months subcutaneously and a control group . no significant difference was seen in survival in the igl groups compared with conventional management ( p = 0.203 ) while , unexpectedly , the highest survival rate was observed in the control group . most importantly , patients were less ill and admission glucose levels were lower in digami-2 than in digami-1 and the investigators did not succeed in normalising glucose levels in the igl groups . additionally , in digami-2 , 14% of the patients in the control group also received insulin infusion during hospital admission , the overall admission glucose levels were lower and reperfusion treatment was given more often ( 78% vs. 50% ) . the more recent hi-5 study did find a lower mortality at 3 and 6 months in favour of igl , which is consistent with digami-1 , although this was not statistically significant . there was also a lower incidence of heart failure during admission ( 12.7% vs. 22.8% ; p = 0.04 ) and reinfarction within 3 months ( 2.4% vs. 6.1% ; p the mortality rates in the hi-5 study were markedly lower than those in the digami studies . this can be explained by the younger population ( 62 vs. 68 years ) , by inclusion of non - diabetic subjects ( 48% ) , increased use of reperfusion therapy ( 67% vs. 50% ) and overall improved care in the more recent time period ( 20012004 vs. 19901993 ) of the hi-5 vs. the digami study . mmol / l or higher , and aimed for values between 7 and 10 mmol / l . after the landmark trial from leuven , in an intensive care population , found a 10.4% absolute difference in mortality in favour of igl targeted to 4.46.1 the most recent nice - sugar trial even reported a higher incidence of death , similar to digami-2 . since hypoglycaemic episodes occurred more frequently in the igl group , this resulted in a modification in the acc / aha guidelines to use 10 mmol / l as a threshold for initiating treatment in stemi patients . mmol / l ; however , these were established before the nice - sugar results appeared . the fear for hypoglycaemia in certain igl protocols seems grounded , and prevention requires frequent measurement or a wider glucose target range . . evaluated insulin protocols in 24 studies ( including six with ami patients ) . the best results were found using a dynamic scale protocol for continuous intravenous insulin infusion , combined with frequent blood glucose measurement and taking into account changes in glucose levels rather than single values . computerised insulin protocols exist [ 2628 ] and can improve protocol compliance and glycaemic regulation ; however , frequent measurements are required . more recently , a closed loop system for glycaemic regulation in the intensive care unit was developed using a continuous glucose sensor . also , the reliability of continuous glucose sensors needs further improvement and validation is necessary in the ami patient population , particularly in patients with heart failure and hypoperfusion of the skin and subcutaneous tissues . done in the pre - reperfusion therapy era ( before 1988 ) showed a lower mortality in the gik - treated group than in controls ( 16% vs. 21% ; p = 0.004 ) . however , in later trials that included patients receiving reperfusion therapy ( either by thrombolysis or mechanical ) , gik did not show beneficial effects . the early randomised trials which were performed between 1994 and 2004 varied in size from 118 to 20,195 and included patients in europe [ 32 , 34 , 35 ] and other continents [ 36 , 37 ] . reperfusion therapy was given as thrombolysis or primary percutaneous coronary intervention in less than 1% to 100% of patients enrolled . all - cause mortality at 3040 days was higher in the gik group in four of the six studies ( reaching statistical significance in pol - gik ) . in three studies that reported longer follow - up ( 612 months ) the mortality difference in favour of the control group remained ( also reaching statistical significance in pol - gik ) . two studies showed a trend in cardiac and all - cause ( gips ) mortality in favour of gik , though these did not reach statistical significance . because in gips a subgroup of patients without heart failure had a lower 30-day mortality in the gik - treated group ( 1.2% vs. 4.2% , p = 0.01 ) , gips-2 was set up excluding patients with symptoms of heart failure , but this study did not show any beneficial effect of gik . the differences in effect of gik between the earlier and more recent studies can be explained by improvements in treatment ( including reperfusion , antiplatelet and -blocker therapy ) , which is reflected in the lower mortality rates of the studies performed after 1987 . in the more recent studies all 3040 day mortality rates were below 16% , which is the mortality rate of gik - treated patients in the meta - analysis of trials from 1965 to 1987 . a parallel could be drawn between gik and the administration of intravenous magnesium in ami patients ; promising beneficial effects in smaller studies were not confirmed in the large randomised magic trial and the concept was abandoned . the concept of systematic gik in patients with elevated or normal glucose levels was not supported by recent trials in the reperfusion era and has been abandoned . more research is needed to investigate the role of computerised insulin protocols and continuous glucose sensors to improve safety and efficacy of igl .

patients with acute myocardial infarction ( ami ) and diabetes mellitus , as well as patients admitted with elevated blood glucose without known diabetes , have impaired outcome . therefore intensive glucose - lowering therapy with insulin ( igl ) has been proposed in diabetic or hyperglycaemic patients and has been shown to improve survival and reduce incidence of adverse events . the current manuscript provides an overview of randomised controlled trials investigating the effect of igl . furthermore , systematic glucose insulin potassium infusion ( gik ) has been studied to improve outcome after ami . in spite of positive findings in some early studies , gik did not show any beneficial effects in recent clinical trials and thus this concept has been abandoned . while igl targeted to achieve normoglycaemia improves outcome in patients with ami , achievement of glucose regulation is difficult and carries the risk of hypoglycaemia . more research is needed to determine the optimal glucose target levels in ami and to investigate whether computerised glucose protocols and continuous glucose sensors can improve safety and efficacy of igl .

in this pilot , multicenter , prospective , open - label , randomized study , we enrolled 90 adult patients admitted to general medicine and surgery services . recruited patients had a known history of t2d with a blood glucose ( bg ) prior to randomization of between 140 and 400 mg / dl and a known history of t2d for > 3 months , were between 18 and 80 years of age , and were treated at home with diet alone , any combination of oral antidiabetic agents , or low - dose insulin therapy at a daily dose 0.4 units / kg prior to admission . on admission , we stopped oral antidiabetic agents and insulin therapy , and bg was measured before meals and bedtime . we excluded patients with any bg between admission and randomization of > 400 mg / dl or with a prior history of hyperglycemic crises ; patients with hyperglycemia but without a known history of diabetes ; patients admitted to or expected to require icu admission or cardiac surgery ; patients with a history of pancreatitis or active gallbladder disease , corticosteroid therapy , clinically relevant hepatic disease , or impaired renal function ( glomerular filtration rate [ gfr ] < 30 ml / min or serum creatinine 3.0 mg / dl ) ; and patients with a history of diabetic ketoacidosis ( 24 ) , pregnancy , or any mental condition rendering the subject unable to give informed consent . patients were randomized according to a 1:1:1 ratio into three regimens : sitagliptin once daily , sitagliptin and basal insulin ( glargine lantus ; sanofi ) once daily , and basal bolus insulin with glargine once daily and lispro before meals ( humalog ; eli lilly and company ) . patients treated with sitagliptin received a single dose of 100 mg / day ( at any time of day ) if gfr > 50 ml / min or 50 mg / day if gfr was between 30 and 50 ml / min . patients in the sitagliptin and basal group received a starting total daily dose ( tdd ) of glargine of 0.25 units / kg / day , except for those patients 70 years of age and/or with a serum creatinine 2.0 mg / dl who received a starting tdd of 0.15 units / kg . patients in the basal bolus group were started at a tdd of 0.5 units / kg divided half as insulin glargine once daily and half as insulin lispro before meals . in patients 70 years of age and/or with a serum creatinine 2.0 mg / dl , the starting tdd in the basal bolus group was reduced to 0.3 units / kg in the basal bolus group . patients in all three groups received supplemental ( correction ) doses of insulin lispro before meals and bedtime for bg > 140 mg / dl . the goal of therapy was to maintain a fasting and premeal glucose concentration between 100 and 140 mg / dl . the doses of insulin were adjusted daily according to protocol ( included in supplementary table 1 ) . treatment failure was arbitrarily defined as an average daily bg > 240 mg / dl or two consecutive values bg > 240 mg / dl ( 11,14 ) . if this occurred , patients in the sitagliptin and sitagliptin plus glargine groups were switched to basal bolus regimen starting at a tdd of 0.5 units / kg . bg was measured before each meal and at bedtime ( or every 6 h if a patient was not eating ) using a point - of - care glucose meter ( accu - check ; roche , indianapolis , in ) . in addition , bg was measured at any time if a patient experienced symptoms of hypoglycemia or if requested by the treating physician . the results of bg values are presented as premeal glucose , bedtime glucose , and mean daily bg during the hospital stay after day 1 . this study was conducted at grady memorial hospital ( atlanta , ga ) , emory university hospital , and university of michigan health system . the study protocol and consent form were approved by the institutional review board at each participating institution . a research pharmacist at each institution according to a computer - generated randomization table coordinated the randomization and treatment assignment . all patients were managed for medical and surgical problem(s ) by their primary care team who received a copy of the assigned treatment protocol . the primary outcome of the study was to determine differences in glycemic control as measured by mean daily bg concentration among treatment groups . secondary outcomes included differences between treatment groups in any of the following measures : number of bg values within range , number of hypoglycemic events ( bg < 70 and < 40 mg / dl ) , number of episodes of hyperglycemia ( bg > 200 mg / dl ) after the first day of treatment , ttd of insulin , length of hospital stay , hospital complications , and differences in glycemic control between medicine and surgery patients . this was a noninferiority study design based on the hypothesis that the difference in mean daily bg between basal plus sitagliptin and basal bolus regimens would be no greater than 18 mg / dl ( 1 mmol / l ) ( 11,14 ) . we compared baseline and clinical characteristics and outcomes , such as mean daily bg after day 1 , occurrence of hypoglycemia , and occurrence of complications , among treatment groups and between medical and surgical patients . the comparisons were made with the use of one - way anova for continuous variables and tests ( or fisher exact test ) for discrete variables . the data were generally presented as mean sd for continuous variables and count ( percentage ) for discrete variables . the primary outcome of the study was to determine differences in glycemic control as measured by mean daily bg concentration among treatment groups . secondary outcomes included differences between treatment groups in any of the following measures : number of bg values within range , number of hypoglycemic events ( bg < 70 and < 40 mg / dl ) , number of episodes of hyperglycemia ( bg > 200 mg / dl ) after the first day of treatment , ttd of insulin , length of hospital stay , hospital complications , and differences in glycemic control between medicine and surgery patients . this was a noninferiority study design based on the hypothesis that the difference in mean daily bg between basal plus sitagliptin and basal bolus regimens would be no greater than 18 mg / dl ( 1 mmol / l ) ( 11,14 ) . we compared baseline and clinical characteristics and outcomes , such as mean daily bg after day 1 , occurrence of hypoglycemia , and occurrence of complications , among treatment groups and between medical and surgical patients . the comparisons were made with the use of one - way anova for continuous variables and tests ( or fisher exact test ) for discrete variables . the data were generally presented as mean sd for continuous variables and count ( percentage ) for discrete variables . a total of 90 patients with t2d were consented ( 55 medicine and 35 surgery ) ; 8 patients were excluded from further analysis because they received < 24 h of insulin treatment , were transferred to the icu , or received corticosteroid therapy . a total of 27 patients in the sitagliptin alone group , 29 patients in the sitagliptin and glargine group , and 26 in the basal bolus group were included in the final analysis . there were no significant differences in the mean age , racial distribution , bmi , duration of diabetes , type of treatment prior to admission , or mean hospital length of stay ( los ) among groups . the most common admitting diagnoses in medicine patients were cardiovascular ( 14% ) , infectious ( 28% ) , and pulmonary ( 22% ) disorders , whereas the most common types of surgery were orthopedic ( 28% ) , urologic ( 19% ) , thoracic ( 16% ) , and abdominal ( 9% ) procedures . clinical characteristics of study patients the admission bg , hba1c concentration , and changes in glycemic control during the hospital stay are shown in table 2 . the mean admission glucose for the entire cohort was 211.9 63 mg / dl and the mean hba1c was 8.2 2% . all treatment regimens resulted in prompt and similar improvement in mean daily bg concentration after the 1st day of therapy ( fig . 1 ) . the percentages of glucose readings within target range between 70 and 140 mg / dl were slightly higher in the sitagliptin and glargine ( 43% ) and basal bolus ( 43% ) regimens compared with sitagliptin ( 36% ) , but results were not statistically significant ( p = 0.53 ) ( table 2 ) . similarly , there were fewer bg readings > 200 mg / dl in the sitagliptin and glargine group compared with basal bolus and sitagliptin alone ( 13 , 21 , and 21% , respectively ) ; however , the difference was not statistically significant ( p = 0.23 ) . in addition , there were no differences in the number of treatment failures ( 8 vs. 11 vs. 10% , respectively , p > 0.99 ) . glycemic control , insulin therapy , and hypoglycemic events in patients treated with sitagliptin alone or in combination with basal insulin and basal bolus regimen differences in glycemic control in medicine and surgery patients with t2d treated with sitagliptin alone or in combination with basal insulin and basal bolus regimen . a : mean daily glucose levels in patients treated with sitagliptin alone or in combination with basal ( glargine ) insulin and basal bolus ( glargine + lispro ) insulin regimens . all groups received supplemental ( correction ) doses of lispro before meals and bedtime for bg > 140 mg / dl . b : mean bg levels before meals and bedtime during the hospital stay in patients treated with sitagliptin alone or in combination with basal insulin and basal bolus insulin regimens . the tdd of insulin ( units / day ) was higher in the basal bolus group ( 39.8 22 units / day ) than in the glargine plus sitagliptin ( 28.2 12 units / day ) and sitagliptin ( 11.5 7 units / day ) groups ( p < 0.001 ) . there were no differences in the total dose of basal insulin between basal bolus ( 17 9 units / day ) and sitagliptin and glargine ( 20 9 units / day ) groups , but patients in the basal bolus group received three times the amount of lispro ( 22.4 15 units / day ) before meals compared with the sitagliptin and glargine ( 7.9 6 units / day ) and sitagliptin ( 11.5 7 units / day ) groups ( p < 0.001 ) ( table 2 ) . most patients received insulin supplements for correction of hyperglycemia during treatment with basal bolus 96% , glargine and sitagliptin 93% , and sitagliptin 100% ( p = 0.65 ) . in addition , patients in the basal bolus group received a higher number of insulin injections per day ( 2.4 0.8 ) than patients in the sitagliptin and glargine and sitagliptin groups ( 1.8 0.9 and 1.8 1.1 , respectively , p < 0.01 ) ( table 2 ) . a bg < 70 mg / dl was reported in one patient in the sitagliptin group ( 4% ) , two patients ( 8% ) in the basal bolus group , and two patients ( 7% ) in the sitagliptin and glargine group ( p = 0.86 ) . there were no patients with severe hypoglycemia ( < 40 mg / dl ) . in all cases , hypoglycemia was corrected with oral dextrose , and none of these episodes were associated with adverse outcomes . the level of glucose at admission or at randomization was found to be a good predictor of glycemic control and treatment response during the hospital stay . compared with patients with glucose 180 mg / dl , those with a bg > 180 mg / dl had significantly higher mean daily glucose levels after the 1st day of therapy ( p < 0.001 ) . there were no differences in mean daily bg concentration or in the number of treatment failures among different treatment groups in patients with a randomization bg < 180 mg / dl ( supplementary fig . 2b ) ; however , patients with a randomization bg > 180 mg / dl treated with sitagliptin alone had higher mean daily bg ( 182.7 30 mg / dl ) compared with patients treated with basal bolus ( 168.1 31 mg / dl ) and sitagliptin plus glargine ( 161.8 31 mg / dl ) ( p = 0.08 ) . this pilot , multicenter , randomized clinical trial compared the efficacy and safety of a daily dose of sitagliptin alone or in combination with glargine insulin to a standard basal bolus regimen in general medicine and surgery patients with t2d . we observed similar improvements in glycemic control in all treatment groups with no differences in the mean daily bg , number of bg readings within target , number of treatment failures , hospital los , or number of hypoglycemic events . in addition , the total daily insulin dose and number of insulin injections were significantly less in the sitagliptin groups compared with the basal bolus regimen . the result of this preliminary study suggests that treatment with sitagliptin alone or in combination with basal insulin is safe and effective for the management of general medicine and surgery patients with t2d . the association between hyperglycemia and increased risk of hospital complications is well established in icu and non - icu patients ( 36,2527 ) . recent guidelines from professional organizations ( 1820 ) recommend the use of subcutaneous insulin as the preferred therapy for glycemic control in hospitalized patients in a non - icu setting . the two most common subcutaneous insulin regimens for inpatient glycemic management are sliding scale regular insulin ( ssi ) and basal bolus insulin therapy in combination with correction insulin scale . the use of basal bolus regimen is preferred as it improves glycemic control and reduces the rate of hospital complications ( 12,13 ) . the rabbit 2 medicine trial ( 11 ) reported that a bg target of < 140 mg / dl was achieved in two - thirds of patients treated with basal bolus regimen , whereas only one - third of those treated with ssi achieved target glycemia . the rabbit surgery trial also reported a higher percentage of glucose readings < 140 mg / dl with basal bolus compared with ssi treatment ( 53 30 vs. 31 28% ) ( 14 ) . in this study , we report that sitagliptin alone or in combination with basal ( glargine ) insulin resulted in similar improvements in glycemic control compared with basal bolus regimen . in agreement with recent reports , the level of glucose at admission or at randomization was found to be a good predictor of glycemic control and treatment response during the hospital stay ( 25 ) . compared with patients with glucose > 180 mg / dl , those with a bg 180 mg / dl had a lower mean daily glucose and less treatment failures , independent of treatment regimen . in patients with an admission or randomization bg 180 mg / dl , we observed no differences in mean daily bg concentration or in the number of treatment failures among patients treated with sitagliptin plus supplements compared with patients treated with sitagliptin and glargine or basal bolus regimens ( p = 0.63 ) . patients with a randomization bg > 180 mg / dl treated with sitagliptin alone had higher mean daily bg compared with sitagliptin and glargine or basal bolus regimens ( p = 0.08 ) . this observation indicates that sitagliptin plus rapid - acting supplements ( correction ) before meals is useful in patients with mild - to - moderate hyperglycemia , whereas treatment with sitagliptin plus basal insulin or basal bolus regimens should be considered in those with more severe hyperglycemia . as previously reported ( 11,14 ) , we show that the use of basal insulin as part of a basal bolus regimen or in combination with sitagliptin is well tolerated with a low rate of hypoglycemia . in the rabbit medicine trial , 3% of patients in the basal bolus group had a bg < 60 mg / dl and no patients had a value < 40 mg / dl ( 11 ) . in the rabbit surgery trial , 12% of patients treated with basal bolus had a bg < 60 mg / dl and 4% had a value < 40 mg / dl ( 14 ) . in the current study , a bg < 70 mg / dl was reported in 7% of patients treated with sitagliptin and glargine and in no patients treated with basal bolus or sitagliptin alone . minimizing hypoglycemic events is of major importance in hospitalized patients because it has been shown to be an independent risk factor of poor outcome ( 26,27 ) . we recruited a relatively small number of patients in this pilot study and excluded a large number of patients , which included those admitted to the icu , with clinically relevant hepatic disease , with pancreatitis , with serum creatinine 3.0 mg / dl or gfr < 30 ml / min , with severe hyperglycemia ( bg > 400 mg / dl ) , and receiving a total dose of insulin > 0.4 units / kg / day prior to admission . in such patients , a standard basal bolus approach may be the preferred approach in achieving glycemic control . in addition , our study was not powered to determine differences in hospital complications across the three groups . a large , prospective , randomized , multicenter trial of glycemic control comparing sitagliptin alone or in combination with basal insulin with the basal bolus approach is needed to address these important issues . in summary , these preliminary results indicate that the inpatient use of sitagliptin alone or in combination with basal insulin resulted in a similar improvement in glycemic control compared with a standard basal bolus insulin regimen . these results indicate that sitagliptin alone or in combination with basal insulin is an effective alternative to the basal bolus insulin regimen for general medicine and surgery patients with t2d .

objectivethis study investigated the safety and efficacy of sitagliptin ( januvia ) for the inpatient management of type 2 diabetes ( t2d ) in general medicine and surgery patients.research design and methodsin this pilot , multicenter , open - label , randomized study , patients ( n = 90 ) with a known history of t2d treated with diet , oral antidiabetic agents , or low total daily dose of insulin ( 0.4 units / kg / day ) were randomized to receive sitagliptin alone or in combination with glargine insulin ( glargine ) or to a basal bolus insulin regimen ( glargine and lispro ) plus supplemental ( correction ) doses of lispro . major study outcomes included differences in daily blood glucose ( bg ) , frequency of treatment failures ( defined as three or more consecutive bg > 240 mg / dl or a mean daily bg > 240 mg / dl ) , and hypoglycemia between groups.resultsglycemic control improved similarly in all treatment groups . there were no differences in the mean daily bg after the 1st day of treatment ( p = 0.23 ) , number of readings within a bg target of 70 and 140 mg / dl ( p = 0.53 ) , number of bg readings > 200 mg / dl ( p = 0.23 ) , and number of treatment failures ( p > 0.99 ) . the total daily insulin dose and number of insulin injections were significantly less in the sitagliptin groups compared with the basal bolus group ( both p < 0.001 ) . there were no differences in length of hospital stay ( p = 0.78 ) or in the number of hypoglycemic events between groups ( p = 0.86).conclusionsresults of this pilot indicate that treatment with sitagliptin alone or in combination with basal insulin is safe and effective for the management of hyperglycemia in general medicine and surgery patients with t2d .

latent m. tuberculosis infection ( ltbi ) represents a considerable reservoir of future active disease and contagion . risk factors include co - infection with human immunodeficiency virus ( hiv ) , diabetes mellitus , low body weight , old age , or use of immunosuppressive medications . in immunocompetent individuals , the annual risk of progression preventing ltbi individuals from reactivation ( before they become symptomatic and infectious ) may constitute a major step towards the elimination of tb . therefore , the revised global plan to stop tb ( 201115 ) has set 2015 as the goal for point - of - care tests that can be used for the accurate detection of preclinical tb . bacterial load is associated with disease risk , and antibody levels against m. tuberculosis components may be biomarkers for load as well as disease risk [ 46 ] . stratification of tb suspects into groups of absent , low ( smear - negative tuberculosis ) , and high ( smear - positive tuberculosis ) bacterial burden showed that antibody levels correlated with bacillary burden . in the same study , data of the macaque model , which reproduces key features of latent tb in humans , showed that infection outcome was reflected by the antibody response in the latent infection group , thereby confirming previous animal studies [ 79 ] . although studies were small , increased specific antibody levels during the ltbi stage in humans also characterized progressors [ 5 , 6 ] . antibody - based tests for the diagnosis of active tb disease are often criticized for their lack of specificity in tb endemic regions , which is due to a high background prevalence of ltbi . clearly , further research is needed to elucidate whether m. tuberculosis specific antibody tests can determine active tb and cases at risk for progression and whether lack of specificity of antibody - based tb tests will turn out to be due to a high risk for an early stage of progression to active tb . the low frequency of reactivation in immunocompetent ltbi individuals poses a challenge for the discovery of prognostic markers . therefore , we chose to investigate the distribution of serologic responses , as a nonrandom distribution may point to a subgroup with higher bacterial load and an increased risk for future progression to disease . in a south african tb endemic population , we evaluated the serodiagnostic reactivity of l - alanine dehydrogenase ( aladh ) ( rv2780 ) , nitrate / nitrite response transcriptional regulator narl ( rv0844c ) , periplasmic phosphate - binding lipoprotein psts3 ( rv0928 ) , 19 kda lipoprotein antigen precursor lpqh ( rv3763 ) , and lipoprotein mpt83 ( rv2873 ) . igg responses to each of the two surface - exposed lipoproteins , 19 kda and mpt83 , are predominantly recognized in active tb sera and not in non - tb disease ( ntbd ) sera . the 19 kda antigen promotes binding to host cells and phagocytosis of mycobacteria , inhibits ifn--induced killing of mycobacteria by macrophages , and induces macrophage apoptosis . mpt83 elicits t cell proliferation of the majority of tb patients and is being considered as future subunit vaccine candidate . the third lipoprotein , m. tuberculosis psts3 , which is involved in active transport of inorganic phosphate across the membrane ( import ) , has not been investigated yet in subjects with ltbi for the serodiagnosis of m. tuberculosis . however , psts3 generates ifn--producing cells in a more potent manner than the closely related 38 kda ( psts1 ) , a major m. tuberculosis antigen [ 4 , 18 , 19 ] . the in tb serodiagnostics newly investigated protein antigen narl is a putative nitrate response regulator involved in the regulation of anaerobic metabolism and is part of the membrane fraction of m. tuberculosis . igg responses to the culture filtrate ( and membrane ) protein aladh are unable to distinguish untreated tb patients and controls in endemic settings . aladh is present in m. tuberculosis but not in the vaccine strain mycobacterium bovis bcg [ 21 , 23 ] . it may play a role in cell wall synthesis as l - alanine is an important constituent of the peptidoglycan layer . we focused on iga antibodies because m. tuberculosis - specific iga antibodies discriminated better than igg antibodies between active tb and tb endemic controls in africa as well as between healthy close contacts of pulmonary tb patients and healthy individuals without such contact . moreover , iga production was reported to be highly t - cell dependent , and a protective role for iga was suggested in several murine models of mycobacterial infection , for example , . sera utilized to probe antibody assays were from a retrospective serum bank collected from individuals in an epidemiological field site in metropolitan cape town in south africa with a population of whom 99.7% are of mixed race . the incidence of new smear - positive tb in this community was 341/100 000 population in 2002 and the majority of people harbours latent infection . in the study community , bcg vaccination ( danish strain , 1331 , statens serum institute , copenhagen , denmark ) is routinely administered at birth since 1971 . the study was approved by the ethics committee of the faculty of health sciences at the stellenbosch university and written informed consent was obtained from all participants or their legal guardians in the case of children . inclusion criteria for all healthy control participants enrolled into the study were residence in the described community , absence of clinical signs of tb or other diseases , absence of prior tb , hiv negativity , and no pregnancy . several parameters were employed to determine tb infection status : the mantoux skin test , two different and independent commercial interferon- release assays ( igras ) [ the quantiferon tb gold in tube ( qft ) ( qiagen , hilden , germany , australia ) and t-spot.tb ( oxford immunotec , abingdon , uk ) ] , chest x - ray ( cxr ) , and sputum afb staining . we used > 15 mm as cut off for mantoux test positivity and alternatively > 5 mm as described in more detail in the results section . two igras were used , as rates of positive results have been reported to differ between t-spot.tb and quantiferon - tb gold ( e.g. [ 31 , 32 ] ) . sixty - four consecutively recruited recent household contacts of active tb patients were part of a larger household contact study , and because the non - ltbi individuals constituted fewer than 20% of the contacts , three additional community controls with no known previous tb exposure were added and underwent the same investigations as the household contacts except the igra assays . the majority of ltbi subjects were followed up for the development of active tb disease within 2 years after their first recruitment , and one progressor ( after 3 months ) was identified according to hospital records . forty - two hiv - negative , ziehl - neelsen sputum smear - positive and bactec sputum culture - positive active pulmonary tb patients with no known multidrug resistance that were part of the same larger study as the controls , of which results have been published recently [ 33 , 34 ] , were included . seven tb patients were excluded due to hiv - seropositivity ( n = 1 ) , ntm infection ( n = 2 ) , or concomitant illness , such as diabetes mellitus ( n = 4 ) . all tb patients were self - reporting , untreated cases , and all except 2 had a first episode of active tb . blood samples of control subjects and tb patients ( at diagnosis prior to initiation of treatment ) were taken . after transport of the blood samples to the laboratory ( within 2 h of collection and at ambient conditions ) , serum was separated by centrifugation ( 1250 g for 7 min ) and stored in aliquots at 80c until use . the production of the functional m. tuberculosis l - alanine dehydrogenase ( aladh ) in the heat - induced strain escherichia ( e. ) coli cag629 ( pmsk12 ) has been described previously . for cloning , the genes of the remaining 4 protein antigens ( table 1 ) were amplified by pcr using primers with integrated restriction sites allowing the site - directed insertion of cleaved pcr - products into pet vectors ( novagen ) . all four genes were fused to sequence coding for 6-fold his - tag ( table 1 ) . the genes were expressed in e. coli bl21(de3 ) or in case of psts3 in e. coli rosetta ( de3 ) . the antigens were purified using standard chromatographic methods ( affinity chromatography , ion exchange chromatography , size exclusion chromatography ) . insoluble antigens were solubilized ( refolded ) from denaturating conditions ( 8 m urea ) into buffers free of choatropic reagents . further details regarding the protein purification are described in the supplements ( see supplementary material available online at http://dx.doi.org/10.1155/2015/364758 ) . microtiter plates were coated with m. tuberculosis antigens and serologic antibody responses were determined using standard procedures as described in the supplements . laboratory personnel performing the serodiagnosis assays were blinded to the clinical status of the patients or the controls . subsequent record reviews were done by clinical staff to classify the individuals into clinical groups without knowledge of the serologic response phenotype . graphpad prism ( graph pad , san diego , ca , usa ) was used to create graphs and statistical analyses were performed using medcalc software ( kagi , berkeley , ca ) . the t - test for independent samples or the mann - whitney test was used for the statistical comparison of 2 groups depending on the fact whether or not the data were normally distributed . given the aim to develop a diagnostic test for tb with a specificity level above at least 90% , the experiments were analyzed by using specificity levels of 90% , as was done by other investigators [ 36 , 37 ] . the spearman rank test was used for correlation analyses . generally , a two - tailed p value of p 0.05 was considered significant . sixty - four healthy household contacts of recently ( within past 2 months ) diagnosed active pulmonary tb patients underwent tst and 2 commercial igra tests , qft , and t-spot.tb . fifty - three household contacts were classified as ltbi due to positivity in tst ( induration 15 mm ) , qft and/or t-spot.tb ( n = 53 ; age range : 1559 years ; 58.2% females ) . as noninfected control individuals were rare in the described tb endemic settings , we added 3 healthy nonhousehold contacts ( community controls with no known previous tb exposure ) with tst - indurations of 0 mm , normal chest x - rays and afb - negative - assisted sputum samples to the healthy control group in order to increase the statistical power ( n = 14 ; age range : 10.755.2 years ; 71.4% females ) . the mantoux induration > 15 mm criterion was used for the definition of ltbi , as a large - scale study performed in a rural african population showed that the local environmental mycobacterial exposure is reflected in mantoux indurations that cluster around 10 mm , whereas the m. tuberculosis exposures are reflected by indurations with a mode at 1517 mm or larger . still , the exact definition of the ltbi status is hampered by the lack of a gold standard . according to centers for disease control and prevention ( cdc ) criteria mantoux induration > 5 mm of recent tb household contacts can be considered as ltbi , and we alternatively considered this cut off for defining a positive tuberculin reaction : of the fourteen members of the original hc group , two subjects had mantoux indurations between 5 mm and 15 mm , and both individuals had negative igra results . therefore , in an alternative grouping of the controls , we transferred the 2 igra - negative individuals with mantoux indurations between 5 and 15 mm from the hc group ( hc : n = 12 ) into the ltbi group ( ltbi : n = 55 ) . the 42 smear - positive active pulmonary tb patients ( age range : 1855 years ; 40.5% females ) included in this study were recruited from the same community as the control participants . the 5 protein antigens narl , aladh , 19 kda , psts3 , and mpt83 ( table 1 ) were cloned and expressed in e. coli and purified using standard chromatographic methods . a coomassie brilliant blue r-250 stained sds - page analysis of the 5 proteins is shown in figure 1 . the 109 serum samples derived from 42 pulmonary tb patients , 53 ltbi controls , and 14 non - ltbi controls were tested in a blinded fashion for iga responses specific to the 5 proteins as well as for igg responses to aladh . among controls , we found that elevated iga levels against the investigated 5 antigens were not randomly distributed but concentrated on a subgroup of 28.4% ( n = 19)with particular high levels in a subgroup of 4.5% ( n = 3 ) , which comprised the progressor from latent infection to active tb . to graphically distinguish between those ltbi individuals with negative serum data ( more than 70% of the controls ) and those with moderately elevated and highly elevated serology , we used the mean ranks of iga signals against the 5 antigens investigated to divide the ltbi subjects into the 3 subgroups ltbi ( low iga ) , ltbi ( medium iga ) and ltbi ( high iga ) ( figure 2 ) . furthermore , we statistically compared the non - ltbi controls with either the active tb patients or the ltbi subgroup with elevated iga signals comprising the 2 subgroups ltbi ( medium iga ) and ltbi ( high iga ) ( table 2 ) . based on a cut - off referring to the 92.9-percentile of the healthy non - ltbi controls [ 92.9% ( 95% ci , 66.199.8% ) specificity ] , the iga response against the novel protein antigen narl achieved with 81% ( 95% ci , 65.991.4% ) the highest sensitivity for the detection of active tb patients , followed by anti - aladh iga with 76.2% ( 95% ci , 60.587.9% ) sensitivity and anti-19 kda iga with 64.3% ( 95% ci , 48.078.4% ) sensitivity ( table 2 ) . moreover , based on a specificity of 100% ( 95% ci , 76.8100% ) , anti - narl iga and anti - aladh iga both detected 84.2% ( 95% ci , 60.496.6% ) of the ltbi subgroup with elevated iga signals , followed by anti-19 kda iga with 78.9% ( 95% ci , 54.493.9% ) sensitivity ( table 2 ) . anti - narl iga , anti-19 kda iga , and anti - aladh iga detected the ltbi ( high iga ) subgroup , which comprised the progressor to active tb , with a distinct signal - to - noise ratio compared to the non - ltbi community controls ( figures 2(a ) , 2(b ) , and 2(f ) ) . in contrast , the ltbi ( high iga ) subgroup was indistinguishable from the non - ltbi community controls when using the igg response to aladh ( figure 2(e ) ) . very similar results to those shown in table 2 were obtained when using the alternative groups hc and ltbi ( table s1 ) . when testing for correlations between the antibody od values in ltbi sera ( n = 53 ) ( table 3 and figure 3 ) , we found strong correlations between the iga responses and the 5 protein antigens [ strongest correlation between anti - aladh iga and anti-19 kda iga : spearman 's r = 0.96 ( 95% ci , 0.930.98 ) ; p < 0.0001 ( figure 3(a ) ) ; weakest correlation between anti - narl iga and anti - psts3 iga : r = 0.82 ( 95% ci , 0.710.89 ) ; p < 0.0001 ( figure 3(d ) ) ] . in contrast , we found no or negligible correlations of any of the iga responses with the exemplarily tested igg response to aladh [ e.g. , no correlation between anti - aladh iga and anti - aladh igg : r = 0.21 ( 95% ci , 0.060.46 ) ; p = 0.128 ( figure 3(c ) ) ] ( table 3 ) . very similar results to those shown in table 3 were obtained when using the alternative group ltbi ( table s2 ) . in recent years it became increasingly clear that antibody - based diagnostics of active tb ( often developed and tested in non - tb endemic countries ) performed poorly in tb endemic settings due to high background signals of antibodies in ltbi individuals . in our study we also found that the presence of ltbi affected identification of active tb by serology . in particular , we found that among controls elevated iga levels against the investigated 5 antigens were not randomly distributed but concentrated on a subgroup of < 30%with particular high levels in a small subgroup of ~5% . the consistently elevated iga levels in a subgroup of controls suggest higher mycobacterial load , a risk factor for progression to active tb . whether high iga and/or igg levels have prognostic potential should be investigated in future large scale studies . in several previous reports it has indeed been suggested , directly or indirectly , that progression to active tb may be predicted by increased specific antibodies levels . certain tb - specific antibody responses decline significantly during successful therapy ( and the time thereafter ) when both bacterial load and risk of disease also decline [ 38 , 39 ] . in inactive tb ( defined by a positive response to the tst , negative sputum cultures , and abnormal but stable cxr findings ) , a special form of latent infection known to have increased risk of active tb , elevated specific antibody responses have been reported [ 40 , 41 ] . several independent studies showed that antibody responses to mycobacterial proteins were detectable months to years prior to the diagnosis of tb in persons infected with hiv , for example , , again suggesting that in vivo m. tuberculosis replication may begin long before progression to active tb becomes clinically detectable . specific antibacterial antibodies have been shown to be present in sera obtained from m. tuberculosis h37rv aerosol - infected rabbits , and guinea pigs both with preclinical tb [ 7 , 8 ] . in mouse models of m. avium infection , susceptibility to infection correlated with increased synthesis of specific anti - bacterial antibodies . integration of macaque and human proteome - scale antibody profiling data revealed dynamic characteristics of the antibody response in relation to bacillary burden and infection outcome . two individuals with elevated specific igg responses originally categorized as ltbi were subsequently diagnosed to have culture - confirmed active tb within a few weeks of the serologic testing . moreover , among apparently healthy professional contacts of tb patients ( or pathological specimens thereof ) , elevated specific igg and/or igm responses to mycobacterial antigens were determined in a subgroup of 9 individuals , of whom 4 ( 44.4% ) developed active tb within one year after serological testing . clearly , future large scale , long term prospective studies in different tb endemic areas are needed to further evaluate and substantiate the validity of the hypothesis that certain specific anti - mycobacterial antibody responses are predictors of future active tb . if true , the reported low specificity of serodiagnostics for the detection of active tb would turn out to be due to a high risk for an early stage of progression to active disease and would then offer new opportunities to interrupt the cycle of transmission . if our findings are confirmed the clinical relevance would be as follows : a corresponding antibody - based test in endemic settings would then not differentiate between active tb cases on the one hand and latent or absent infection on the other hand . instead , it would distinguish a high - risk group for preclinical or active tb from a group that is unlikely to progress to clinical disease . this would be of particular significance as such antibody tests could be developed into low - cost , point of care tests that may be used as screening tools , particularly in high tb burden low - resource settings . in such a clinical approach , the pre - screened high risk individuals could then be investigated using established assays such as x - ray , igra , sputum smear , sputum culture and genexpert mtb / rif for final clinical assessment [ 3 , 42 ] . concerning the serodiagnostic results , those ltbi individuals showing the highest iga or igg antibody signals might have the highest risk to progress to active tb as their higher antibody levels may indicate higher bacterial loads . the detection of preclinical and early tb would have considerable clinical impact , as the identification , close monitoring , or early treatment of those ltbi with incipient disease would offer an opportunity to break the cycle of transmission and to prevent more serious lung destruction . in addition , early treatment of ltbi may decrease the emergence of multidrug resistance- ( mdr- ) tb strains , as the low numbers and slow turnover of mycobacteria in ltbi presumably present less opportunity to develop resistance . in the present study the ltbi subject with the highest iga levels against narl , aladh and 19 kda developed active tb within 3 months after recruitment . although this is in line with the working hypothesis , one has to be very careful with its interpretation as we are dealing only with a single case . clearly , as stated above , further research is needed to investigate whether antibody responses to mycobacterial antigens hold any prognostic significance for subsequent development of active tb in individuals with ltbi as previously suggested [ 46 , 8 ] . though a recent metaanalysis showed that so far neither igras nor the tst can discriminate the ~90% of persons with true ltbi from the ~10% who will develop active tb , promising tendencies are noteworthy also in this field [ 45 , 46 ] . subjects with high risk for preclinical tb might be monitored more closely in defined time intervals using the currently best available resources and should be treated where applicable . due to the low frequency of reactivation in immunocompetent ltbi individuals even in tb endemic settings , large - scale longitudinal human studies would have to be conducted to further investigate whether serologic responses to mycobacterial antigens hold any prognostic significance for subsequent development of active tb in individuals with ltbi ( correlates of risk ) . the results of this study and previous human and animal studies [ 4 , 5 , 79 , 3841 ] suggest that this is a promising approach , particularly as the serological tests could be performed on existing stored sera from previous studies . our results suggest that for these investigations besides igg also iga serology should be considered . due to the important role of iga antibodies in the lung the development of point - of - care tests that can be used as correlates of risk or as diagnostic for active tb would constitute a major advance . in conclusion , as suggested in human studies and several animal models [ 4 , 5 , 79 , 3841 ] , it remains a promising hypothesis that those latently infected individuals with antibody responses resembling those of active tb subjects are more prone to progression to active tb . our finding of a nonrandom distribution of antibody responses among ltbi subjects suggests that the development of serodiagnostic kits for the determination of a high - risk group for preclinical or active tb in endemic settings may be possible . furthermore , our results encourage the further investigation of iga besides igg responses in the field of serodiagnosis of active tb and possibly preclinical tb . moreover , iga antibodies against the novel antigen narl were able to determine , besides the putative high - risk subgroup for preclinical tb ( including highest levels for the actual progressor ) , the highest proportion of active tb patients .

elevated antibody responses to mycobacterium tuberculosis antigens in individuals with latent infection ( ltbi ) have previously been linked to an increased risk for progression to active disease . studies in the field focussed mainly on igg antibodies . in the present study , iga and/or igg responses to the mycobacterial protein antigens aladh , narl , 19 kda , psts3 , and mpt83 were determined in a blinded fashion in sera from 53 ltbi controls , 14 healthy controls , and 42 active tb subjects . among controls , we found that elevated iga levels against all investigated antigens were not randomly distributed but concentrated on a subgroup of < 30%with particular high levels in a small subgroup of ~5% comprising one progressor to active tb . based on a specificity of 100% , anti - narl iga antibodies achieved with 78.6% sensitivity the highest accuracy for the detection of active tb compared to healthy controls . in conclusion , the consistently elevated iga levels in a subgroup of controls suggest higher mycobacterial load , a risk factor for progression to active tb , and together with high igg levels may have prognostic potential and should be investigated in future large scale studies . the novel antigen narl may also be promising for the antibody - based diagnosis of active tb cases .

high - pressure techniques have been successfully employed to investigate the polymorphism of a number of different molecular organic solids . the exploration using high - pressure techniques allows for a greater range of phase space to be investigated for new polymorphs of materials . the observation of polymorphism at high pressure is an important factor for the exploration of polymerization , as different three - dimensional arrangements of molecules will have an effect on the structure of the polymer that is produced . a number of groups have been investigating the use of high pressure ( 1100 gpa ) to investigate the chemical reaction of a number of systems via the crystalline state including simple systems such as ethylene , benzene , and even co2 . as an example of the pressures required to induce chemical reactions , chelazzi and co - workers subjected ethylene to 3.0 gpa to produce polyethylene , while santoro and co - workers observed the transition of co2 into an amorphous nonmolecular phase between 40 and 48 gpa . many of these studies have used spectroscopic techniques to elucidate the changes in structure before and after the polymerization reaction . a recent and novel extension of the work is to combine polymerization with crystal engineering , the ability to manipulate intermolecular interactions to create multicomponent materials . by using crystal engineering strategies , goroff s team manipulated intermolecular interactions between oxalamides and diiodobutadiyne to ensure that the carbon carbon triple bonds of diiodobutadiyne aligned in a specific manner so as to aid the pressure - induced polymerization process . by doing so , the authors followed the polymerization process , including changes to the crystalline state , via x - ray diffraction due to the maintenance of long - range order in the overall structure . further work has extended the investigation of high - pressure polymerization to ring systems such as l , l - lactide and carnosine . in this article , we have investigated the room - temperature behavior of acrylic acid ( scheme 1 ) at high pressure using neutron diffraction . acrylic acid is the simplest unsaturated carboxylic acid and is the precursor to poly(acrylic acid ) ( paa ) . paa is used in a wide range of research and industrial applications , ranging from superabsorbent materials to drug delivery vehicles . recent studies by murli and song and our own group have investigated acrylic acid - h4 under high - pressure conditions and have revealed the existence of the new polymorph of pure acrylic acid by rapid compression to 3.3 gpa or through the use of a solution of acrylic acid in a pressure transmitting medium ( ptm ) of 4:1 methanol : ethanol ( 50% v / v ) at 0.61 gpa . the discovery of a new polymorph at high pressure is an important finding , as the spatial arrangement of molecules in the crystal structure may have a significant effect on the structure of the polymer that is created via this polymorph . in our own work , there was an indication that the x - ray radiation was in fact polymerizing the material , as the diffraction pattern started to deteriorate after 15 h of data collection . for this reason , we decided to investigate the changes that occur to the crystal structure before the polymerization process via neutron diffraction due to its noninvasive nature . herein , we present the results of this study , as well as further spectroscopic analysis of the polymerization product . high - pressure neutron powder diffraction data were collected for acrylic acid - d4 using the pearl diffractometer at the u.k . neutron data associated with the research published in this paper can be requested from the corresponding author . perdeuteration is required to avoid the large backgrounds that would be observed in hydrogenous materials due to the large incoherant scattering cross section of hydrogen . as a point of note , there have been a number of studies that have explored the use of neutron diffraction for the hydrogenous materials to negate the requirement to deuterate samples . the sample was first mixed with 20% 4:1 methanol - d4:ethanol - d6 before being added dropwise using a glass capillary into a standard ti zr alloy capsule gasket filled with loosely packed ground silica wool which was used to inhibit the formation of large crystallites . the methanol : ethanol mixture was used as a ptm to provide quasi - hydrostatic conditions during the compression . calcium fluoride was mixed with the silica wool to act as a suitable pressure marker . the resulting capsule assembly was then compressed within a type v3b paris - edinburgh ( p - e ) press equipped with standard profile anvils with cores fabricated from zirconia toughened alumina ( zta ) . the p - e cell ram pressure was monitored and controlled by means of an automated hydraulic system . time - of - flight ( tof ) neutron powder diffraction data suitable for structure refinement were obtained by electronically focusing the 702 individual detector element spectra of the main 2 = 90 detector bank . the summed pattern was then normalized with respect to the incident beam monitor and the scattering from a spherical vanadium standard sample . lastly , the diffraction pattern intensities were corrected for the wavelength and scattering - angle dependence of the neutron attenuation by the p - e cell anvil and gasket ( ti zr ) materials . sample pressures were calculated from the refined caf2 lattice parameters and the known room - temperature equation of state . structures were refined in topasacademic using a z - matrix model parametrized in terms of the intramolecular bond distances , angles , and torsions and the molecular position and orientation . the dft - optimized structures ( see below ) were used to formulate restraints which were then applied to the rietveld refinements , as described in ref ( 29 ) . figure 1 shows two rietveld fits of data at 0.32 gpa ( phase i ) and 0.87 gpa ( phase ii ) , and the corresponding crystallographic data for selected pressures is shown in table 1 . a plot of the diffraction at various pressures ( figure es1 ) as well as the crystallographic information for all pressures ( table es1 ) can be found in the supporting information . figure 2 shows the changes in unit cell parameters and molecular volume with increasing pressure . the phase transition from phase i to phase ii can clearly be observed at 0.8 gpa . during the compression of acrylic acid , it was noted that the sample pressure continued to increase slowly after a finite change of pressure before eventually stabilizing after an interval of 30 min . we have attributed this unusual behavior to the solubility of acrylic acid in the ptm and that during compression there is an initial solubilization of the acrylic acid before it recrystallizes back out of solution , causing the creep in pressure . this effect became less pronounced at higher pressures , which would be consistent with the expected decrease in solubility with pressure . the rietveld refinements of phase i at 0.33 gpa ( upper ) and phase ii at 0.87 gpa ( lower ) . geometry optimizations were performed by periodic density functional theory ( dft ) using the dmol code as part of the materials studio modeling suite . the dnp numerical basis set was used in combination with the pbe functional with the tkatchenko the unit cell dimensions were held fixed at the values obtained in pawley refinements of the neutron powder diffraction data described above , and coordinates were allowed to optimize . convergence was defined when the maximum changes in total energy , displacement , and gradient were 10 ha , 5 10 , and 2 10 ha , respectively . the raman modes for acrylic acid - d4 were calculated using the plane - wave - pseudopotential castep v4.2 program as implemented in materials studio , employing the generalized gradient approximation ( gga ) functional pw91 using the optimized phase i structure at 0.32 gpa . norm - conserving pseudopotentials optimized for gga dft methods with a basis set cutoff energy of 830 ev were used . lattice parameters remained fixed , but the atomic positions were optimized according to the following criteria : total energy convergence 5 10 ev / atom , maximum force on any atom 0.01 ev / , stress 0.02 gpa , and atomic displacements 5 10 . the unit cell parameters as a function of pressure ( upper ) . the molecular volume of acrylic acid as a function of pressure ( lower ) . the dotted line is representative of the phase boundary between the two phases . using the optimized structures from the dft calculations , the lattice energies and molecule molecule interaction energies were calculated using the pixelc module in the clp package by gavezotti . electron densities were calculated at the mp2/6 - 31 g * * level using gaussian 09 . calculation of the energies using the refined data showed a similar trend but not as smooth as those observed with the optimized structures highlighting the limitations of refining powder data even with the implementation of restraints ; hence , the optimized structures were used . during the creation of the z - matrix , bond lengths to deuterium atoms were normalized to neutron values and thus these values were retained for the energy calculations . table es2 ( supporting information ) provides the total lattice energy as well as the breakdown of intermolecular interactions into coulombic , electrostatic , dispersion , and repulsion terms . acrylic acid phase i crystallizes in a layered structure , with the layers perpendicular to the c - axis ( space group ibam ) , where the acid groups hydrogen bond via a dimer motif ( figure 3 ) ; this is the same phase that is observed in the h4 system . the dimers are arranged in the layer so that the cd groups of c2 and c3 are in close contact with the oxygen atoms of a neighboring dimer . pixel calculations of phase i show that by far the most favorable molecule molecule interaction is , unsurprisingly , between the hydrogen bonded molecules ( interaction 1 ) with the dispersive and coulombic terms showing the greatest stabilizing contribution ( figure 4a ; table es2 , supporting information ) . interaction 2 represents the interaction between the central molecule and molecules directly above and below ( in different layers ) where the carbonyl groups of the acid are arranged in an antiparallel arrangement ( figure 4b ) . this type of interaction has been investigated for its potential stabilizing contribution to the crystalline state by allen et al . they showed through the use of the cambridge structural database ( csd ) and ab initio molecular - orbital calculations that carbonyl carbonyl interactions can be comparable to medium - strength hydrogen bonds albeit that they are a little weaker in this case . while the energies calculated from pixel can not be broken down into individual interactions , there is a significant contribution from the dispersive component in interaction 2 which is known to contribute to carbonyl carbonyl interactions . interaction 4 is the second most attractive interaction displayed in phase i , where neighboring dimers in the layer interact through close contacts between the cd2 and cd4 with o1 and o2 . due to the layered nature of the structure , this interaction will be important as the acrylic acid is subjected to an applied pressure . a layer of molecules lying perpendicular to the c - axis as observed in phase i and phase ii of acrylic acid with the molecules hydrogen bonding through a typical carboxylic acid dimer . atom colors are assigned as follows : gray , carbon ; red , oxygen ; white , hydrogen . six most imporatant interactions for the acrylic acid polymorphs : ( a ) int . ( b ) the interlayer interactions observed in phase i. ( c ) the interlayer interactions observed in phase ii . similar interactions have been identified between polymorphs and their descriptor kept constant despite the structural changes between phases , e.g. , int . atom colors are assigned as follows : gray , carbon ; red , oxygen ; white , hydrogen . on compression to 0.69 gpa , the unit cell parameters of phase i compress by 0.5 , 0.27 , and 2.73% for the a- , b- , and c - axis , respectively , with the molecular volume reducing by 4.47% ( figure 2 ) . increasing the pressure to 0.86 gpa initiates a sluggish transition to a new phase ( phase ii , p21/c ) , similar to that of acrylic acid - h4 , which is complete after 30 min . this pressure is slightly higher than was originally quoted in our previous paper of acrylic acid - h4 ( 0.6 gpa , ) but this may be due to either the different ratios of acrylic acid to ptm that was used in this study compared with our original study ( 20% v / v ptm this study ; 50% v / v previous work ) , a deuteration effect , or the sample environment that has been used in this study . the phase transition was also monitored in a diamond anvil cell ( dac ) where the reconstructive nature can clearly be observed ( figure 5 ) . the longer time for full conversion in the dac ( 60 min ) was probably due to the larger crystallites involved compared with the deliberately fine powder that is observed in the p - e cell during the neutron experiment ; the quality of the powder was monitored indirectly through the analysis of the patterns from different detector banks . over the phase transition , the a - axis approximately halves , the b - axis maintains its compression rate , i.e. relatively unchanged , and the c - axis elongates . these changes to the unit cell result in a small change in the molecular volume ( 2.9% reduction ; cf . 2.0% from 0.52 to 0.68 gpa in phase i ) , but it does lead to a significant reduction in the void volume from 30 to 4 ( as calculated by materials mercury ) . this is an 87% reduction in void volume compared with a 47% reduction that is observed from 0.31 to 0.68 gpa in phase i. images of the i ii transition in acrylic acid - d4 observed in a diamond anvil cell taken at various times after a pressure increase to 0.8 gpa . ( a ) 0 min , ( b ) 12 min , ( c ) 18 min , ( d ) 26 min , ( e ) 30 min , ( f ) 40 min , ( g ) 48 min , ( h ) 56 min , and ( i ) 60 min . the packing of molecules within the layers is similar in both phases , but in phase ii , the layers are puckered and their relative positions with respect to neighboring layers also change . there is a 5.52 , 7.26 , and 10.94% compression of the a- , b- , and c - axes , respectively , that culminates in a 21.41% compression of the unit cell volume from 0.86 to 7.2 gpa . the bulk modulus for phase ii was determined to be 6.6(7 ) gpa with a v0 value of 371(3 ) using a murnaghan equation of state ( eos ) in eosfit5.2 . the data were difficult to fit due to the compressibility of the material and the lack of low pressure data or reliable ambient pressure volume ( acrylic acid is liquid under ambient pressure and temperature ) . these were the best values calculated from a number of different eos ( birch murnaghan , vinet and murnaghan ) . the v0 value seems reasonable given the molecular volumes of the two phases are very close to one another ; hence , using the value at 0.33 gpa , one obtains a v0 value of 364 . up to this pressure , acrylic acid remains molecular and no polymerization occurs , consistent with the previously observed onset of polymerization at 8 gpa with loss of raman modes . we have also carried out raman studies on the h4 form to 8.2 gpa , and we see little change in the spectrum except around the c c torsion ( 350 cm ) and c = c c bend ( 380 cm ) which may be signifying that the polymerization process is imminent . due to the experimental setup ( zta anvils for a better signal - to - noise ) and peculiar behavior of the sample , we were unable to compress the sample beyond 7.21 gpa . the six most important molecular interactions ( > 4 kj mol ) in phase ii are shown in figure 4 , in which analogous interactions in phases i and ii are illustrated . the interactions within the layers remain the same ( interactions 1 and 4 ) even though neighboring dimers are now observed at a slight angle to each other due to the puckering of the layer . after the change of 10 kj mol over the phase transition , interaction 1 does not vary significantly until 4.1 gpa , where the total energy for the interaction starts to become more negative overall . at this point , there is an increase in the rate of change of the coulombic term versus centroid distance , which may account for the effect on the overall energy of interaction . toward 7.2 gpa , the energy of the interaction reaches a plateau , signifying that if the pressure were increased any further then either the repulsion between the molecules would overcome any attractive forces or the compression would be taken up elsewhere in the structure . a search of the csd shows that the c1c1 ( carbonyl carbon ) distance observed at 7.2 gpa is at the minimum of those represented in the database , reflecting , perhaps , the lower limit that the hydrogen bonded dimer can be sustained before another molecular arrangement is required ( figure es2 , supporting information ) . interaction 2 still possesses an antiparallel interaction in phase ii despite being in a slightly different orientation , which emphasizes the importance of this interaction in stabilizing the structure . as further pressure is applied to the structure , the total energy remains fairly constant before becoming less favorable at a value of 3.2 between the centroids of the molecules . in terms of the specific interaction between the carbonyl groups , the distance between the carbon and oxygen at this pressure is 3.15(5 ) , which is close to the energy minimum observed by allen et al . for this type of interaction ( 3.04 ) . thus , while the pixel calculations calculate molecule molecule interactions , the trends in the energy associated with interaction 2 are consistent with previous literature detailing the interaction between carbonyl groups . the total energy only starts to become more repulsive at 4.9 between the centroids , which equates to a contact distance of 2.16 between d2 and o2 which is somewhat surprising given that this interaction is within the layer and therefore should have less room for compression in comparison to interactions between the layers . interaction 5 , between molecules in different layers , shows the greatest change during the compression , with the overall energy changing by 13 kj mol . the molecules are aligned so that the alkene moieties are in close proximity to one another and thus will be associated with the polymerization reaction . the distance between the alkene groups compresses to 3.519 which is close to the optimal distance that is observed for polymerization reactions using diynes and therefore is close enough to initiate the reaction . plot of intercentroid distance and energy for each of the six highlighted interactions in phases i ( filled symbols ) and ii ( open symbols ) of acrylic acid . the behavior of acrylic acid on decompression was not investigated using neutron diffraction ; however , the experiment was repeated using raman spectroscopy and a diamond anvil cell as the pressure vessel . a few drops of the exact same solution of acrylic acid - d4 in perdeuterated methanol : ethanol ptm were loaded into the dac and compressed to 6.3 gpa ( a similar pressure to the neutron experiment ) ( figure 7 , upper ) . this is significantly shorter than the neutron experiment ; however , previous research by murli and song suggested that the high pressure phase was stable for over one month at 4.5 gpa . the presence of the c = c stretch ( 1571 cm ) throughout the compression series shows that the molecular nature is retained to 6.3 gpa , which is consistent with the neutron data . this result also confirms that the energy of the laser used ( 532 nm ) for pressure determination and raman spectroscopy was not high enough to induce polymerization , as has been observed in other molecular systems at high pressure . raman modes of the perdeuterated sample were calculated using castep ( figure 7 , upper ) and show that deuteration has had a significant effect on the frequencies of specific stretches ; e.g. , the c = c bond , identified by murli and song , is at 1644 cm for the hydrogenated sample , whereas we observe this frequency at 1571 cm for the d4 sample . the castep spectrum was calculated using the 0.33 gpa data after geometry optimization ( upper ) ; raman spectra on decompression ( lower ) . note the changes to the raman peak at 1635 and 1685 cm between 2.2 and 0.2 gpa . on decompression , the sample remains in the molecular phase to 2.2 gpa ; however , further decompression to 0.2 gpa shows a significant relative increase in the vibrations at 1635 and 1685 cm when compared with the c = c stretch , indicating that polymerization has occurred . this delay in polymerization on decompression has been observed in other systems , such as benzene , where the reaction may be initiated at high pressure , but it is only on the release of pressure that sufficient volume is available for the reaction to reach completion . the drop from 2.0 to 0.2 gpa straddles the i ii phase transition , which would also allow freedom for the molecules to move , thereby permitting polymerization to occur . we have conducted subsequent experiments where we have loaded the exact same sample from the neutron experiment into a diamond anvil cell , compressed it to 1 gpa , and left it at this pressure for a week . even at this low pressure , the material had started to polymerize which would help to support our theory with respect to the polymerization mechanism . experimental details for the ft - ir measurements can be found in the supporting information . upon opening the ti zr gasket from the p - e cell neutron diffraction experiment , there was an odor of residual unreacted acrylic acid , but the vast majority was polymerized which bound together the other content of the capsule ( caf2 and glass wool ) . d / o d region ( 22502750 cm ) and the o h region ( 32503750 cm ) . the o h stretch will be due to water present in the sample adsorbed after opening the gasket to the atmosphere due to the hygroscopic nature of poly(acrylic acid ) ; paa has been used as a hydrogel in the past . the c = o stretch remains in a similar position to other paa samples of different molecular weights ( purchased from sigma - aldrich , figure es3 , supporting information ) ( 1700 cm ) ; however , the stretch does not possess a shoulder toward higher wavenumbers that appears in the other higher molecular weight polymers . this may be due to a greater proportion of the acid moieties being engaged in hydrogen bonding ; therefore , the environments around the carbonyl groups are less varied than in a polymer produced through more conventional routes ; i.e. , the molecules are fully hydrogen bonded in the solid state , and this is translated into the polymer product . this would also affect the bands around 1178 and 1248 cm which have been characterized as c o stretching coupled with oh bending . the polymer produced during the neutron experiment shows very small absorbances in this region compared with the high molecular weight polymers , but there is a more substantial absorbance between 800 and 1100 cm that could be partially due to the c o stretch in a highly hydrogen - bonded system but this assignment is cautious as the si o stretch from the glass wool also appears at 891 cm . the sample was heated at a rate of 10 k min from 300 to 650 k ( figure es4 , supporting information ) . the trace shows two main endothermic events at 330 and 503 k which represent dehydration and the formation of poly(acrylic acid ) anhydride . these temperatures are in agreement with the study by moharram and allam , indicating that , despite the observed changes in the raman and ir , the thermal properties remain consistent with polymers synthesized by standard procedures . we have confirmed that the acrylic acid - d4 undergoes a polymorphic transition at 0.87 gpa using neutron diffraction . we have identified that acrylic acid retains its molecular nature up to a static pressure of 7.21 gpa but then undergoes polymerization between 0.2 and 2.2 gpa on decompression , which can be attributed to the increase in molecular volume that allows the polymerization reaction to proceed unhindered . we have also shown that one can also initiate polymerization by applying pressures of 1 gpa for a period of a week . while the dsc trace shows previously identified thermal events , the raman and ir present peaks are different from those observed for polymers synthesized under conventional polymerization conditions , thereby indicating the novel polymeric structure obtained via high pressure techniques .

this article details the exploration of perdeuterated acrylic acid at high pressure using neutron diffraction . the structural changes that occur in acrylic acid - d4 are followed via diffraction and rationalized using the pixel method . acrylic acid undergoes a reconstructive phase transition to a new phase at 0.8 gpa and remains molecular to 7.2 gpa before polymerizing on decompression to ambient pressure . the resulting product is analyzed via raman and ft - ir spectroscopy and differential scanning calorimetry and found to possess a different molecular structure compared with polymers produced via traditional routes .

-synuclein is a 140 amino acid brain protein , mainly localized in presynaptic terminals [ 1 , 2 ] . although the detailed physiological functions of -synuclein are still elusive , recent studies suggest that it plays a key role in synaptic functions cooperated with cysteine - string protein- ( csp ) , which contains a typical domain for hsp40-type molecular cochaperones . in the subgroup of neurodegenerative disorders termed synucleinopathies , -synuclein is known to polymerize into fibrils and to accumulate in pathologic hallmark inclusions , such as lewy body ( lb ) , lewy neuritis ( ln ) , and glial cytoplasmic inclusions ( gcis ) . the lb and ln are characteristic of parkinson 's disease ( pd ) , and point mutations or gene multiplications of -synuclein are responsible for familial pd [ 46 ] . moreover , transgenic flies overexpressing mutated human -synuclein showed progressive locomotor disability with dopaminergic neuronal cell death with intracytoplasmic inclusions . these findings suggest that abnormal -synuclein metabolism plays a key role in neurodegenerative processes in pd and other synucleinopathies , but the precise underlying mechanisms still remain unknown . to elucidate the possible roles of -synuclein in neurodegeneration , we have developed cells that overexpress wild - type or mutant -synucleins in dopaminergic or inducible catechol - quinone producing cell lines [ 9 , 10 ] . the inclusions in synucleinopathies were proved to be composed of -sheet rich fibrils formed by nitrated species of -synuclein . several lines of evidence suggested that reactive oxygen species ( ros ) play a key role in the conformational change of -synuclein and the following aggregate formation [ 1215 ] . we developed human dopaminergic sh - sy5y cells overexpressing wild - type or mutant -synucleins , and established experimental models of intracellular aggregate formation following the exposure to various ros . the aggregates thus formed were immunopositive for ubiquitin , nitrotyrosine , and dityrosine , and positive for thioflavin s staining , which was in good agreement with the pathological features of inclusion bodies in synucleinopathies . the -tubulin and molecular chaperones coexisted as well , suggesting that the aggregate formation was associated with the intracellular transport system for protein turnover responses against the toxic effects of misfolded proteins . such mechanisms are called aggresome and are suggested to represent one of the cytoprotective responses [ 1618 ] . interestingly , the recent study on huntingtin showed that inclusion body formation reduced the risk of neuronal death . however , it is still controversial whether the aggregate formation of -synuclein has cytotoxicity in the neuronal cell or sequesters toxic species . we established a cellular model in which intracellular -synuclein aggregations were efficiently formed in response to various types of ros exposure [ 9 , 19 ] . under these conditions , a significant number of cells showed caspase 3 activation . to explore possible relationships between the aggregate formation and apoptosis , first we investigated whether -synuclein aggregates colocalized with activated caspase-3 using a double immunostaining method . following the combined exposure of the cells to a no donor and rotenone surprisingly , immunocytochemical analyses revealed that the aggregate positive cells did not show any caspase 3 activations and , conversely , that caspase 3 activated cells did not contain any -synuclein aggregates . iron was able to induce -synuclein aggregates more effectively than any other ros inducers and no donors , suggesting the iron plays a key role in the aggregate formation . when using both ros and no inducers , the addition of ferric iron triggered further aggregate formation , but cells positive for activated caspase 3 were not coincident with aggregate positive cells . in quantification experiments , it was revealed that caspase 3-positive cells were decreased by the addition of ferric iron . on the other hand , by chelating ferric iron , the aggregate formation was decreased with concomitant increases of caspase 3 activation . these data suggest that the ferric iron plays a key role in the -synuclein aggregation . furthermore , these data also imply that the aggregate formation may be cytoprotective against various cellular insults including oxidative stress [ 19 , 20 ] . since -synuclein is ubiquitously expressed at high levels in all brain regions , the mechanisms responsible for the preferential and selective neurodegeneration of dopaminergic neurons in the substantia nigra remain to be determined . previous studies suggested that the specific vulnerability of dopaminergic neurons may be linked to the cytotoxic oxidative potential of dopamine . highly reactive oxygen species ( ros ) are generated not only in dopamine oxidation but also during the decay of catechol - derived orthoquinones which covalently incorporate into a variety of molecules including proteins and nucleic acids . on the other hand , previous reports demonstrated that -synuclein might regulate dopamine metabolism by direct interaction with the tyrosine hydroxylase , the dopamine transporter and vesicular monoamine transporter ( vmat2 ) , key proteins in the regulation of the dopamine content within nerve terminals [ 26 , 27 ] . therefore , the pathological metabolism of -synuclein may be closely linked to the misregulation of dopamine , consequently leading to neuronal death . in support of this notion , catechol - derived orthoquinones ( eg , dopamine - quinone or dopa - quinone ) accelerate and stabilize the formation of -synuclein protofibrils by inhibiting the conversion of toxic protofibrils into fibrils [ 28 , 29 ] . to shed light on the pathophysiological mechanisms underlying -synuclein - mediated neurodegeneration in dopamine neurons , we developed novel neuronal cell lines coexpressing -synuclein ( wild - type or a53 t ) and tyrosinase that produces catecholamines and their oxidized metabolites [ 30 , 31 ] . investigating the effects of wild - type or mutant -synuclein expression , we found that the coexpression of wild - type and a53 t mutant -synuclein in tyrosinase - overexpressing cells exacerbated dna damage and successive apoptotic cell death compared to the cells overexpressing cat or antisense -synuclein . both wild - type and a53 t mutant -synucleins coexpressed with tyrosinase resulted in the gradual accumulation of high - molecular weight complexes immunopositive for -synuclein . this band , possibly representing oligomerized forms , corresponded to the size of -synuclein tetramer and was also detected by the nbt / glycinate redox - cycling staining , suggesting that it was modified by quinones . moreover , during these processes , the mitochondrial membrane potential was specifically decreased without the activation of map kinases . although the underlying mechanism(s ) of neuronal cell death following the coexpression of tyrosinase and -synuclein are still elusive , it is likely that -synuclein modified by the oxidized catechol metabolites forms cytotoxic intermediates , that is , recent reports suggested that protofibrillar -synuclein tightly binds to lipid bilayers and increases the membrane permeability by forming pore - like structures [ 3335 ] . while the membranous structures damaged by protofibrils in dopaminergic nerve terminals remain unknown , intracellular organelles , such as synaptic vesicles and mitochondria , are possible candidates . in this regard , disruption of synaptic vesicle membranes would result in an increase of the cytoplasmic dopamine levels that would trigger the further accumulation of dopamine - quinone and dopamine - derived oxyradicals and thus lead to a vicious cycle . likewise , mitochondrial enzymes in the electron transport chain and the functional permeability transition pores are impaired by dopamine oxidation products making it plausible that the early damage of mitochondria observed in this cellular model reflects the actions of -synuclein protofibrils and the subsequent increase of the membrane permeability in the presence of oxidized catecholamine metabolites . we further analyzed the resting membrane potential and whole - cell membrane conductance using the ramp voltage in the cell lines expressing wild - type or mutant -synuclein . interestingly , the cells expressing a53 t -synuclein have the most depolarized membrane potential . by the application of the ramp voltage under the whole cell voltage - clamp condition , we obtained an almost linear current - voltage ( i - v ) relationship in each cell line . the slope of the i - v relationship in the cells expressing mutant -synuclein was significantly steeper than that in the cells expressing the vector alone or wild - type -synuclein , indicating that the expression of mutant -synuclein results in higher ion permeability of the plasma membrane . because it has been suggested that abnormal intracellular calcium homeostasis plays a crucial role in the pathogenesis of neurodegenerative disorders , the intracellular free calcium concentrations in -synuclein - transfected cells were quantified using a calcium indicator dye , fura-2 . notably , both the intracellular calcium concentrations under basal conditions and after depolarization induced by potassium chloride application were significantly higher in the mutant -synuclein expressing cells than in cells expressing the empty vector or wild - type -synuclein . these results suggest that mutant -synuclein is involved in the perturbation of the intracellular calcium homeostasis . taken together , our data from cellular models of synucleinopathy suggest that oligomer or protofibril , but not aggregate or fibril , formation of -synuclein plays a key role in the pathomechanisms of synucleinopathy ( figure 1 ) . the iron specifically triggers the aggregate formation of -synuclein , but this seems to be a cytoprotective process . the cytotoxic protofibril formation may be facilitated by not only gene mutations , but also the modification of -synuclein by catechol - derived quinones [ 28 , 32 ] . the cellular membranes are the primary targets injured by protofibrils , and the mitochondrial dysfunction seems to be an initial step in the neurodegeneration of synucleinopathy . obviously , protofibrils or oligomers of -synuclein are heterogeneous in size and stability and exist as mixtures in the cytosol . therefore , at present it is difficult to specify the detailed molecular structures that may be responsible for the cellular injuries . however , from these data , it is plausible that the reduction of the protofibril pool may rescue neurons from death ( figure 1 ) . if this is the case , not only the acceleration of degradation ( c in figure 1 ) but also the facilitation of aggregate formation ( d in figure 1 ) , may be a novel strategy for the treatment of synucleinopathy . of course , the most important way would be to decrease the input ( a in figure 1 ) into the protofibril pool . the inclusions in synucleinopathies were proved to be composed of -sheet rich fibrils formed by nitrated species of -synuclein . several lines of evidence suggested that reactive oxygen species ( ros ) play a key role in the conformational change of -synuclein and the following aggregate formation [ 1215 ] . we developed human dopaminergic sh - sy5y cells overexpressing wild - type or mutant -synucleins , and established experimental models of intracellular aggregate formation following the exposure to various ros . the aggregates thus formed were immunopositive for ubiquitin , nitrotyrosine , and dityrosine , and positive for thioflavin s staining , which was in good agreement with the pathological features of inclusion bodies in synucleinopathies . the -tubulin and molecular chaperones coexisted as well , suggesting that the aggregate formation was associated with the intracellular transport system for protein turnover responses against the toxic effects of misfolded proteins . such mechanisms are called aggresome and are suggested to represent one of the cytoprotective responses [ 1618 ] . interestingly , the recent study on huntingtin showed that inclusion body formation reduced the risk of neuronal death . however , it is still controversial whether the aggregate formation of -synuclein has cytotoxicity in the neuronal cell or sequesters toxic species . we established a cellular model in which intracellular -synuclein aggregations were efficiently formed in response to various types of ros exposure [ 9 , 19 ] . under these conditions , a significant number of cells showed caspase 3 activation . to explore possible relationships between the aggregate formation and apoptosis , first we investigated whether -synuclein aggregates colocalized with activated caspase-3 using a double immunostaining method . following the combined exposure of the cells to a no donor and rotenone , -synuclein aggregates were efficiently formed in the cytoplasm as previously reported . surprisingly , immunocytochemical analyses revealed that the aggregate positive cells did not show any caspase 3 activations and , conversely , that caspase 3 activated cells did not contain any -synuclein aggregates . iron was able to induce -synuclein aggregates more effectively than any other ros inducers and no donors , suggesting the iron plays a key role in the aggregate formation . when using both ros and no inducers , the addition of ferric iron triggered further aggregate formation , but cells positive for activated caspase 3 were not coincident with aggregate positive cells . in quantification experiments , it was revealed that caspase 3-positive cells were decreased by the addition of ferric iron . on the other hand , by chelating ferric iron , the aggregate formation was decreased with concomitant increases of caspase 3 activation . these data suggest that the ferric iron plays a key role in the -synuclein aggregation . furthermore , these data also imply that the aggregate formation may be cytoprotective against various cellular insults including oxidative stress [ 19 , 20 ] . since -synuclein is ubiquitously expressed at high levels in all brain regions , the mechanisms responsible for the preferential and selective neurodegeneration of dopaminergic neurons in the substantia nigra remain to be determined . previous studies suggested that the specific vulnerability of dopaminergic neurons may be linked to the cytotoxic oxidative potential of dopamine . highly reactive oxygen species ( ros ) are generated not only in dopamine oxidation but also during the decay of catechol - derived orthoquinones which covalently incorporate into a variety of molecules including proteins and nucleic acids . on the other hand , previous reports demonstrated that -synuclein might regulate dopamine metabolism by direct interaction with the tyrosine hydroxylase , the dopamine transporter and vesicular monoamine transporter ( vmat2 ) , key proteins in the regulation of the dopamine content within nerve terminals [ 26 , 27 ] . therefore , the pathological metabolism of -synuclein may be closely linked to the misregulation of dopamine , consequently leading to neuronal death . in support of this notion , catechol - derived orthoquinones ( eg , dopamine - quinone or dopa - quinone ) accelerate and stabilize the formation of -synuclein protofibrils by inhibiting the conversion of toxic protofibrils into fibrils [ 28 , 29 ] . to shed light on the pathophysiological mechanisms underlying -synuclein - mediated neurodegeneration in dopamine neurons , we developed novel neuronal cell lines coexpressing -synuclein ( wild - type or a53 t ) and tyrosinase that produces catecholamines and their oxidized metabolites [ 30 , 31 ] . investigating the effects of wild - type or mutant -synuclein expression , we found that the coexpression of wild - type and a53 t mutant -synuclein in tyrosinase - overexpressing cells exacerbated dna damage and successive apoptotic cell death compared to the cells overexpressing cat or antisense -synuclein . both wild - type and a53 t mutant -synucleins coexpressed with tyrosinase resulted in the gradual accumulation of high - molecular weight complexes immunopositive for -synuclein . this band , possibly representing oligomerized forms , corresponded to the size of -synuclein tetramer and was also detected by the nbt / glycinate redox - cycling staining , suggesting that it was modified by quinones . moreover , during these processes , the mitochondrial membrane potential was specifically decreased without the activation of map kinases . although the underlying mechanism(s ) of neuronal cell death following the coexpression of tyrosinase and -synuclein are still elusive , it is likely that -synuclein modified by the oxidized catechol metabolites forms cytotoxic intermediates , that is , protofibrils . recent reports suggested that protofibrillar -synuclein tightly binds to lipid bilayers and increases the membrane permeability by forming pore - like structures [ 3335 ] . while the membranous structures damaged by protofibrils in dopaminergic nerve terminals remain unknown , intracellular organelles , such as synaptic vesicles and mitochondria , are possible candidates . in this regard , disruption of synaptic vesicle membranes would result in an increase of the cytoplasmic dopamine levels that would trigger the further accumulation of dopamine - quinone and dopamine - derived oxyradicals and thus lead to a vicious cycle . likewise , mitochondrial enzymes in the electron transport chain and the functional permeability transition pores are impaired by dopamine oxidation products making it plausible that the early damage of mitochondria observed in this cellular model reflects the actions of -synuclein protofibrils and the subsequent increase of the membrane permeability in the presence of oxidized catecholamine metabolites . we further analyzed the resting membrane potential and whole - cell membrane conductance using the ramp voltage in the cell lines expressing wild - type or mutant -synuclein . interestingly , the cells expressing a53 t -synuclein have the most depolarized membrane potential . by the application of the ramp voltage under the whole cell voltage - clamp condition , we obtained an almost linear current - voltage ( i - v ) relationship in each cell line . the slope of the i - v relationship in the cells expressing mutant -synuclein was significantly steeper than that in the cells expressing the vector alone or wild - type -synuclein , indicating that the expression of mutant -synuclein results in higher ion permeability of the plasma membrane . because it has been suggested that abnormal intracellular calcium homeostasis plays a crucial role in the pathogenesis of neurodegenerative disorders , the intracellular free calcium concentrations in -synuclein - transfected cells were quantified using a calcium indicator dye , fura-2 . notably , both the intracellular calcium concentrations under basal conditions and after depolarization induced by potassium chloride application were significantly higher in the mutant -synuclein expressing cells than in cells expressing the empty vector or wild - type -synuclein . these results suggest that mutant -synuclein is involved in the perturbation of the intracellular calcium homeostasis . taken together , our data from cellular models of synucleinopathy suggest that oligomer or protofibril , but not aggregate or fibril , formation of -synuclein plays a key role in the pathomechanisms of synucleinopathy ( figure 1 ) . the iron specifically triggers the aggregate formation of -synuclein , but this seems to be a cytoprotective process . the cytotoxic protofibril formation may be facilitated by not only gene mutations , but also the modification of -synuclein by catechol - derived quinones [ 28 , 32 ] . the cellular membranes are the primary targets injured by protofibrils , and the mitochondrial dysfunction seems to be an initial step in the neurodegeneration of synucleinopathy . obviously , protofibrils or oligomers of -synuclein are heterogeneous in size and stability and exist as mixtures in the cytosol . therefore , at present it is difficult to specify the detailed molecular structures that may be responsible for the cellular injuries . however , from these data , it is plausible that the reduction of the protofibril pool may rescue neurons from death ( figure 1 ) . if this is the case , not only the acceleration of degradation ( c in figure 1 ) but also the facilitation of aggregate formation ( d in figure 1 ) , may be a novel strategy for the treatment of synucleinopathy . of course , the most important way would be to decrease the input ( a in figure 1 ) into the protofibril pool .

-synuclein is a key molecule in the pathogenesis of synucleinopathy including parkinson 's disease and multiple system atrophy . in this mini - review , we mainly focus on recent data obtained from cellular models of synucleinopathy and discuss the possible mechanisms of neurodegeneration . recent progress suggests that the aggregate formation of -synuclein is cytoprotective and that its precursor oligomer ( protofibril ) may be cytotoxic . the catechol - derived quinones are the candidate molecules that facilitate the oligomer formation of -synuclein . furthermore , the cellular membranes are shown to be the primary targets injured by mutant -synucleins , and the mitochondrial dysfunction seems to be an initial step in the neuronal death .

since the inception of our specialty , radiologists have served as consultants to physicians of various disciplines , and we continue to diagnose , inform treatment decisions and guide management across the spectrum of medical disease . while the role of the radiologist in the multidisciplinary approach to care is little changed over time , our communication with ordering providers has evolved dramatically . widely available technologies , including the picture archiving communication system ( pacs ) , appropriateness criteria , and critical results communication systems , amongst others , have had the potential to decrease personalized , one - on - one interactions with referring providers . the means by which we fulfill our consultant role have changed , whereby information / result delivery is facilitated but free exchange of pertinent details is limited in any single clinical scenario . a variety of radiology consultation services have been described in the literature , aimed at facilitating interactions between radiologists and clinical providers to optimize patient care . consultation model types include clinical decision support , patient - centric , subspecialty interpretation , and/or some combination of these . in the clinical decision support model , radiologists are consulted by referring clinicians for assistance in radiologic procedure selection and planning . in the patient - centric model , patients are encouraged to interact directly with radiologists , for discussion of results or questions related to their imaging evaluation . the second opinion / subspecialty interpretation is generally sought out by referring clinicians or healthcare teams , but in some instances , is available directly to patients ( in the latter instance , typically for a fee ) ( table 1 ) ( 1 ) . in oncology care in particular , nuanced , yet critical clinical details may not be readily available in study indications from electronic systems . in tumor board conferences and formal or informal consultation , radiologists play an integral role in multidisciplinary discussions centered on diagnosis , staging , approach to tissue sampling and definitive versus palliative management . at referral centers , outside imaging studies are reviewed in consultation to broaden the team 's understanding of the prior workup , minimize duplication of services , and plan subsequent treatments . imaging studies performed at the home institution are also re - examined to answer specific clinical questions , which may be related to patient symptomatology , underlying tumor genomics , targeted therapy selection , predicted treatment response and/or prognosis . radiologists as consultants have both an opportunity and an obligation to demonstrate true added value to optimized treatment plans . to explore the utility and impact of radiology consultation services in the academic setting , this article will further describe existing consultation models and the circumstances that precipitated their development . in addition , the contributions of a consultant radiologist in breast cancer care are reviewed as the archetype of radiology consultation services provided to oncology practitioners . the concept of a radiology consultation service was first reported in 1979 , when shuman and heilman ( 2 ) described their 2 year experience offering formal radiologic consults to clinicians anticipating complex radiologic work - ups . referring clinicians would utilize the standard consultation form used for their services , which was then forwarded to the radiology resident assigned to consult that month . the resident would carefully review the patient 's medical record , discuss issues with the appropriate services , and when indicated , perform a physical exam , and examine all prior pertinent imaging studies . a comprehensive radiologic evaluation plan was devised , discussed with the appropriate attending radiologist , and then conveyed to the requesting clinician . the impetus for starting this service arose from the rising cost of healthcare , with often indiscriminate use of expensive imaging tests , and the simultaneous explosion of technological advances in radiology , as well as marked growth of interventional radiology procedures . the authors reasoned that radiologists are most knowledgeable about varied imaging modalities and their best utilization , as well as the capabilities of radiologic intervention , and should thus be consulted when making imaging decisions regarding complex cases . they describe the mixed responses of both the referring clinicians ( some disliking the intrusion into their diagnostic arena ) , and the radiology residents and staff ( some of whom were uneasy with the expansion of clinical interactions ) , but promoted the concept as deserving of further evaluation . several other academic radiology departments have instituted various forms of consultation , using the clinical decision support model , in some cases , mandating approval , and in others , offering it as an optional service and educational tool . baker ( 3 ) analyzed several different scenarios where consult radiologists would provide guidance to clinicians in selecting appropriate radiologic studies and/or in planning imaging workups , and showed utility when compared to a control group without access to radiologic consultation . in a pilot study of patients presenting with biliary tract disease , non - emergent gastrointestinal bleeding or abdominal mass , they reported that actively structuring the diagnostic evaluation by a consultant radiologist resulted in a 64% reduction in time to diagnosis and 32% reduction in number of studies performed ( 3 ) . by embedding a radiology consultant in daily medical rounds on an acute care medical ward , whereby all radiology requests were discussed daily , they reported significantly fewer radiologic examinations were performed , while house staff were educated on proper utilization of imaging tests , a potential means of cost containment ( 4 ) . in a third study , on an acute care surgical ward , a consultant radiologist reviewed all radiographic studies performed , discussing their limitations , pertinence and cost , and future examination were discussed . over the study period , the number of ct scans , ultrasound studies and barium studies decreased significantly , and the average length of patient stay decreased by 2.8 days ( 5 ) . ( 6 ) reported a study whereby all body ct scans required consultation with a radiologist prior to scheduling . they noted 95% cooperation by referring clinicians , and described benefits of obtaining more detailed clinical information , thus providing improved scanning protocols , fewer post - ct diagnostic tests , and improved rapport between the referring clinicians and radiologists . with the ever present reminder of cost containment looming , the role of the radiologist as a consultant offering clinical decision support has been further investigated using several study designs , including optional vs. mandatory , assisting house staff vs. all referring clinicians , incorporated into rounds vs. a stand - alone service ( 789 ) . in most institutions it was generally well received , but has had variable success in decreasing the number of studies ordered . ( 8) found that mandatory radiology precertification in an inpatient setting did not reduce radiology resource utilization . they noted that although participating physicians were supportive , internal medicine ward physicians did not reduce their test ordering , and radiologists , trained in the interpretation of exams , rather than assessment of appropriateness , were reluctant to perform precertification . the authors suggested that practice guidelines might help standardize the consultation process , and that financial incentives may motivate physicians to reduce ancillary tests . with the advent of pacs , direct interactions with radiologists in reading rooms has declined , in one study , by 82% for general radiography , and 44% for cross sectional imaging studies , despite an increase in overall volume ( 10 ) . as consultation has been shown to improve cost containment and allow for a more rapid diagnosis , improved communication between referring clinicians and radiologists is imperative . tillack and borgstede ( 11 ) looked at the impact of embedding radiology reading rooms in the clinic , noting a significantly greater number of visits by referring providers to embedded reading rooms ( though the study was slightly confounded by comparing embedded versus non - embedded reading rooms of different specialties , with inherent differences in consultation patterns ) . the present emphasis on value ( over volume ) in health imaging is strongly touted by the american college of radiology ( acr ) , as noted in imaging 3.0 , which encourages radiologists to assist providers with clinical decision support , to discuss results and provide actionable , meaningful reports ( 12 ) . it is felt that clinical decision support helps to eliminate unnecessary examinations and increases the radiologists ' relevance , as well as provides radiologists with better detailed patient information , which allows for more directed protocols and accurate coding for billing . in 1966 , sherman ( 13 ) asked , is it not really the patient we are obligated to serve above all others ? , and suggested that patients ( not their clinicians ) are entitled to results of their exams . in 1990 , the beginning of malpractice litigation that alleged failure of communication of radiologic results was brought to light in the american college of radiology ( acr ) bulletin ( 14 ) . berlin noted that soon after the expansion of radiologist 's duty to directly convey significant radiologic findings to the referring clinician , their role was further extended to include direct communication to patients , fueled by the judicial system , the federal government , the medical community and patients ( 15 ) . he cites numerous lawsuits where radiologists were found to be negligent for not communicating significant results directly to patients . the united states government 's involvement is evident in the mammography quality standards act , published in october 1992 , mandating that all patients receive a summary of their mammographic findings , in layman 's terms , within 30 days of their mammogram . berlin further notes that both the american medical association and acr have language in their bylaws and guidelines , describing the responsibility of the radiologist ( and all consulting physicians ) to communicate results directly to patients , and answer any questions they might have . however , he goes on to discuss the various difficulties that might arise , including selecting the format of transmissions , the affect on relationships with referring physicians , placing the radiologist in the unenviable position of being asked to advise on treatment options in which he / she is not involved and possibly less knowledgeable . several older studies that surveyed patients undergoing radiologic imaging reported patient preferences to hear imaging results from the radiologists interpreting their exams . a small study of 79 cancer patients undergoing ct imaging at a dedicated cancer center reported that 70% wanted the radiologist to disclose their results ( 16 ) . in a survey of patient preferences of 261 consecutive patients undergoing radiologic evaluation in a large university hospital , the overwhelming majority ( 92% for normal findings , 87% for abnormal findings ) also preferred to get results from the radiologist at the time of the procedure , rather than later , from their referring clinician ( 17 ) . a follow - up survey of referring clinicians and radiologists concluded that radiologists and referring physicians both tended to support the proposition that , if asked by patients , radiologists should disclose the results of their imaging studies ( 18 ) . however , given the uncertainties of many , direct patient radiologist communication has not become the standard of care in most general radiology practices . recently , the consultation radiologist role has resurfaced in the literature , as a means of increasing the radiologists ' visibility to patients . in most scenarios , the ordering physician conveys results to the patient , who is often unaware that a radiologist has played any role in providing his or her care . several institutions have revisited the exploration of patient preferences in receiving results of radiologic studies . a survey of 129 patients undergoing ct or mri at an academic medical center outpatient facility revealed that speed of report delivery was the most important factor in patient satisfaction , without an overwhelming preference for which physician provided the results ( 19 ) . in another study of 86 patients undergoing ct or ultrasound evaluation , patients preferred hearing results from both their referring clinicians and the interpreting radiologist , and found the latter consultation beneficial ( 20 ) . however , at least 2 other studies noted that most patients preferred to hear from their referring clinicians . 642 responses to a survey of patients undergoing ct or mri revealed that for both normal and abnormal results , the preferred mode of communication was a phone call from the referring physician ( 34.1% and 49.8% , respectively ) ( 21 ) . patients also preferred detailed radiology reports , with some requesting access to key images . in a similar study of patients undergoing ct or mri at an academic medical center ( 77% ) or a county hospital ( 23% ) , among 617 surveys , 63% revealed preference for receiving results from their ordering providers ; 64% desired access to their imaging report , and 85% wished to see their images ( 22 ) . mangano et al . ( 23 ) have piloted a patient - centered radiology consultation area , where patients meet with a radiologist to discuss their imaging study results . the impetus in piloting this service was to increase the visibility of the radiologist , and increase awareness of the critical role imagers play in healthcare . this has been among the goals of several acr outreach campaigns , including the face of radiology campaign ( 24 ) . another academic medical center created a unique consultation service , devised to address the ambiguous information legally mandated to be shared with patients following mammograms that reveal dense breast tissue . following mammography , patients are presented with data regarding breast density and cancer , though without evidence - based guidelines for further evaluation . sullivan et al . ( 25 ) report their experience in providing consultation services to these patients ( and referring clinicians ) , by employing a registered radiology assistant ( rra ) , who they define as a mid - level provider who has received advanced education and clinical experience in radiology . the rra receives the requests , collects pertinent information and prepares the consultation , thus improving efficiency and optimizing the radiologist 's time spent in direct consultation with the patient . although the consultation service was only utilized by 138 of 7131 ( 2% ) of their dense breast patient population ( plus an additional 14 patients whom heard about the service from other sources ) , over 70% felt sure of their best choice for additional screening and over 78% felt they had gained enough information to make a decision regarding screening following their consultations . the authors concluded that their service provides education , and ultimately improves quality of care . the subspecialty interpretation model is based on a general consensus that subspecialists provide better care in their area of expertise than generalists . although a study evaluating interpretation of pediatric plain radiographs at a rural hospital did not show a statistically significant difference in interpretation between pediatric radiologists and generalists , the authors note that the subspecialists were more accurate ( 26 ) . in another study , the detection of extracapsular extension of prostate cancer on mri was significantly affected by the reader 's subspecialty experience ( 27 ) . several tertiary care centers have looked at the quantitative effect of radiology consultations on management of cancer patients . one center reported the addition of significant new information in 49% of patients reviewed at their division of oncology radiology conference , resulting in major changes in management in 37% ( 28 ) . two different centers specifically looked at reinterpretation of imaging studies in head and neck cancer patients . the first reported a change in interpretation of 41% , with statistically significant changes in tnm staging in 34% of patients , which altered treatment plans in 98% and affected prognosis in 95% ( 29 ) . changes were attributed to lack of subspecialization in head and neck imaging at the outside institution , and to often incomplete clinical information of the original interpreting radiologist . in a similar study , fellowship trained neuroradiologists provided formal second opinions on head and neck imaging studies , resulting in changes in stage in 56% and management changes in 38% , noting 93% accuracy based on pathologic staging as gold standard ( 30 ) . a recent study at our cancer center evaluated the influence of a second opinion / subspecialty consultation on the surgical management of breast cancer patients ( 31 ) . they reported changes in 11.7% of patients undergoing surgery for breast cancer . given the variability in quality of outside studies and their interpretations , and the differences that a comprehensive evaluation may make in a patient 's prognosis and treatment strategy , review of outside studies by a specialized oncologic imager is critically important . changes in patient care due to direct subspecialty radiology consultation have recently been reported in disciplines other than oncology . ( 32 ) reviewed over 2000 second opinion subspecialty consultations in musculoskeletal radiology , and noted clinically important differences in interpretation in 26.2% overall , and discrepant interpretations in 36.3% of oncologic cases . based on the final pathologic diagnosis , they reported that second opinion consultations were correct in 82.0% of cases where discrepancies were likely to change patient management . dickerson et al . ( 33 ) also reported significant alterations in surgical decision making when acute care surgeons collaborated directly and in - person with radiologists . they reported that surgeons ' diagnostic impressions and medical and/or surgical planning were altered in 43% of cases , and concluded that the in - person collaboration promoted a shared mental model that facilitates the exchange of complex information . of note , some departments have taken a different approach , choosing to issue a formal dictation of outside studies rather than a verbal consultation and bill for a second opinion read . yousem ( 34 ) chose to upload outside neuroradiology studies to their electronic archive and provide an official dictation , noting the benefits to the referring clinicians including less hassle in bringing studies to radiologists , increased ease in making images available to operating rooms and conference rooms ( via pacs ) , provision of subspecialty interpretations , access to the outside preoperative studies when evaluating for residual disease postoperatively , decreased interruption to workflow ( as studies are now read from a pacs system work list ) , and monetary reimbursement . given their success in neuroradiology , they have now initiated this approach throughout their department . at our tertiary care cancer center , imaging plays an integral role in optimizing patient care by guiding decisions regarding diagnosis , staging , tissue sampling , trial eligibility and treatment . since pacs enables remote access to imaging studies , radiologists are commonly called upon to lend expertise in viewing both our own and outside imaging studies . a large study at our own institution over a decade ago looked at the volume and impact of second opinion consultations , noting an overall increase in daily workload of 18% ( 35 ) . the impact on workflow , finances , and compensation in terms of relative value units were all considered . based on the strict reimbursement requirements ( which include a written report and documentation of medical necessity ) , as well as the variable quality of outside imaging studies and time needed to thoroughly review and redictate a study , it was deemed not cost effective to issue formal reports on outside consultation cases . however , referring clinicians strongly favored and valued this service , therefore institute administrators deemed it worthwhile to contract with the imaging department to provide consultation services by a radiologist located within the clinic . our decision to participate was not only a matter of convenience , profit , and productivity , but one that has been shown to greatly benefit clinicians and patients alike . monetary compensation for this dedicated radiologist allowed the hiring of an additional full time employee , allowing for fewer interruptions to the radiologists interpreting our home institution 's studies , and incidentally increasing productivity overall . although our survey only captured consultations on outside studies or on our studies that required further comparison , the consult radiologist is responsible for reviewing both on - site studies , as well as those from associated institutions , and is available to perform tumor measurements for clinical trial patients . at our institution , the success of the consultation service and increasing patient volume has resulted in expanded general and subspecialty oncologic imaging consultation services . the consult radiologist provides several services , including ( though not limited to ) clinical decision support , interpretations , advice regarding biopsy or treatment planning , and determination of measurable disease to confirm trial eligibility . given the ease of access and face - to - face interaction , we have noted increased dependence on our interpretation of all studies , including studies from both outside and inside the network , and also our daily services from within the institution . most importantly , however , the interactions between imagers and oncologists , surgeons , radiation oncologists , nurses and physician assistants , have grown more collegial , and in fact have facilitated research opportunities / collaborative efforts . thus , radiologists have become more integrated into the multidisciplinary approach which is so critical in caring for cancer patients ( 36 ) . ( 37 ) describe this integrated consultation service as only a part of their vision for the role of imaging in optimizing cancer care . they support the use of imaging in pre - clinical studies to facilitate translational research , and also note the importance of effective , specialized training programs for future oncologic imagers . a brief evaluation of various treatment decisions in a breast cancer patient 's medical journey elucidates the critical role that radiologists play as consultants to surgeons , radiation oncologists and medical oncologists . after a breast cancer diagnosis although a patient may opt for a lumpectomy versus mastectomy by personal choice , the radiologist , upon evaluation of the breast imaging studies , will assist the surgeon in determining whether breast conservation is feasible . additional sites or wider extent of disease seen with mammography , ultrasound , or mri , may preclude planned breast conservation surgery . the breast imager is not infrequently consulted by the radiation oncologist when planning radiation therapy for breast cancer patients . the presence of internal mammary lymphadenopathy may change the radiation field , and therefore the radiologist is sought out to provide an opinion regarding the presence of internal mammary lymph nodes and their exact location on mri and ct , so that radiation treatment can be accurately planned . patients with a diagnosis of inflammatory breast cancer undergo additional staging with pet - ct imaging , which in a small study at our institution altered the radiation treatment planning in 24% ( fig . 1 ) ( 38 ) . knowledge of a patient 's breast tumor pathology will also affect the consult radiologist 's approach to disease evaluation and recommendations for future imaging . in cases of invasive lobular cancer , which are often mammographically occult , sonography or mri may be important in better defining tumor margins . given the knowledge of different patterns of metastatic disease spread with lobular breast cancer ( versus invasive ductal cancer ) , a subspecialized oncologic radiologist is more attentive to subtle peritoneal thickening , bowel wall thickening or adnexal changes , that may herald early disease spread , thus suggesting biopsies or close follow - up ( fig . the radiologist is often consulted by the oncologist to evaluate changes , especially those not readily evaluated with standard response criteria ( such as response evaluation criteria in solid tumors [ recist ] ) . for example , the presence of increased sclerosis in a breast cancer patient with known osseous metastases at a site of previously radiologically occult disease may actually represent response , rather than disease progression as described in the university of texas md anderson cancer center criteria ( mda criteria ) , and must be viewed in light of other changes ( fig . 3 ) ( 39 ) . many , increasing , radiologic response criteria are not necessarily known to many referring clinicians . the combination of imaging expertise , knowledge of disease presentation and patterns of spread , as well as responses to therapeutic options with their potential toxicities and complications make the consultant radiologist vitally important to patient care in the oncologic setting . it is often the radiologist who will first detect signs of drug toxicity or will note disease that will preclude the use of certain medications . as newer therapies have been developed , response criteria have changed to address the varied appearances of tumor response , and different patterns of drug toxicity have been noted . therefore , it is critical that radiologists interact personally with oncologists and other members of the oncology care team to be made aware of the newest therapies . together , care teams can learn about their resultant imaging findings , as well as their potential complications and toxicities . this relationship is mutually beneficial , affording imagers the ability to stay abreast of continuously changing therapeutic advances and the opportunity to acquire information about individual patients and their specific radiologic queries , as well as providing referring clinicians with radiologic expertise to help provide the best care to their patients . in the last several years , there has been much focus on the value of imaging in healthcare , with campaigns sponsored by the acr , and multiple publications urging radiologists to take a more active role in reaching out to clinicians and patients as a consultant ( 404142 ) . this report summarizes several different models of consultation services which address various end goals including optimization of interdisciplinary care foremost . at our tertiary center , a hybrid model has been incorporated to serve the specific needs of our patients and providers , aimed at cultivating these relationships and leveraging the strengths of our cancer imaging practice . in coming years , it will be important to quantify the added value of radiology consultation , as well as secure buy - in from the stakeholders on a broader scale .

the purpose of the article is to describe the various radiology consultation models in the era of precision medicine . since the inception of our specialty , radiologists have served as consultants to physicians of various disciplines . a variety of radiology consultation services have been described in the literature , including clinical decision support , patient - centric , subspecialty interpretation , and/or some combination of these . in oncology care in particular , case complexity often merits open dialogue with clinical providers . to explore the utility and impact of radiology consultation services in the academic setting , this article will further describe existing consultation models and the circumstances that precipitated their development . the hybrid model successful at our tertiary cancer center is discussed . in addition , the contributions of a consultant radiologist in breast cancer care are reviewed as the archetype of radiology consultation services provided to oncology practitioners .